Deliberately introduced dung beetles in Australia: 12 years of occurrence and abundance records from 2001 to 2022.

Since 1968, the Australian Dung Beetle Project has carried out field releases of 43 deliberately introduced dung beetle species for the biological control of livestock dung and dung-breeding pests. Of these, 23 species are known to have become established. For most of these species, sufficient time has elapsed for population expansion to fill the extent of their potential geographic range through both natural and human-assisted dispersal. Consequently, over the last 20 years, extensive efforts have been made to quantify the current distribution of these introduced dung beetles, as well as the seasonal and spatial variation in their activity levels. Much of these data and their associated metadata have remained unpublished, and they have not previously been synthesized into a cohesive dataset. Here, we collate and report data from the three largest dung beetle monitoring projects from 2001 to 2022. Together, these projects encompass data collected from across Australia, and include records for all 23 species of established dung beetles introduced for biocontrol purposes. In total, these data include 22,718 presence records and 213,538 absence records collected during 10,272 sampling events at 546 locations. Most presence records (97%) include abundance data. In total, 1,752,807 dung beetles were identified as part of these data. The distributional occurrence and abundance data can be used to explore questions such as factors influencing dung beetle species distributions, dung beetle biocontrol, and insect-mediated ecosystem services. These data are provided under a CC-BY-NC 4.0 license and users are encouraged to cite this data paper when using the data.

Effects of Mixed Pasture Legume Phytoestrogens on Superovulatory Response and Embryo Quality in Angus Cows.

Ovulation and artificial insemination rates have been observed to decrease in sheep and cows when exposed to dietary phytoestrogens at concentrations greater than 25 mg/kg DM. A grazing trial was undertaken to investigate the effects of coumestrol and other key phytoestrogens on the superovulatory response, embryo numbers and quality in beef cows grazing legume pastures. A 7-week controlled grazing trial was conducted with legume and ryegrass pasture treatments, with cows exposed to legumes at two timed treatments, 4 and 7 weeks. Twenty Angus cows were subjected to a conventional estrus synchronization and superovulation protocol. Embryos were recovered via conventional uterine body flushing 7 days post artificial insemination (AI). Numerous phytoestrogens were identified in both pasture and plasma samples, including coumestrol and formononetin. Concentrations of phytoestrogens in the pasture ranged from 0.001 to 47.5 mg/kg DM and 0 to 2.6 ng/mL in plasma. Approximately 50% of cows produced viable embryos 7 days post AI. A significant interaction between the effect of treatment groups on the embryo stage was observed (p < 0.05). The results suggest that concentrations of >25 mg/kg DM of phytoestrogens less than 20 days preceding AI may negatively affect oocyte developmental competence, reduce progesterone production and thus contribute to early embryonic loss.

Field evaluation of electrophysiologically-active dung volatiles as chemical lures for trapping of dung beetles.

Dung beetles are economically important beneficial insects that process dung. To locate this source, they use volatile organic compounds (VOCs). The objectives of the study were to evaluate the attractiveness of ten electrophysiologically-active dung volatiles (phenol, skatole, indole, p-cresol, butanone, butyric acid, eucalyptol, dimethyl sulphide, dimethyl disulphide, and toluene) to dung beetles in the field and to investigate how the composition of volatile blends influences efficacy as lures for use in traps. Six combinations of the compounds were compared with field collected cattle dung bait and a negative control, across three seasons. Both dung and synthetic baits captured all exotic dung beetle species present in the study area. A six-compound mix (M1), comprising major dung volatiles, served as an attractive chemical mixture. The addition of dimethyl sulphide, dimethyl disulphide (M2) and toluene (M4) enhanced attractancy of M1 for dung beetles, while eucalyptol (M3) decreased the attractancy. The degree of attraction by various dung beetle species to synthetic baits varied, but baits proved to be effective, especially for summer trapping. The trap design used in this study presented a convenient and practical way to sample dung beetle and other associated scarabs from open pastures. The attraction of introduced dung beetle species to synthetic baits is documented here for the first time in Australia. In addition, necrophagous Omorgus sp. is reported here for the first time to be attracted to synthetic baits. They showed a significant attraction to the mixture containing dimethyl sulphide and dimethyl disulphide (M2). The current study represents a promising first step towards formulating a synthetic chemical lure for dung beetles, offering a consistent, standardised, and bio-secure trapping method compared to use of naturally occurring dung baits, especially as a multi-species lure.

Characterisation of Antennal Sensilla and Electroantennography Responses of the Dung Beetles Bubas bison, Onitis aygulus and Geotrupes spiniger (Coleoptera: Scarabaeoidea) to Dung Volatile Organic Compounds.

Locating sporadically distributed food resources and mate finding are strongly aided by volatile cues for most insects, including dung beetles. However, there is limited information on the olfactory ecology of dung beetles. We conducted a scanning electron microscopy study on the morphology and distribution of the antennal sensilla of three introduced dung beetle species in Australia: Geotrupes spiniger (Coleoptera: Geotrupidae), Bubas bison and Onitis aygulus (Coleoptera: Scarabaeidae). Three main morphological types of antennal sensilla were identified: sensilla trichodea (ST), sensilla basiconica (SB) and sensilla chaetica (SCh). Distinct variations of SB distribution were observed in B. bison and G. spiniger and on different lamellar surfaces in both sexes of all three species. Sexual dimorphism in antennal sensilla distribution or their abundance was not evident. To complement the morphological characterisation of sensilla, electroantennography (EAG) was carried out to construct EAG response profiles of the three species to selected dung volatiles. An initial study revealed that antennae of all species were sensitive to a mix of phenol, skatole, indole, p-cresol, butanone and butyric acid, common components of livestock dung headspace. In addition to these six compounds, dimethyl sulfide, dimethyl disulfide, eucalyptol and toluene were tested for antennal activity. All compounds evoked measurable EAG responses, confirming antennal sensitivity. Geotrupes spiniger exhibited significant responses to all the compounds compared to the control, whereas B. bison and O. aygulus only responded to a subset of compounds. A comparison of relative EAG amplitudes revealed highly significant responses to p-cresol in G. spiniger and to skatole in B. bison. Geotrupes spiniger displayed differential responses to all the compounds. Pooled EAG data suggest highly significant differences in responses among the three species and among compounds. Our findings suggest that a blend of volatiles may offer potential for the trapping of dung beetles, thereby avoiding the use of dung baits that are inconvenient, inconsistent and may pose a threat to farm biosecurity.

Contrasting Volatilomes of Livestock Dung Drive Preference of the Dung Beetle Bubas bison (Coleoptera: Scarabaeidae).

Volatile cues can play a significant role in the location and discrimination of food resources by insects. Dung beetles have been reported to discriminate among dung types produced by different species, thereby exhibiting behavioral preferences. However, the role of volatile organic compounds (VOCs) in dung localization and preference remains largely unexplored in dung beetles. Here we performed several studies: firstly, cage olfactometer bioassays were performed to evaluate the behavioral responses of Bubas bison (Coleoptera: Scarabaeidae) to VOCs emanating from fresh horse, sheep, and cattle dung; secondly, concurrent volatilome analysis was performed to characterize volatilomes of these dung types. Bubas bison adults exhibited greater attraction to horse dung and less attraction to cattle dung, and they preferred dung from horses fed a pasture-based diet over dung from those fed lucerne hay. Volatilomes of the corresponding dung samples from each livestock species contained a diverse group of alkanes, alkenes, alkynes, alcohols, aldehydes, ketones, esters, phenols, and sulfurous compounds, but the composition and abundance of annotated VOCs varied with dung type and livestock diet. The volatilome of horse dung was the most chemically diverse. Results from a third study evaluating electroantennogram response and supplementary olfactometry provided strong evidence that indole, butyric acid, butanone, p-cresol, skatole, and phenol, as well as toluene, are involved in the attraction of B. bison to dung, with a mixture of these components significantly more attractive than individual constituents.

Discovery, validation, and diagnostic ability of multiple protein-based biomarkers in saliva and gingival crevicular fluid to distinguish between health and periodontal diseases.

AIM: To discover and validate differential protein biomarker expression in saliva and gingival crevicular fluid (GCF) to discriminate objectively between periodontal health and plaque-induced periodontal disease states. MATERIALS AND METHODS: One-hundred and ninety participants were recruited from two centres (Birmingham and Newcastle upon Tyne, UK) comprising healthy, gingivitis, periodontitis, and edentulous donors. Samples from the Birmingham cohort were analysed by quantitative mass spectrometry proteomics for biomarker discovery. Shortlisted candidate proteins were then verified by enzyme-linked immunosorbent assay in both cohorts. Leave-one-out cross validation logistic regression analysis was used to identify the best performing biomarker panels. RESULTS: Ninety-five proteins were identified in both GCF and saliva samples, and 15 candidate proteins were selected based upon differences discovered between the donor groups. The best performing panels to distinguish between: health or gingivitis and periodontitis contained matrix metalloproteinase-9 (MMP9), S100A8, alpha-1-acid glycoprotein (A1AGP), pyruvate kinase, and age (area under the curve [AUC] 0.970); health and gingivitis contained MMP9, S100A8, A1AGP, and pyruvate kinase, but not age (AUC 0.768); and mild to moderate and advanced periodontitis contained MMP9, S100A8, A1AGP, pyruvate kinase, and age (AUC 0.789). CONCLUSIONS: Biomarker panels containing four proteins with and without age as a further parameter can distinguish between periodontal health and disease states.

Evaluation of Barley Cultivars for Competitive Traits in Southern New South Wales.

The potential of multi-purpose barley (Hordeum vulgare L.) cultivars to suppress weeds while maintaining optimal yield and grain quality has been reported but not recently evaluated in replicated field trials performed under southern Australian field conditions. Therefore, to investigate this potential, aboveground competitive traits were assessed in nine genetically diverse commercial barley cultivars in 2015, 2016 and 2017, in two locations in the Riverina region of NSW in replicated field trials performed in the absence of pre-emergent herbicide treatment. Crop and weed establishment, early vigour, leaf area index, photosynthetically active radiation (PAR) and biomass were assessed at various crop growth stages, including early growth, vegetative, flowering, grain fill and harvest. Cultivar differences in crop and weed biomass accumulation at ~50, 100 and 150 days after planting were noted at both locations. Early barley biomass accumulation was inversely related to weed biomass in both locations and most years, suggesting strong (over 90%) potential for heritable competitive barley interference against weeds. The current study also observed a positive relationship between PAR light interception and crop biomass in all three years at both locations, suggesting that PAR light interception contributed positively to crop biomass accumulation by directly increasing photosynthesis (50-70%) and growth or indirectly influencing weed biomass accumulation (10-15%) and weed interference (50-75). Partial least squares modelling was performed with 2015 and 2016 datasets to assess the interactions between crop developmental traits and weed suppression. Cultivars exhibiting enhanced early vigour and PAR light interception were generally more weed suppressive under optimal higher soil moisture conditions. Our results indicate that the choice of barley cultivar has a significant impact on weed establishment, fecundity and seedbank dynamics. The use of competitive barley genotypes is, thus, a cost-effective strategy to reduce weed seedbank numbers over time and may reduce potential herbicide use.

Path Meander of Male Codling Moths (Cydia pomonella) Foraging for Sex Pheromone Plumes: Field Validation of a Novel Method for Quantifying Path Meander of Random Movers Developed Using Computer Simulations.

Measures of path meander are highly relevant to studies of optimal foraging by animals. However, directly recording paths of small animals such as insects can be difficult because of small size or crepuscular activity. Computer simulations of correlated random walkers demonstrated that the rates of decay in captures across a rectangular grid of traps when movers were released at its corner can be used to produce calibration curves for quantifying path meander indirectly. Simulations using spatial parameters matching those previously documented for male codling moths (Cydia pomonella (L.)) foraging for female pheromone plumes in the field predicted that meander, as measured in circular standard deviation (c.s.d.) of turn angles between track segments, should be ca. 50° and 30° when the target population density is high vs. low, respectively. Thus, if optimized, the mean value measured for C. pomonella populations encountering an unknown target density should fall between these limits. We recorded decay in C. pomonella catch across a 5 × 5 grid of pheromone-baited traps each separated by 15 m on 39 occasions where batches of ca. 800 males were released 10 m outside the corner of trapping grids arranged in five large Michigan apple orchards. This decay constant was translated into mean c.s.d value for path meander using the standard curve generated by the computer simulations. The measured decay constant for C. pomonella males was negative 0.99 ± 0.02 (S.E.M.), which translates to a path meander of 37 ± 2° c.s.d. Thus, the measured path meander of 37° fell between the 50° and 30° values optimal for dense and sparse populations, respectively. In addition to providing a rare documented example of optimal foraging for odor plumes, this research offers proof-of-concept for a novel approach to quantifying path meander of movers that could prove useful across diverse taxa.

Toxic Potential and Metabolic Profiling of Two Australian Biotypes of the Invasive Plant Parthenium Weed (Parthenium hysterophorus L.).

Parthenium weed (Parthenium hysterophorus L.) is an invasive plant species in around 50 countries and a 'Weed of National Significance' in Australia. This study investigated the relative toxicity of the leaf, shoot and root extracts of two geographically separate and morphologically distinct biotypes of parthenium weed in Queensland, Australia. Parthenium weed exhibited higher phytotoxic, cytotoxic and photocytotoxic activity in leaf tissue extracts in contrast to shoot and root. The germination and seedling growth of a dicot species (garden cress) were inhibited more than those of a monocot species (annual ryegrass) using a phytotoxicity bioassay. The cytotoxicity of leaf extracts was assessed in a mouse fibroblast cell suspension assay and increased under high ultraviolet A(UV-A) radiation. A major secondary metabolite, parthenin, was found in abundance in leaf extracts and was positively correlated with cytotoxicity but not with photocytotoxicity or phytotoxicity. Ambrosin and chlorogenic acid were also detected and were positively correlated with germination inhibition and the inhibition of radicle elongation, respectively. In addition, other currently unidentified compounds in the leaf extracts were positively correlated with phytotoxicity, cytotoxicity and photocytotoxicity with two to three molecules strongly correlated in each case. Both parthenium weed biotypes investigated did not differ with respect to their relative toxicity, despite their reported differences in invasive potential in the field. This suggests that secondary chemistry plays a limited role in their invasion success.

Rehmannia glutinosa Replant Issues: Root Exudate-Rhizobiome Interactions Clearly Influence Replant Success.

Production of medicinal tubers of Rehmannia glutinosa is severely hindered by replanting issues. However, a mechanistic understanding of the plant-soil factors associated with replant problems is currently limited. Thus, we aimed to identify the R. glutinosa root exudates, evaluate their potential phytotoxicity and profile the interactions between the plant and its associated rhizobiome. Stereomicroscopy and liquid chromatography coupled to a quadrupole/time of flight mass spectrometer were used to monitor and identify secreted metabolites, respectively. Seedling bioassays were used to evaluate the phytotoxicity of R. glutinosa root exudates. Two complimentary experiments were performed to investigate allelochemical fate in rhizosphere soil and profile the associated microbiota. Root specific microbes were further isolated from R. glutinosa rhizosphere. Impacts of isolated strains were evaluated by co-cultivation on plate and on seedlings in tissue culture, with a focus on their pathogenicity. Interactions between key R. glutinosa root exudates and isolated rhizobiomes were investigated to understand the potential for plant-soil feedbacks. Quantification and phytotoxic analysis of metabolites released from R. glutinosa indicated catalpol was the most abundant and bioactive metabolite in root exudates. Subsequent microbial profiling in soil containing accumulated and ecologically significant levels of catalpol identified several taxa (e.g., Agromyces, Lysobacter, Pseudomonas, Fusarium) that were specifically shifted. Isolation of R. glutinosa rhizobiomes obtained several root specific strains. A significant antagonistic effect between strain Rh7 (Pseudomonas aeruginosa) and two pathogenic strains Rf1 (Fusarium oxysporum) and Rf2 (Fusarium solani) was observed. Notably, the growth of strain Rh7 and catalpol concentration showed a hormesis-like effect. Field investigation further indicated catalpol was increasingly accumulated in the rhizosphere of replanted R. glutinosa, suggesting that interactions of biocontrol agents and pathogens are likely regulated by the presence of bioactive root exudates and in turn impact the rhizo-ecological process. In summary, this research successfully monitored the release of R. glutinosa root exudates, identified several abundant bioactive R. glutinosa secreted metabolites, profiled associated root specific microbes, and investigated the plant-soil feedbacks potentially regulated by catalpol and associated rhizobiomes. Our findings provide new perspectives toward an enhanced understanding R. glutinosa replant problems.

Metabolic Profiling Provides Unique Insights to Accumulation and Biosynthesis of Key Secondary Metabolites in Annual Pasture Legumes of Mediterranean Origin.

Annual legumes from the Mediterranean region are receiving attention in Australia as alternatives to traditional pasture species. The current study employed novel metabolic profiling approaches to quantify key secondary metabolites including phytoestrogens to better understand their biosynthetic regulation in a range of field-grown annual pasture legumes. In addition, total polyphenol and proanthocyanidins were quantified using Folin-Ciocalteu and vanillin assays, respectively. Metabolic profiling coupled with biochemical assay results demonstrated marked differences in the abundance of coumestans, flavonoids, polyphenols, and proanthocyanidins in annual pasture legume species. Genetically related pasture legumes segregated similarly from a chemotaxonomic perspective. A strong and positive association was observed between the concentration of phytoestrogens and upregulation of the flavonoid biosynthetic pathway in annual pasture legumes. Our findings suggest that evolutionary differences in metabolic dynamics and biosynthetic regulation of secondary metabolites have logically occurred over time in various species of annual pasture legumes resulting in enhanced plant defense.

PGRMC1 effects on metabolism, genomic mutation and CpG methylation imply crucial roles in animal biology and disease.

BACKGROUND: Progesterone receptor membrane component 1 (PGRMC1) is often elevated in cancers, and exists in alternative states of phosphorylation. A motif centered on PGRMC1 Y180 was evolutionarily acquired concurrently with the embryological gastrulation organizer that orchestrates vertebrate tissue differentiation. RESULTS: Here, we show that mutagenic manipulation of PGRMC1 phosphorylation alters cell metabolism, genomic stability, and CpG methylation. Each of several mutants elicited distinct patterns of genomic CpG methylation. Mutation of S57A/Y180/S181A led to increased net hypermethylation, reminiscent of embryonic stem cells. Pathways enrichment analysis suggested modulation of processes related to animal cell differentiation status and tissue identity, as well as cell cycle control and ATM/ATR DNA damage repair regulation. We detected different genomic mutation rates in culture. CONCLUSIONS: A companion manuscript shows that these cell states dramatically affect protein abundances, cell and mitochondrial morphology, and glycolytic metabolism. We propose that PGRMC1 phosphorylation status modulates cellular plasticity mechanisms relevant to early embryological tissue differentiation.

Production of pyrrolizidine alkaloids and shikonins in Echium plantagineum L. in response to various plant stressors.

BACKGROUND: Echium plantagineum, a native of Europe and Africa, is a noxious invasive weed in Australia forming monocultural stands in pastures and rangelands. It produces a complex mixture of bioactive secondary metabolites, including toxic pyrrolizidine alkaloids (PAs), that protect the plant from insect and livestock herbivory and naphthoquinones (NQs), which suppress competition from weeds, insects and pathogens, and also influence invasion success. However, the extent to which allelochemical production is impacted by environmental factors, thereby influencing plant defense against pests, remains unclear. RESULTS: Following plant stress induced by drought, herbivory and high temperature, extracts of E. plantagineum shoots and roots were subjected to metabolic profiling by UPLC-MS-DAD- QToF mass spectrometry. Abundance of NQs, especially deoxyshikonin, shikonin and dimethylacrylshikonin, rapidly increased in roots exposed to elevated temperatures. Water withholding initially increased NQ abundance, but prolonged drought resulted in reduced total PAs and NQs. Intraspecific competition elevated the production of NQs, whereas simulated herbivory had no initial effect on NQs. Following herbivory, the abundance of the PA 3'-O-acetylechimidine-N-oxide in seedling shoots was increased. CONCLUSIONS: Differential accumulation of defense metabolites by E. plantagineum following exposure to various stressors suggested stress-dependent biosynthetic regulation, particularly with respect to NQ production, which was rapidly induced following drought, intraspecific competition and high temperature treatment, thereby positively impacting resistance or defense against herbivores, weeds and pathogens. We propose that trade-offs between above- and below-ground metabolism in E. plantagineum may facilitate allelochemical production in response to stress, rendering plants with an enhanced ability to defend against other neighboring plants, insects and microbes, with allelochemical production further facilitated by catabolic recycling following lengthier exposure to stress. © 2019 Society of Chemical Industry.

Plant Density and Rhizosphere Chemistry: Does Marigold Root Exudate Composition Respond to Intra- and Interspecific Competition?

The development of techniques to non-destructively monitor allelochemical dynamics in soil using polydimethylsiloxane (PDMS) microtubing (silicone tubing microextraction, or STME) provides a means to test important ecological hypotheses regarding the roles of these compounds in plant-plant interactions. The objective of this study was to investigate the impact of intra- and interspecific competition on the exudation of thiophenes by marigolds (Tagetes patula L.). Marigolds were grown at a density of 1, 3 and 5 plants in pots (8.75 × 8.75 cm) containing two STME samplers. An additional treatment included one marigold surrounded by four velvetleaf (Abutilon theophrasti L.) plants. Marigold roots released two primary thiophenes, 3-buten-1-ynyl)-2,2'-bithienyl and α-terthienyl, which are readily absorbed by silicone microtubing. Thiophene exudation was monitored over the period 15-36 days after planting, at 2-5 day intervals. At the end of the study, root and soil samples were also analyzed for thiophene content. Thiophene production per plant increased over time, and thiophene release was strongly correlated with plant size. These results indicate that thiophene release in this study was passively controlled by resource availability. However, poor growth of velvetleaf plants competing with marigold suggests that thiophenes negatively influenced velvetleaf growth. This study, then, provides indirect evidence that thiophene exudation is insensitive to neighbor identity but differentially effective in inhibiting the growth of heterospecific neighbors.

Chemometric analysis of Amaranthus retroflexus in relation to livestock toxicity in southern Australia.

Amaranthus retroflexus L., an introduced invasive weed in southern Australia, has been associated with acute renal failure and/or mortality in a number of livestock species. While its leaves, flowers and stems are generally reported to contain high levels of nitrogen, few studies have fully characterised the chemical composition of A. retroflexus foliage with respect to mammalian toxicity. We performed extensive metabolic profiling of stems, leaves, roots and inflorescence tissues of A. retroflexus collected from three spatially and/or temporally distinct toxicity outbreaks, and report on the 1) composition of primary and secondary metabolites in methanolic extracts of A. retroflexus tissues using HPLC and HPLC-MS QToF and 2) chemometric analysis of A. retroflexus extracts in relation to the associated toxin(s). All tissues of A. retroflexus possessed an abundance of N-containing metabolites, particularly quaternary ammonium compounds which were identified as betaines, two of which (valine betaine and isoleucine betaine) are rarely encountered in plants. Cytotoxicity to murine fibroblasts was highest in extracts of leaf tissue and was associated with a single, a small modified peptide with high similarity to N-acetyl-L-α-aspartyl-L-alanyl-L-α-aspartyl-L-α-glutamyl-O-(carboxymethyl)-L-tyrosyl-L-leucinamide, a synthetic phosphotyrosyl mimic involved in cell signaling processes. One possible mode of action leading to acute renal failure in grazing livestock by a modified peptide such as this is proposed.

Metabolic Profiling and Identification of Shikonins in Root Periderm of Two Invasive Echium spp. Weeds in Australia.

Metabolic profiling can be successfully implemented to analyse a living system's response to environmental conditions by providing critical information on an organism's physiological state at a particular point in time and allowing for both quantitative and qualitative assessment of a specific subset(s) of key metabolites. Shikonins are highly reactive chemicals that affect various cell signalling pathways and possess antifungal, antibacterial and allelopathic activity. Based on previous bioassay results, bioactive shikonins, are likely to play important roles in the regulation of rhizosphere interactions with neighbouring plants, microbes and herbivores. An effective platform allowing for rapid identification and accurate profiling of numerous structurally similar, difficult-to-separate bioactive isohexenylnaphthazarins (shikonins) was developed using UHPLC Q-TOF MS. Root periderm tissues of the invasive Australian weeds Echium plantagineum and its congener E. vulgare were extracted overnight in ethanol for shikonin profiling. Shikonin production was evaluated at seedling, rosette and flowering stages. Five populations of each species were compared for qualitative and quantitative differences in shikonin formation. Each species showed little populational variation in qualitative shikonin production; however, shikonin was considerably low in one population of E. plantagineum from Western New South Wales. Seedlings of all populations produced the bioactive metabolite acetylshikonin and production was upregulated over time. Mature plants of both species produced significantly higher total levels of shikonins and isovalerylshikonin > dimethylacrylshikonin > shikonin > acetylshikonin in mature E. plantagineum. Although qualitative metabolic profiles in both Echium spp. were nearly identical, shikonin abundance in mature plant periderm was approximately 2.5 times higher in perennial E. vulgare extracts in comparison to those of the annual E. plantagineum. These findings contribute to our understanding of the biosynthesis of shikonins in roots of two related invasive plants and their expression in relation to plant phenological stage.

Ecology and genetics affect relative invasion success of two Echium species in southern Australia.

Echium plantagineum and E. vulgare are congeneric exotics first introduced to Australia in the early 1800 s. There, E. plantagineum is now highly invasive, whereas E. vulgare has a limited distribution. Studies were conducted to evaluate distribution, ecology, genetics and secondary chemistry to shed light on factors associated with their respective invasive success. When sampled across geographically diverse locales, E. plantagineum was widespread and exhibited a small genome size (1 C = 0.34 pg), an annual life cycle, and greater genetic diversity as assessed by DNA sequence analysis. It was found frequently in areas with temperature extremes and low rainfall. In contrast, E. vulgare exhibited a larger genome size (1 C = 0.43 pg), a perennial lifecycle, less chloroplast genetic diversity, and occurred in areas with lower temperatures and higher rainfall. Twelve chloroplast haplotypes of E. plantagineum were evident and incidence aligned well with reported historical introduction events. In contrast, E. vulgare exhibited two haplotypes and was found only sporadically at higher elevations. Echium plantagineum possessed significantly higher levels of numerous pyrrolizidine alkaloids involved in plant defence. We conclude that elevated genetic diversity, tolerance to environmental stress and capacity for producing defensive secondary metabolites have contributed to the successful invasion of E. plantagineum in Australia.

WITHDRAWN: Occlusal interventions for periodontitis in adults.

BACKGROUND: Occlusal interventions may be used in adults with periodontitis. At present there is little consensus regarding the indications and effectiveness of occlusal interventions in periodontal patients. OBJECTIVES: To identify and analyse the evidence for the effect of occlusal interventions on adults who have periodontitis in relation to tooth loss, probing depths, clinical attachment level, adverse effects and patient-centred outcomes. SEARCH METHODS: The search was last conducted in April 2008. We searched the Cochrane Oral Health Group's Trials Register (to 30th April 2008); the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2008, Issue 1); MEDLINE (1966 to 30th April 2008); and EMBASE (1980 to 30th April 2008). There were no language restrictions. SELECTION CRITERIA: We included randomised controlled trials (RCTs) assessing occlusal interventions in patients with periodontitis with a follow up of at least 3 months. DATA COLLECTION AND ANALYSIS: Screening of eligible studies, assessment of the methodological quality of the trials and data extraction were conducted in duplicate and independently by two review authors. Any disagreements between the review authors were resolved by discussion. The main investigator of the included trial was contacted to obtain missing information. The Cochrane Collaboration statistical guidelines were to be followed for data synthesis. MAIN RESULTS: Abstracts of 54 papers were identified by the search. One paper was eligible for inclusion. This paper studied the effect of occlusal adjustment against no occlusal adjustment in patients who were treated with non-surgical and surgical periodontal therapy. Methodological quality assessment of the included paper revealed that randomisation of the patients into the treatment groups was adequate. Allocation concealment, masking of patients and clinicians were not reported and no response to author contact was received.Mean change in attachment level and mean pocket depth were reported in the included trial. Mean difference in clinical attachment level between occlusal intervention and control in the non-surgical group amounted to 0.38 mm (95% confidence interval (CI) 0.04 to 0.72) favouring the occlusal intervention group and was statistically significant. In the surgical group the mean difference in clinical attachment level between occlusal intervention and control amounted to 0.40 mm (95% CI 0.05 to 0.75) favouring the occlusal intervention group and was also statistically significant. The difference in mean pocket depth reduction between the occlusal intervention and control in both the surgical and non-surgical groups was less than 0.1 mm and was not statistically significant. Tooth loss, patient-centred affects and adverse effects were not reported. Meta-analysis was not possible due to the inclusion of only one study. AUTHORS' CONCLUSIONS: There is only one randomised trial that has addressed this question. The data from this study are inconclusive. We therefore conclude there is no evidence for or against the use of occlusal interventions in clinical practice. This question can only be addressed by adequately powered bias-protected randomised controlled trials.

Identification and localization of bioactive naphthoquinones in the roots and rhizosphere of Paterson's curse (Echium plantagineum), a noxious invader.

Bioactive plant secondary products are frequently the drivers of complex rhizosphere interactions, including those with other plants, herbivores and microbiota. These chemically diverse molecules typically accumulate in a highly regulated manner in specialized plant tissues and organelles. We studied the production and localization of bioactive naphthoquinones (NQs) in the roots of Echium plantagineum, an invasive endemic weed in Australia. Roots of E. plantagineum produced red-coloured NQs in the periderm of primary and secondary roots, while seedling root hairs exuded NQs in copious quantities. Confocal imaging and microspectrofluorimetry confirmed that bioactive NQs were deposited in the outer layer of periderm cells in mature roots, resulting in red colouration. Intracellular examination revealed that periderm cells contained numerous small red vesicles for storage and intracellular transport of shikonins, followed by subsequent extracellular deposition. Periderm and root hair extracts of field- and phytotron-grown plants were analysed by UHPLC/Q-ToF MS (ultra high pressure liquid chromatography coupled to quadrupole time of flight mass spectrometry) and contained more than nine individual NQs, with dimethylacrylshikonin, and phytotoxic shikonin, deoxyshikonin and acetylshikonin predominating. In seedlings, shikonins were first found 48h following germination in the root-hypocotyl junction, as well as in root hair exudates. In contrast, the root cortices of both seedling and mature root tissues were devoid of NQs. SPRE (solid phase root zone extraction) microprobes strategically placed in soil surrounding living E. plantagineum plants successfully extracted significant levels of bioactive shikonins from living roots, rhizosphere and bulk soil surrounding roots. These findings suggest important roles for accumulation of shikonins in the root periderm and subsequent rhizodeposition in plant defence, interference, and invasion success.