Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Spatial and temporal foraging patterns of Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), for protein and implications for management

Fruit flies require protein for reproductive development and actively feed upon protein sources in the field. Liquid protein baits mixed with insecticide are used routinely to manage pest fruit flies, such as Bactrocera tryoni (Froggatt). However, there are still some gaps in the underpinning science required to improve the efficacy of bait spray technology. The spatial and temporal foraging behaviour of B. tryoni in response to protein was investigated in the field. A series of linked trials using either wild flies in the open field or laboratory-reared flies in field cages and a netted orchard were undertaken using nectarines and guavas. Key questions investigated were the fly's response to protein relative to: height of protein within the canopy, fruiting status of the tree, time of day, season and size of the experimental arena. Canopy height had a significant response on B. tryoni foraging, with more flies foraging on protein in the mid to upper canopy. Fruiting status also had a significant effect on foraging, with most flies responding to protein when applied to fruiting hosts. B. tryoni demonstrated a repeatable diurnal response pattern to protein, with the peak response being between 12:0016:00 h. Season showed significant but unpredictable effects on fruit fly response to protein in the subtropical environment where the work was undertaken. Relative humidity, but not temperature or rainfall, was positively correlated with protein response. The number of B. tryoni responding to protein decreased dramatically as the spatial scale increased from field cage through to the open field. Based on these results, it is recommend that, to be most effective, protein bait sprays should be applied to the mid to upper canopies of fruiting hosts. Overall, the results show that the protein used, an industry standard, has very low attractancy to B. tryoni and that further work is urgently needed to develop more volatile protein baits.

Differentiation of Indian, East Timorese, Papuan and Floridian Candidatus Liberibacter asiaticus' isolates on the basis of simple sequence repeat and single nucleotide polymorphism profiles at 25 loci

Japanese isolates of Candidatus Liberibacter asiaticus have been shown to be clearly differentiated by simple sequence repeat (SSR) profiles at four loci. In this study, 25 SSR loci, including these four loci, were selected from the whole-genome sequence and were used to differentiate non-Japanese samples of Ca. Liberibacter asiaticus (13 Indian, 3 East Timorese, 1 Papuan and 8 Floridian samples). Out of the 25 SSR loci, 13 were polymorphic. Dendrogram analysis using SSR loci showed that the clusters were mostly consistent with the geographical origins of the isolates. When single nucleotide polymorphisms (SNPs) were searched around these 25 loci, only the upstream region of locus 091 exhibited polymorphism. Phylogenetic tree analysis of the SNPs in the upstream region of locus 091 showed that Floridian samples were clustered into one group as shown by dendrogram analysis using SSR loci. The differences in nucleotide sequences were not associated with differences in the citrus hosts (lime, mandarin, lemon and sour orange) from which the isolates were originally derived.

Spatial and temporal effects of grazing management and rainfall on the vertebrate fauna of a tropical savanna

Grazing by domestic livestock is one of the most widespread uses of the rangelands of Australia. There is limited information on the effects of grazing by domestic livestock on the vertebrate fauna of Australia and the establishment of a long-term grazing experiment in north-eastern Queensland at Wambiana provided an opportunity to attempt an examination of the changes in vertebrate fauna as a consequence of the manipulation of stocking rates. The aim was to identify what the relative effects of vegetation type, stocking rate and other landscape-scale environmental factors were on the patterns recorded. Sixteen 1-ha sites were established within three replicated treatments (moderate, heavy and variable stocking rates). The sites were sampled in the wet and dry seasons in 1999-2000 (T-0) and again in 2003-04 (T-1). All paddocks of the treatments were burnt in 1999. Average annual rainfall declined markedly between the two sampling periods, which made interpretation of the data difficult. A total of 127 species of vertebrate fauna comprising five amphibian, 83 bird, 27 reptile and 12 mammal species were recorded. There was strong separation in faunal composition from T-0 to T-1 although changes in mean compositional dissimilarity between the grazing stocking rate treatments were less well defined. There was a relative change in abundance of 24 bird, four mammal and five reptile species from T-0 to T-1. The generalised linear modelling identified that, in the T-1 data, there was significant variation in the abundance of 16 species explained by the grazing and vegetation factors. This study demonstrated that vertebrate fauna assemblage did change and that these changes were attributable to the interplay between the stocking rates, the vegetation types on the sites surveyed, the burning of the experimental paddocks and the decrease in rainfall over the course of the two surveys. It is recommended that the experiment is sampled again but that the focus should be on a rapid survey of abundant taxa (i.e. birds and reptiles) to allow an increase in the frequency of sampling and replication of the data. This would help to articulate more clearly the trajectory of vertebrate change due to the relative effects of stocking rates compared with wider landscape environmental changes. Given the increasing focus on pastoral development in northern Australia, any opportunity to incorporate the collection of data on biodiversity into grazing manipulation experiments should be taken for the assessment of the effects of land management on faunal species.

Sequence variation in the putative effector gene SIX8 facilitates molecular differentiation of Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc), causal agent of fusarium wilt of banana, is among the most destructive pathogens of banana and plantain. The development of a molecular diagnostic capable of reliably distinguishing between the various races of the pathogen is of key importance to disease management. However, attempts to distinguish isolates using the standard molecular loci typically used for fungal phylogenetics have been complicated by a poor correlation between phylogeny and pathogenicity. Among the available alternative loci are several putative effector genes, known as SIX genes, which have been successfully used to differentiate the three races of F. oxysporum f. sp. lycopersici. In this study, an international collection of Foc isolates was screened for the presence of the putative effector SIX8. Using a PCR and sequencing approach, variation in Foc-SIX8 was identified which allowed race 4 to be differentiated from race 1 and 2 isolates, and tropical and subtropical race 4 isolates to be distinguished from one another.

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses.

Temporal and spatial patterns of soil water extraction and drought resistance among genotypes of a perennial C-4 grass

The objective of this study was to investigate patterns of soil water extraction and drought resistance among genotypes of bermudagrass (Cynodon spp.) a perennial C-4 grass. Four wild Australian ecotypes (1-1, 25a1, 40-1, and 81-1) and four cultivars (CT2, Grand Prix, Legend, and Wintergreen) were examined in field experiments with rainfall excluded to monitor soil water extraction at 30-190 cm depths. In the study we defined drought resistance as the ability to maintain green canopy cover under drought. The most drought resistant genotypes (40-1 and 25a1) maintained more green cover (55-85% vs 5-10%) during water deficit and extracted more soil water (120-160 mm vs 77-107 mm) than drought sensitive genotypes, especially at depths from 50 to 110 cm, though all genotypes extracted water to 190 cm. The maintenance of green cover and higher soil water extraction were associated with higher stomatal conductance, photosynthetic rate and relative water content. For all genotypes, the pattern of water use as a percentage of total water use was similar across depth and time We propose the observed genetic variation was related to different root characteristics (root length density, hydraulic conductivity, root activity) although shoot sensitivity to drying soil cannot be ruled out.

Sustainable grazing management for temporal and spatial variability in north Australian rangelands – a synthesis of the latest evidence and recommendations

Rainfall variability is a major challenge to sustainable grazing management in northern Australia, with management often complicated further by large, spatially-heterogeneous paddocks. This paper presents the latest grazing research and associated bio-economic modelling from northern Australia and assesses the extent to which current recommendations to manage for these issues are supported. Overall, stocking around the safe long-term carrying capacity will maintain land condition and maximise long-term profitability. However, stocking rates should be varied in a risk-averse manner as pasture availability varies between years. Periodic wet-season spelling is also essential to maintain pasture condition and allow recovery of overgrazed areas. Uneven grazing distributions can be partially managed through fencing, providing additional water-points and in some cases patch-burning, although the economics of infrastructure development are extremely context-dependent. Overall, complex multi-paddock grazing systems do not appear justified in northern Australia. Provided the key management principles outlined above are applied in an active, adaptive manner, acceptable economic and environmental outcomes will be achieved irrespective of the grazing system applied.

Large-scale characterization of drought pattern: a continent-wide modelling approach applied to the Australian wheatbelt – spatial and temporal trends

* Plant response to drought is complex, so that traits adapted to a specific drought type can confer disadvantage in another drought type. Understanding which type(s) of drought to target is of prime importance for crop improvement. * Modelling was used to quantify seasonal drought patterns for a check variety across the Australian wheatbelt, using 123 yr of weather data for representative locations and managements. Two other genotypes were used to simulate the impact of maturity on drought pattern. * Four major environment types summarized the variability in drought pattern over time and space. Severe stress beginning before flowering was common (44% of occurrences), with (24%) or without (20%) relief during grain filling. High variability occurred from year to year, differing with geographical region. With few exceptions, all four environment types occurred in most seasons, for each location, management system and genotype. * Applications of such environment characterization are proposed to assist breeding and research to focus on germplasm, traits and genes of interest for target environments. The method was applied at a continental scale to highly variable environments and could be extended to other crops, to other drought-prone regions around the world, and to quantify potential changes in drought patterns under future climates.

Aspects and Applications of Pulse Sequence Design for Solution-State Nuclear Magnetic Resonance Spectroscopy

NMR spectroscopy enables the study of biomolecules from peptides and carbohydrates to proteins at atomic resolution. The technique uniquely allows for structure determination of molecules in solution-state. It also gives insights into dynamics and intermolecular interactions important for determining biological function. Detailed molecular information is entangled in the nuclear spin states. The information can be extracted by pulse sequences designed to measure the desired molecular parameters. Advancement of pulse sequence methodology therefore plays a key role in the development of biomolecular NMR spectroscopy. A range of novel pulse sequences for solution-state NMR spectroscopy are presented in this thesis. The pulse sequences are described in relation to the molecular information they provide. The pulse sequence experiments represent several advances in NMR spectroscopy with particular emphasis on applications for proteins. Some of the novel methods are focusing on methyl-containing amino acids which are pivotal for structure determination. Methyl-specific assignment schemes are introduced for increasing the size range of 13C,15N labeled proteins amenable to structure determination without resolving to more elaborate labeling schemes. Furthermore, cost-effective means are presented for monitoring amide and methyl correlations simultaneously. Residual dipolar couplings can be applied for structure refinement as well as for studying dynamics. Accurate methods for measuring residual dipolar couplings in small proteins are devised along with special techniques applicable when proteins require high pH or high temperature solvent conditions. Finally, a new technique is demonstrated to diminish strong-coupling induced artifacts in HMBC, a routine experiment for establishing long-range correlations in unlabeled molecules. The presented experiments facilitate structural studies of biomolecules by NMR spectroscopy.

Nucleotide sequence of initiator tRNA from Mycobacterium smegmatis

Abstract is not available.

Monitoring temporal changes in use of two cathinones in a large urban catchment in Queensland, Australia

Wastewater analysis was used to examine prevalence and temporal trends in the use of two cathinones, methylone and mephedrone, in an urban population (>200,000 people) in South East Queensland, Australia. Wastewater samples were collected from the inlet of the sewage treatment plant that serviced the catchment from 2011 to 2013. Liquid chromatography coupled with tandem mass spectrometry was used to measure mephedrone and methylone in wastewater sample using direct injection mode. Mephedrone was not detected in any samples while methylone was detected in 45% of the samples. Daily mass loads of methylone were normalized to the population and used to evaluate methylone use in the catchment. Methylone mass loads peaked in 2012 but there was no clear temporal trend over the monitoring period. The prevalence of methylone use in the catchment was associated with the use of MDMA, the more popular analogue of methylone, as indicated by other complementary sources. Methylone use was stable in the study catchment during the monitoring period whereas mephedrone use has been declining after its peak in 2010. More research is needed on the pharmacokinetics of emerging illicit drugs to improve the applicability of wastewater analysis in monitoring their use in the population.

Spatial and temporal variability in midge (Nematocera) assemblages in shallow Finnish lakes (60−70 °N) : community-based modelling of past environmental change

Multi- and intralake datasets of fossil midge assemblages in surface sediments of small shallow lakes in Finland were studied to determine the most important environmental factors explaining trends in midge distribution and abundance. The aim was to develop palaeoenvironmental calibration models for the most important environmental variables for the purpose of reconstructing past environmental conditions. The developed models were applied to three high-resolution fossil midge stratigraphies from southern and eastern Finland to interpret environmental variability over the past 2000 years, with special focus on the Medieval Climate Anomaly (MCA), the Little Ice Age (LIA) and recent anthropogenic changes. The midge-based results were compared with physical properties of the sediment, historical evidence and environmental reconstructions based on diatoms (Bacillariophyta), cladocerans (Crustacea: Cladocera) and tree rings. The results showed that the most important environmental factor controlling midge distribution and abundance along a latitudinal gradient in Finland was the mean July air temperature (TJul). However, when the dataset was environmentally screened to include only pristine lakes, water depth at the sampling site became more important. Furthermore, when the dataset was geographically scaled to southern Finland, hypolimnetic oxygen conditions became the dominant environmental factor. The results from an intralake dataset from eastern Finland showed that the most important environmental factors controlling midge distribution within a lake basin were river contribution, water depth and submerged vegetation patterns. In addition, the results of the intralake dataset showed that the fossil midge assemblages represent fauna that lived in close proximity to the sampling sites, thus enabling the exploration of within-lake gradients in midge assemblages. Importantly, this within-lake heterogeneity in midge assemblages may have effects on midge-based temperature estimations, because samples taken from the deepest point of a lake basin may infer considerably colder temperatures than expected, as shown by the present test results. Therefore, it is suggested here that the samples in fossil midge studies involving shallow boreal lakes should be taken from the sublittoral, where the assemblages are most representative of the whole lake fauna. Transfer functions between midge assemblages and the environmental forcing factors that were significantly related with the assemblages, including mean air TJul, water depth, hypolimnetic oxygen, stream flow and distance to littoral vegetation, were developed using weighted averaging (WA) and weighted averaging-partial least squares (WA-PLS) techniques, which outperformed all the other tested numerical approaches. Application of the models in downcore studies showed mostly consistent trends. Based on the present results, which agreed with previous studies and historical evidence, the Medieval Climate Anomaly between ca. 800 and 1300 AD in eastern Finland was characterized by warm temperature conditions and dry summers, but probably humid winters. The Little Ice Age (LIA) prevailed in southern Finland from ca. 1550 to 1850 AD, with the coldest conditions occurring at ca. 1700 AD, whereas in eastern Finland the cold conditions prevailed over a longer time period, from ca. 1300 until 1900 AD. The recent climatic warming was clearly represented in all of the temperature reconstructions. In the terms of long-term climatology, the present results provide support for the concept that the North Atlantic Oscillation (NAO) index has a positive correlation with winter precipitation and annual temperature and a negative correlation with summer precipitation in eastern Finland. In general, the results indicate a relatively warm climate with dry summers but snowy winters during the MCA and a cool climate with rainy summers and dry winters during the LIA. The results of the present reconstructions and the forthcoming applications of the models can be used in assessments of long-term environmental dynamics to refine the understanding of past environmental reference conditions and natural variability required by environmental scientists, ecologists and policy makers to make decisions concerning the presently occurring global, regional and local changes. The developed midge-based models for temperature, hypolimnetic oxygen, water depth, littoral vegetation shift and stream flow, presented in this thesis, are open for scientific use on request.

First complete genome sequence of a capsicum chlorosis tospovirus isolate from Australia with an unusually large S RNA intergenic region

The first complete genome sequence of capsicum chlorosis virus (CaCV) from Australia was determined using a combination of Illumina HiSeq RNA and Sanger sequencing technologies. Australian CaCV had a tripartite genome structure like other CaCV isolates. The large (L) RNA was 8913 nucleotides (nt) in length and contained a single open reading frame (ORF) of 8634 nt encoding a predicted RNA-dependent RNA polymerase (RdRp) in the viral-complementary (vc) sense. The medium (M) and small (S) RNA segments were 4846 and 3944 nt in length, respectively, each containing two non-overlapping ORFs in ambisense orientation, separated by intergenic regions (IGR). The M segment contained ORFs encoding the predicted non-structural movement protein (NSm; 927 nt) and precursor of glycoproteins (GP; 3366 nt) in the viral sense (v) and vc strand, respectively, separated by a 449-nt IGR. The S segment coded for the predicted nucleocapsid (N) protein (828 nt) and non-structural suppressor of silencing protein (NSs; 1320 nt) in the vc and v strand, respectively. The S RNA contained an IGR of 1663 nt, being the largest IGR of all CaCV isolates sequenced so far. Comparison of the Australian CaCV genome with complete CaCV genome sequences from other geographic regions showed highest sequence identity with a Taiwanese isolate. Genome sequence comparisons and phylogeny of all available CaCV isolates provided evidence for at least two highly diverged groups of CaCV isolates that may warrant re-classification of AIT-Thailand and CP-China isolates as unique tospoviruses, separate from CaCV.