Relative Abundance Measurements Research Articles

Characterization of Isotopic Abundance Measurements in High Resolution FT-ICR and Orbitrap Mass Spectra for Improved Confidence of Metabolite Identification

Currently there is limited information available on the accuracy and precision of relative isotopic abundance (RIA) measurements using high-resolution direct-infusion mass spectrometry (HR DIMS), and it is unclear if this information can benefit automated peak annotation in metabolomics. Here we characterize the accuracy of RIA measurements on the Thermo LTQ FT Ultra (resolution of 100,000-750,000) and LTQ Orbitrap (R = 100,000) mass spectrometers. This first involved reoptimizing the SIM-stitching method (Southam, A. D. Anal. Chem. 2007, 79, 4595-4602) for the LTQ FT Ultra, which achieved a ca. 3-fold sensitivity increase compared to the original method while maintaining a root-mean-squared mass error of 0.16 ppm. Using this method, we show the quality of RIA measurements is highly dependent on signal-to-noise ratio (SNR), with RIA accuracy increasing with higher SNR. Furthermore, a negative offset between the theoretical and empirically calculated numbers of carbon atoms was observed for both mass spectrometers. Increasing the resolution of the LTQ FT Ultra lowered both the sensitivity and the quality of RIA measurements. Overall, although the errors in the empirically calculated number of carbons can be large (e.g., 10 carbons), we demonstrate that RIA measurements do improve automated peak annotation, increasing the number of single empirical formula assignments by >3-fold compared to using accurate mass alone.

Improving Precision in Resonance Ionization Mass Spectrometry: Influence of Laser Bandwidth in Uranium Isotope Ratio Measurements

The use of broad bandwidth lasers with automated feedback control of wavelength was applied to the measurement of (235)U/(238)U ratios by resonance ionization mass spectrometry (RIMS) to decrease laser-induced isotopic fractionation. By broadening the bandwidth of the first laser in a three-color, three-photon ionization process from a bandwidth of 1.8 GHz to about 10 GHz, the variation in sequential relative isotope abundance measurements decreased from 10% to less than 0.5%. This procedure was demonstrated for the direct interrogation of uranium oxide targets with essentially no sample preparation.

In Situ Proteomic Analysis of Human Breast Cancer Epithelial Cells Using Laser Capture Microdissection: Annotation by Protein Set Enrichment Analysis and Gene Ontology

Identification of molecular signatures that allow detection of the transition from normal breast epithelial cells to malignant invasive cells is a critical component in the development of diagnostic, therapeutic, and preventative strategies for human breast cancer. Substantial efforts have been devoted to deciphering breast cancer etiology at the genome level, but only a limited number of studies have appeared at the proteome level. In this work, we compared individual in situ proteome profiles of nonpatient matched nine noncancerous, normal breast epithelial (NBE) samples with nine estrogen receptor (ER)-positive (luminal subtype), invasive malignant breast epithelial (MBE) samples by combining laser capture microdissection (LCM) and quantitative shotgun proteomics. A total of 12,970 unique peptides were identified from the 18 samples, and 1623 proteins were selected for quantitative analysis using spectral index (SpI) as a measure of protein abundance. A total of 298 proteins were differentially expressed between NBE and MBE at 95% confidence level, and this differential expression correlated well with immunohistochemistry (IHC) results reported in the Human Protein Atlas (HPA) database. To assess pathway level patterns in the observed expression changes, we developed protein set enrichment analysis (PSEA), a modification of a well-known approach in gene expression analysis, Gene Set Enrichment Analysis (GSEA). Unlike single gene-based functional term enrichment analyses that only examines pathway overrepresentation of proteins above a given significance threshold, PSEA applies a weighted running sum statistic to the entire expression data to discover significantly enriched protein groups. Application of PSEA to the expression data in this study revealed not only well-known ER-dependent and cellular morphology-dependent protein abundance changes, but also significant alterations of downstream targets for multiple transcription factors (TFs), suggesting a role for specific gene regulatory pathways in breast tumorigenesis. A parallel GOMiner analysis revealed both confirmatory and complementary data to PSEA. The combination of the two annotation approaches yielded extensive biological feature mapping for in depth analysis of the quantitative proteomic data.

Catch rates and size composition of blue sharks (Prionace glauca) caught by the Brazilian pelagic longline fleet in the southwestern Atlantic Ocean

Distribution and relative abundance of blue sharks ( Prionace glauca ) in the southwestern Atlantic Ocean was modeled based on catch-per-unit-effort (CPUE) per 1000 hooks and length frequencies of blue sharks caught by the Brazilian pelagic tuna longline fleet. As a measure of relative abundance, CPUE of blue sharks caught in 58 238 fishing sets by the Brazilian pelagic tuna longline fleet (national and chartered), from 1978 to 2009, was standardized by a Generalized Linear Model (GLM) using three different approaches: i) a negative binomial error structure (log link); ii) the traditional delta lognormal model; and iii) the Tweedie distribution, recently proposed to adjust models with a high proportion of zeros. A cluster analysis using the K-means method was used to identify target species and incorporate it as a factor into the GLM. Cluster analysis grouped the data into six different fishing clusters according to the percentage of target species. Target factor (cluster) was the most important factor explaining the variance in all three CPUE models. The Tweedie model showed a relatively better fit compared to the other models. Blue shark nominal and standardized CPUE showed a relatively stable trend from 1978 to 1995. From 1995 onwards, however, there was an increasing trend in the standardized CPUE, up to a maximum value in 2008. In general, nominal CPUE and standardized CPUE tracked well up until 2000, after which standardized CPUE’s values were at a noticeably lower level than nominal CPUE. Length frequency data were analyzed for 11 932 blue sharks measured as part of the Brazilian onboard observer program operating on the pelagic tuna longline fleet between 2006 and 2008, with sizes ranging from 91 to 224 cm fork length. Overall, blue shark size data showed clear spatial and seasonal distributions for males and females in the southwestern Atlantic Ocean, with juveniles predominantly concentrated in the most southerly latitudes.

Localization of genes for V+LDL plasma cholesterol levels on two diets in the opossum Monodelphis domestica

Plasma cholesterol levels among individuals vary considerably in response to diet. However, the genes that influence this response are largely unknown. Non-HDL (V+LDL) cholesterol levels vary dramatically among gray, short-tailed opossums fed an atherogenic diet, and we previously reported that two quantitative trait loci (QTLs) influenced V+LDL cholesterol on two diets. We used hypothesis-free, genome-wide linkage analyses on data from 325 pedigreed opossums and located one QTL for V+LDL cholesterol on the basal diet on opossum chromosome 1q [logarithm of the odds (LOD) = 3.11, genomic P = 0.019] and another QTL for V+LDL on the atherogenic diet (i.e., high levels of cholesterol and fat) on chromosome 8 (LOD = 9.88, genomic P = 5 x 10(-9)). We then employed a novel strategy involving combined analyses of genomic resources, expression analysis, sequencing, and genotyping to identify candidate genes for the chromosome 8 QTL. A polymorphism in ABCB4 was strongly associated (P = 9 x 10(-14)) with the plasma V+LDL cholesterol concentrations on the high-cholesterol, high-fat diet. The results of this study indicate that genetic variation in ABCB4, or closely linked genes, is responsible for the dramatic differences among opossums in their V+LDL cholesterol response to an atherogenic diet.

SPECIES DIVERSITY AND SEASONAL ABUNDANCE OF FRUIT PIERCING MOTH COMPLEX IN TAMIL NADU

ABSTRACT Based on the survey made at different localities in Tamil Nadu, five species of primary fruit piercers belonging to two genera viz., Othreis materna (L.), O. fullonia (Clerck), O. homaena Hubner, O. salaminia (Cram.) and Rhytia hypermnestra (Stoll) were found to feed on guava and citrus fruits. Among the five species, O. materna was the predominant piercer followed by O. fullonia and O. homaena. The species viz., O. salaminia and R. hypermnestra were very less abundant in all the localities surveyed. Species richness of fruit piercing moth was higher in Periyakulam (2.173), low in Mettupalayam (1.103). The overall measure of diversity and relative abundance of fruit piercing moths was high at Periyakulam and low in Mettupalayam. Regarding seasonal abundance, the activity of O. materna found from the second fortnight of July to till January and O. fullonia and O. homaena was observed from the first week of September and continuing up to first forthnight of January. The larvae of R. hypermnestra were collected during first week of October and moth activity was recorded from the second fortnight of September.

Evaluation of Accurate Mass and Relative Isotopic Abundance Measurements in the LTQ-Orbitrap Mass Spectrometer for Further Metabolomics Database Building

Recently, high-resolution mass spectrometry has been largely employed for compound identification, thanks to accurate mass measurements. As additional information, relative isotope abundance (RIA) is often needed to reduce the number of candidates prior to tandem MS(n). Here, we report on the evaluation of the LTQ-Orbitrap, in terms of accurate mass and RIA measurements for building further metabolomics spectral databases. Accurate mass measurements were achieved in the ppm range, using external calibration within 24 h, and remained at <5 ppm over a one-week period. The experimental relative abundances of (M+1) isotopic ions were evaluated in different data sets. First of all, 137 solutions of commercial compounds were analyzed by flow injection analysis in both the positive and negative ion modes. It was found that the ion abundance was the main factor impacting the accuracy of RIA measurements. It was possible to define some intensity thresholds above which errors were systematically <20% of their theoretical values. The same type of results were obtained with analyses from two biological media. Otherwise, no significant effect of ion transmission between the LTQ ion trap and the Orbitrap analyzer on RIA measurement errors was found, whereas the reliability of RIA measurements was dramatically improved by reducing the mass detection window. It was also observed that the signal integration method had a significant impact on RIA measurement errors, with the most-reliable results being obtained with peak height integrations. Finally, automatic integrations using the data preprocessing software XCMS and MZmine gave results similar to those obtained by manual integration, suggesting that it is relevant to use the RIA information in automatic elemental composition determination software from metabolomic peak tables.

High accuracy, high-resolution prevalence measurement for the majority of locally expressed regulatory genes in early sea urchin development

Accurate measurements of transcript abundance are a prerequisite to understand gene activity in development. Using the NanoString nCounter, an RNA counting device, we measured the prevalence of 172 transcription factors and signaling molecules in early sea urchin development. These measurements show high fidelity over more than five orders of magnitude down to a few transcripts per embryo. Most of the genes included are locally restricted in their spatial expression, and contribute to the divergent regulatory states of cells in the developing embryo. In order to obtain high-resolution expression profiles from fertilization to late gastrulation samples were collected at hourly intervals. The measured time courses agree well with, and substantially extend, prior relative abundance measurements obtained by quantitative PCR. High temporal resolution permits sequences of successively activated genes to be precisely delineated providing an ancillary tool for assembling maps of gene regulatory networks. The data are available via an interactive website for quick plotting of selected time courses.

Do DNA Microarrays Tell the Story of Gene Expression?

Poor reproducibility of microarray measurements is a major obstacle to their application as an instrument for clinical diagnostics. In this paper, several aspects of poor reproducibility are analyzed. All of them belong to the category of interpretive weaknesses of DNA microarray technology. First, the attention is drawn to the fact that absence of the information regarding post-transcriptional mRNA stability makes it impossible to evaluate the level of gene activity from the relative mRNA abundances, the quantities available from microarray measurements. Second, irreducible intracellular variability with persistent patterns of stochasticity and burstiness put natural limits to reproducibility. Third, strong interactions within intracellular biomolecular networks make it highly problematic to build a bridge between transcription rates of individual genes and structural fidelity of their genetic codes. For these reasons, the microarray measurements of relative mRNA abundances are more appropriate in laboratory settings as a tool for scientific research, hypotheses generating and producing the leads for subsequent validation through more sophisticated technologies. As to clinical settings, where firm conclusive diagnoses, not the leads for further experimentation, are required, microarrays still have a long way to go until they become a reliable instrument in patient-related decision making.

Concerns regarding the use of amphibians as metrics of critical biological thresholds: a comment on Welsh &amp; Hodgson (2008)

Summary1. Welsh &amp; Hodgson (2008) argued that stream‐associated amphibians (Ascaphus truei, Dicamptodon tenebrosus and Rhyacotriton variegatus) are reliable indicators of ecosystem health in the Pacific Northwest (PNW), U.S.A. We demonstrate that their assertions rely on circular reasoning as well as logical and empirical assumptions that have not received rigorous evaluation.2. Welsh &amp; Hodgson (2008) based their arguments on data collected in northwestern California, U.S.A. However, all three taxa occur across a wide range of environmental gradients in the PNW, and other research results suggest that the taxa respond differentially to environmental perturbations across their geographical distribution. Hence, differences in abundance are not always reliable indicators of the state of an ecosystem, as population numbers can be influenced by factors that may not be associated with habitat quality (e.g. predation and disease).3. Welsh &amp; Hodgson (2008) present indices (rather than estimates) of abundance, which contain an unquantifiable amount of uncertainty not reliably attributable to either environmental or management factors. Evaluating parameters such as reproductive success and survival are more likely to provide strong inference about factors influencing population size than correlative studies that associate some measure of relative abundance with individual habitat features.4. We argue that direct measurement of physical features (water temperature, sediment and wood), for which Welsh &amp; Hodgson (2008) suggest using amphibians as surrogates, is more accurate, efficient and cost effective compared with monitoring cryptic species with methods that are unable to quantify spatial and temporal uncertainty in biological responses.5. Headwater streams are critical components of catchments in the PNW, and all three amphibian taxa show strong associations with low‐order streams. However, we think research and monitoring of headwater streams should be guided by hypotheses that evaluate causal mechanisms, rather than relying on descriptive patterns that are limited in geographical scope and which support only weak inference about ecosystem processes. Other studies provide direct links between physical features and responses of target populations (i.e. fish). Adding a second correlative link (i.e. amphibians) provides no added benefit, and increases uncertainty in the association between management actions and changes in population size.

A Bayesian approach to estimating Atlantic salmon fry densities using a rapid sampling technique

Abstract Removal sampling by electric fishing is widely used for assessing fish population size. As it is manpower consuming, less‐demanding techniques have been proposed to increase the number of sites covered with the same human resources. These techniques essentially provide relative abundance measures. To be used for absolute abundance estimation, they need to be inter‐calibrated with another method such as removal sampling. Here, hierarchical Bayesian modelling framework is used for this inter‐calibration because it allows an estimate of absolute abundance from abundance index data alone while accounting for the main sources of uncertainty. It is applied to a 0+ juvenile Atlantic salmon, Salmo salar L. data set from 21 sites on the River Faughan, Northern Ireland. A positive relationship was found between the abundance index (number of fish caught in 5 min of actual electric fishing) and the density. The estimates from the index of abundance alone are fairly imprecise, but still allow differentiation of contrasting levels of fish density.

Host Selection by Culex pipiens Mosquitoes and West Nile Virus Amplification

Recent field studies have suggested that the dynamics of West Nile virus (WNV) transmission are influenced strongly by a few key super spreader bird species that function both as primary blood hosts of the vector mosquitoes (in particular Culex pipiens) and as reservoir-competent virus hosts. It has been hypothesized that human cases result from a shift in mosquito feeding from these key bird species to humans after abundance of the key birds species decreases. To test this paradigm, we performed a mosquito blood meal analysis integrating host-feeding patterns of Cx. pipiens, the principal vector of WNV in the eastern United States north of the latitude 36 degrees N and other mosquito species with robust measures of host availability, to determine host selection in a WNV-endemic area of suburban Chicago, Illinois, during 2005-2007. Results showed that Cx. pipiens fed predominantly (83%) on birds with a high diversity of species used as hosts (25 species). American robins (Turdus migratorius) were marginally overused and several species were underused on the basis of relative abundance measures, including the common grackle (Quiscalus quiscula), house sparrow (Passer domesticus), and European starling (Sturnus vulgaris). Culex pipiens also fed substantially on mammals (19%; 7 species with humans representing 16%). West Nile virus transmission intensified in July of both years at times when American robins were heavily fed upon, and then decreased when robin abundance decreased, after which other birds species were selected as hosts. There was no shift in feeding from birds to mammals coincident with emergence of human cases. Rather, bird feeding predominated when the onset of the human cases occurred. Measures of host abundance and competence and Cx. pipiens feeding preference were combined to estimate the amplification fractions of the different bird species. Predictions were that approximately 66% of WNV-infectious Cx. pipiens became infected from feeding on just a few species of birds, including American robins (35%), blue jays (17%, Cyanocitta cristata), and house finches (15%, Carpodacus mexicanus).

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.

Modelling and measuring single cell RNA expression levels find considerable transcriptional differences among phenotypically identical cells.

BackgroundPhenotypically identical cells demonstrate predictable, robust behaviours. However, there is uncertainty as to whether phenotypically identical cells are equally similar at the underlying transcriptional level or if cellular systems are inherently noisy. To answer this question, it is essential to distinguish between technical noise and true variation in transcript levels. A critical issue is the contribution of sampling effects, introduced by the requirement to globally amplify the single cell mRNA population, to observed measurements of relative transcript abundance.ResultsWe used single cell microarray data to develop simple mathematical models, ran Monte Carlo simulations of the impact of technical and sampling effects on single cell expression data, and compared these with experimental microarray data generated from single embryonic neural stem cells in vivo. We show that the actual distribution of measured gene expression ratios for pairs of neural stem cells is much broader than that predicted from our sampling effect model.ConclusionOur results confirm that significant differences in gene expression levels exist between phenotypically identical cells in vivo, and that these differences exceed any noise contribution from global mRNA amplification.

SPECIES RESPONSES TO NITROGEN FERTILIZATION IN HERBACEOUS PLANT COMMUNITIES, AND ASSOCIATED SPECIES TRAITSEcological ArchivesE089-070

This synthetic data set contains plant species relative abundance measures from 35 nitrogen (N) fertilization experiments conducted at 10 sites across North America. The data set encompasses the fertilization responses of 575 taxa from 1159 experimental plots. The methodology varied among experiments, in particular with regard to the type and amount of N added, plot size, species composition measure (biomass harvest, pin count, or percent cover), additional experimental manipulations, and experimental duration. At each site, each species has been classified according to a number of easily identified categorical functional traits, including life history, life form, the number of cotyledons, height relative to the canopy, potential for clonal growth, and nativity to the United States. Additional data are available for many sites, indicated by references to publications and web sites. Analyses of these data have shown that N enrichment significantly alters community composition in ways that are predictable on the basis of plant functional traits as well as environmental context. This data set could be used to answer a variety of questions about how plant community composition and structure respond to environmental changes.

The Impact of Long‐Term Physical Activity on Age‐Related Changes in Protein and Gene Expression

Age‐related mitochondrial dysfunction is paralleled by reductions in gene transcripts and expression of proteins involved in cellular ATP production. Short‐term endurance exercise has been shown to enhance muscle protein synthesis in the elderly. We determined the impact of long‐term endurance exercise on gene expression and protein abundance of numerous proteins involved in cellular energetics. Vastus lateralis tissue was biopsied from 11 young sedentary (YS), 10 old sedentary (OS), 11 young trained (YT), and 11 old trained (OT) subjects. Trained subjects vigorously exercised ¡Ý 1 hr/d, ¡Ý five d/wk for ¡Ý 4 years. Large‐scale measurement of relative protein abundance was performed using isobaric tags for relative and absolute quantitation (iTRAQ) reagents and tandem mass spectrometry. Gene transcript levels were measured by high‐density oligonucleotide microarrays. Ingenuity Pathway Analysis was used to determine canonical pathways that were altered by age. Of the 33 proteins involved in cellular ATP production 27 were less abundant in OS vs. YS, while only 4 of these proteins were less abundant in OT vs. YT. Genes involved in glycolysis and oxidative phosphorylation were underexpressed in OS vs. YS, with no effects of age in OT vs. YT. CONCLUSIONS: Long‐term vigorous endurance exercise ameliorates the detrimental effects of old age on muscle gene and protein expression in humans.

Proteomics in the Study of Qualitative Platelet Defects: Validation of the Approach in the Gray Platelet Syndrome and Quebec Platelet Disorder.

Qualitative platelet defects are a heterogeneous group of disorders characterized by abnormal platelet function. Currently available laboratory assays lack sensitivity and specificity for detecting Storage Pool and Platelet Secretion defects. The Gray Platelet Syndrome (GPS) is an autosomal recessive disorder characterized by a decrease or absence of alpha granule contents. The Quebec Platelet Disorder (QPD) results from proteolysis of alpha granule proteins due to increased amounts of platelet urokinase. A decrease or deficiency of multimerin is characteristic. Proteomics is a rapidly developing field with great promise in the study of pathophysiology and biomarker development. Recent advances have made measurement of relative protein abundance in multiple samples feasible. We used one such approach, “isotope Tagging for Relative and Absolute Quantitation” (iTRAQ) to characterize the platelet proteome of healthy volunteers and patients with GPS and QPD. iTRAQ allows analysis of up to 4 different samples simultaneously, and differences in protein abundance as low as 20% are reliably detected. The platelet proteomes of 4 healthy volunteers were analyzed. Platelet proteome analysis of study patients was as follows;a.2 GPS patients compared to 2 controls;b.2 heterozygous GPS patients compared to a homozygote and a control; andc.3 QPD patients compared to a control.For comparison, platelet proteins were grouped by location into 3 groups - receptor, alpha granule and cytoskeletal proteins. The results are shown in the figure. Protein abundance in healthy volunteers was very consistent, with inter-individual variability ≤ 20%. Distribution of receptor and cytoskeletal proteins in GPS patients mirrored the controls, but alpha granule proteins were significantly decreased as expected. Comparison of homozygous to heterozygous GPS platelets showed this to be true only in the homozygotes, again, as expected (figure inset). Variable decreases in alpha granule proteins were observed in QPD patients but this was less profound. Multimerin was detected in QPD patients, but at levels significantly lower than controls. While urokinase was not detected in QPD platelets in this study, it was detected in a parallel study evaluating platelet releasates. The iTRAQ technique was reproducible and yielded consistent results, but currently is a relatively expensive and time-consuming approach. In summary, our results suggest that the platelet proteome is tightly regulated and very stable in healthy individuals. A significant decrease in alpha granule proteins was noted in patients with GPS but this was less remarkable in the QPD group, probably because peptides resulting from urokinase-mediated proteolysis are still labeled and quantified. [Display omitted]

Peptide correlation: A means to identify high quality quantitative information in large‐scale proteomic studies

A major challenge of proteomic studies is the accurate quantitation of proteins. LC-MS/MS-based methods are especially suited for profiling proteins in large sample sets. In this setup, the measurement of relative protein abundance relies on the correct quantitation of tryptic peptides. However, peptide intensities often do not unequivocally reflect the abundance of the native proteins in the sample. In this study, we show that peptides that accurately reflect relative protein abundances in large-scale sample sets can be selected based on the correlation to each other. This strategy was tested in a well-controlled experiment using a set of spiked serum samples as well as 55 clinical serum samples from schizophrenia patients and healthy volunteers. The peptide correlation analysis we present here provides an intuitive and simple procedure to obtain a high quality quantitative information from proteomics data.

Quantifying herbivory across a coral reef depth gradient

Despite the widely accepted importance of herbivory as a determinant of reef benthic community structure, few studies have examined the relative contributions of individual species to ecosystem processes at the whole reef scale. This study quantifies the grazing impact of individual species of roving herbivorous fishes across an inner shelf fringing reef at Orpheus Island, Great Barrier Reef, Australia. Estimates of roving herbivore impact based on dawn to dusk observations of feeding rates, measurement of bite sizes and relative abundance revealed that the Orpheus Island system was dominated by 3 species: Scarus rivulatus, Chlorurus microrhinos and Siganus doliatus. The estimated impact of all 3 species varied significantly across the reef depth gradient, with the rate of disturbance peaking at the crest and decreasing significantly down the slope and across the reef flat. The estimated species-specific disturbance levels suggested that during the course of a single month 104% of a square metre area of the reef crest is grazed by S. rivulatus while 40% is subject to grazing by C. microrhinos. A total of 26 cm3 of algal material is removed from the same area by S. doliatus. Overall, there was a 240-fold decrease in grazing activity across the reef flat from that at the crest. The pattern of grazing impact of the numerically dominant siganid and scarid fishes was negatively correlated with the distribution of macroalgae across the same reef gradient. The results of the current study provide support for the hypothesis that algal community structure is shaped by levels of herbivory.

Genome organization of the Chelonus inanitus polydnavirus: excision sites, spacers and abundance of proviral and excised segments

Polydnaviruses are only found in symbiotic association with parasitic wasps within the families Ichneumonidae and Braconidae (ichnoviruses and bracoviruses). They have a segmented genome consisting of circular double-stranded DNA. In the proviral linear form they are integrated in the wasp's genome; in two bracoviruses, segments were found to be clustered. Proviral segments have direct terminal repeats. Segment excision has been proposed to occur through juxtaposition of these repeats by formation of a loop and recombination; one copy of the repeat then ends up in the circular segment and one in the rejoined DNA. Here we analysed the excision/circularization site of four segments of the Chelonus inanitus bracovirus (CiV) and found that they are similar to the two already known sites; on the basis of the combined data an extended excision site motif was found. Analyses of segment flanking sequences led to the first identification of one complete and several partial spacers between proviral segments in a polydnavirus. The spacer between the proviral segments CiV14 and CiV22.5 has a length of 2065 bp; the terminal repeats of CiV14 and CiV22.5 were seen to have an opposite orientation and from this a model on the spacial organization of the loops of the proviral cluster is proposed. Through various approaches it was shown that spacers are not excised or injected into the host. Measurement of relative abundances of various segments in proviral and excised form indicates for the first time that abundant segments are present in multiple copies in the proviral form.