Michael A Nash
University of Melbourne, Zoology, Department Member
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Pesticide application is the dominant control method for arthropod pests in broad-acre arable systems. In Australia, organophosphate pesticides are often applied either prophylactically, or reactively, including at higher concentrations,... more
Pesticide application is the dominant control method for arthropod pests in broad-acre arable systems. In Australia, organophosphate pesticides are often applied either prophylactically, or reactively, including at higher concentrations, to control crop establishment pests such as false wireworms and earth mite species. Organophosphates are reported to be disruptive to beneficial species, such as natural enemies, but this has not been widely assessed in Australian systems. Neither has the risk that secondary outbreaks may occur if the natural enemy community composition or function is altered. We examine the abundance of ground-dwelling invertebrate communities in an arable field over successive seasons under rotation; barley, two years of wheat, then canola. Two organophosphates (chlorpyrifos and methidathion) were initially applied at recommended rates. After no discernible impact on target pest species, the rate for chlorpyrifos was doubled to elicit a definitive response to a le...
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Enzyme engineering is an important biotechnological process capable of generating tailored biocatalysts for applications in industrial chemical conversion and biopharma.
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We used single-molecule AFM force spectroscopy (AFM-SMFS) to screen residues along the backbone of a non-antibody protein binding scaffold (lipocalin/anticalin), and determine the optimal anchor point that maximizes binding strength of... more
We used single-molecule AFM force spectroscopy (AFM-SMFS) to screen residues along the backbone of a non-antibody protein binding scaffold (lipocalin/anticalin), and determine the optimal anchor point that maximizes binding strength of the interaction with its target (CTLA-4). By incorporating non-canonical amino acids into anticalin, and using click chemistry to attach an Fgβ peptide at internal sequence positions, we were able to mechanically dissociate anticalin from CTLA-4 by pulling from eight different anchoring residues using an AFM cantilever tip. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2-3 orders of magnitude over a force range of 50-200 pN. Five of the six internal pulling points tested were significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1-10 nN sec-1. Anisotropic network modelling and molecular dynamics simulations ...
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Fluorination of proteins by cotranslational incorporation of non-canonical amino acids is a valuable tool for enhancing biophysical stability. Despite many prior studies investigating the effects of fluorination on equilibrium stability,... more
Fluorination of proteins by cotranslational incorporation of non-canonical amino acids is a valuable tool for enhancing biophysical stability. Despite many prior studies investigating the effects of fluorination on equilibrium stability, its influence on non-equilibrium mechanical stability remains unknown. Here, we used single-molecule force spectroscopy (SMFS) with the atomic force microscope (AFM) to investigate the influence of fluorination on unfolding and unbinding pathways of a mechanically ultrastable bacterial adhesion complex. We assembled modular polyproteins comprising the tandem dyad XModule-Dockerin (XMod-Doc) bound to a globular Cohesin (Coh) domain. By applying tension across the binding interface, and quantifying single-molecule unfolding and rupture events, we mapped the energy landscapes governing the unfolding and unbinding reactions. We then used sense codon suppression to substitute trifluoroleucine (TFL) in place of canonical leucine (LEU) globally in XMod-Doc...
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Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble targets under hydrodynamic flow. Here we report the molecular mechanism behind an mechanostable protein complex responsible for resisting high... more
Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble targets under hydrodynamic flow. Here we report the molecular mechanism behind an mechanostable protein complex responsible for resisting high shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolved two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin-Cohesin (Doc-Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at ~500 pN and the other at ~200 pN at loading rates from 1-100 nN/sec. A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the low-force pathways at high loading rates, giving rise to a new mechanism of catch bonding that manifests under ...
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Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this... more
Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4'-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of mu...
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The idea that noncrop habitat enhances pest control and represents a win-win opportunity to conserve biodiversity and bolster yields has emerged as an agroecological paradigm. However, while noncrop habitat in landscapes surrounding farms... more
The idea that noncrop habitat enhances pest control and represents a win-win opportunity to conserve biodiversity and bolster yields has emerged as an agroecological paradigm. However, while noncrop habitat in landscapes surrounding farms sometimes benefits pest predators, natural enemy responses remain heterogeneous across studies and effects on pests are inconclusive. The observed heterogeneity in species responses to noncrop habitat may be biological in origin or could result from variation in how habitat and biocontrol are measured. Here, we use a pest-control database encompassing 132 studies and 6,759 sites worldwide to model natural enemy and pest abundances, predation rates, and crop damage as a function of landscape composition. Our results showed that although landscape composition explained significant variation within studies, pest and enemy abundances, predation rates, crop damage, and yields each exhibited different responses across studies, sometimes increasing and so...
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Cellulosomes are polyprotein machineries that efficiently degrade cellulosic material. Crucial to their function are scaffolds consisting of highly homologous cohesin domains, which serve a dual role by coordinating a multiplicity of... more
Cellulosomes are polyprotein machineries that efficiently degrade cellulosic material. Crucial to their function are scaffolds consisting of highly homologous cohesin domains, which serve a dual role by coordinating a multiplicity of enzymes as well as anchoring the microbe to its substrate. Here we combined two approaches to elucidate the mechanical properties of the main scaffold ScaA of Acetivibrio cellulolyticus. A newly developed parallelized one-pot in vitro transcription-translation and protein pull-down protocol enabled high-throughput atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) measurements of all cohesins from ScaA with a single cantilever, thus promising improved relative force comparability. Albeit very similar in sequence, the hanging cohesins showed considerably lower unfolding forces than the bridging cohesins, which are subjected to force when the microbe is anchored to its substrate. Additionally, all-atom steered molecular dynamics...
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Single-molecule force spectroscopy sheds light onto the free energy landscapes governing protein folding and molecular recognition. Since only a single molecule or single molecular complex is probed at any given point in time, the... more
Single-molecule force spectroscopy sheds light onto the free energy landscapes governing protein folding and molecular recognition. Since only a single molecule or single molecular complex is probed at any given point in time, the technique is capable of identifying low-probability conformations within a large ensemble of possibilities. It furthermore allows choosing certain unbinding pathways through careful selection of the points at which the force acts on the protein or molecular complex. This review focuses on recent innovations in construct design, site-specific bioconjugation, measurement techniques, instrumental advances, and data analysis methods for improving workflow, throughput, and data yield of AFM-based single-molecule force spectroscopy experiments. Current trends that we highlight include customized fingerprint domains, peptide tags for site-specific covalent surface attachment, and polyproteins that are formed through mechanostable receptor-ligand interactions. Rec...
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Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed... more
Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that wild-type Doc from Clostridium th...
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Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential... more
Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for self-assembly of the cellulosomal components into the multienzyme complex. The observed cohesin–dockerin rupture forces (>120 pN) are among the highest reported for a receptor–ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ...
Research Interests: Biophysics, Thermodynamics, Chemistry, Calcium, Atomic Force Microscopy, and 11 moreMedicine, Multidisciplinary, Macromolecular X-Ray Crystallography, Protein Stability, Unfolded Protein Response, Hydrogen Bonding, Cohesin, Amino Acid Substitution Rates, Cell Cycle Proteins, Site-directed Mutagenesis, and Clostridium thermocellum
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The composition and ecology of the millipede fauna of Victoria remain poorly understood. We collected millipedes as part of a series of ecological arthropod surveys across south-eastern Australia, focusing mainly on Victoria. These... more
The composition and ecology of the millipede fauna of Victoria remain poorly understood. We collected millipedes as part of a series of ecological arthropod surveys across south-eastern Australia, focusing mainly on Victoria. These samples almost exclusively contained millipedes from the introduced order Julida. We pursued species identification of the julids when it became apparent there were species other than the well-recorded Ommatoiulus moreleti (Lucas, 1860) (Portuguese millipede) in the samples. The majority of specimens were O. moreleti, but we also detected at least one species of Cylindroiulus Verhoeff, 1894, as well as an Ophyiulus Berlese, 1884, species, specimens of which have been identified as Ophyiulus cf. targionii. These are the first Ophyiulus records for Victoria to our knowledge. We present preliminary data on the abundance through the year of Ophyiulus. This is the first study to examine this species in Victoria and little is currently known about its likely im...
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Decomposition of organic matter is an essential process regulating fluxes of energy and matter within ecosystems. Although soil microbes drive decomposition, this is often facilitated by detritivores through comminution. The contribution... more
Decomposition of organic matter is an essential process regulating fluxes of energy and matter within ecosystems. Although soil microbes drive decomposition, this is often facilitated by detritivores through comminution. The contribution of detritivores and microbes to comminution and decomposition processes is likely to be affected by the habitat complexity. In urban ecosystems, human activities and management of vegetation and litter and soil components determine habitat complexities unobserved in natural ecosystems. Therefore, we investigated the effect of habitat complexity of low- and high-complexity urban parks and high-complexity woodland remnants on microbial decomposition and detritivore comminution using litter bags of differing mesh size. Detritivores were sampled using pitfall traps and their assemblage structure related to rates of comminution. Habitats of lower complexity had significantly lower decomposition and comminution rates. In more complex habitats, site histor...
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Habitat complexity is a major determinant of structure and diversity of ant assemblages. Following the size-grain hypothesis, smaller ant species are likely to be advantaged in more complex habitats compared to larger species. Habitat... more
Habitat complexity is a major determinant of structure and diversity of ant assemblages. Following the size-grain hypothesis, smaller ant species are likely to be advantaged in more complex habitats compared to larger species. Habitat complexity can act as an environmental filter based on species size and morphological traits, therefore affecting the overall structure and diversity of ant assemblages. In natural and semi-natural ecosystems, habitat complexity is principally regulated by ecological successions or disturbance such as fire and grazing. Urban ecosystems provide an opportunity to test relationships between habitat, ant assemblage structure and ant traits using novel combinations of habitat complexity generated and sustained by human management. We sampled ant assemblages in low-complexity and high-complexity parks, and high-complexity woodland remnants, hypothesizing that (i) ant abundance and species richness would be higher in high-complexity urban habitats, (ii) ant a...
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Alpine species are distributed across steep environmental gradients and turnover of closely related species along these clines is common. Thermal tolerance is frequently inferred as a proximal driver of both high- and low-elevation range... more
Alpine species are distributed across steep environmental gradients and turnover of closely related species along these clines is common. Thermal tolerance is frequently inferred as a proximal driver of both high- and low-elevation range limits across a wide range of species, as temperature decreases rapidly with elevation. Furthermore, locally adaptive genetic variation within species can be maintained where populations exist along an environmental gradient. Three species of alpine-endemic grasshopper (Kosciuscola genus) occupy overlapping elevation zones in the mountains of southeastern Australia. We explored role of thermal tolerance in shaping distribution patterns of the Kosciuscola in the NSW alpine region, and the potential for local adaptation in thermal limits. All species showed remarkable thermal tolerance ranges of over 50°C, reflecting the highly variable and unpredictable climate of the Australian alpine region. There were marked differences in cold tolerance between s...
Canola crops grown across southern Australia are subject to attack by over forty invertebrate pests, existing as a complex species, with threats often changing between seasons. A comparison of pest outbreak reports in canola crops from... more
Canola crops grown across southern Australia are subject to attack by over forty invertebrate pests, existing as a complex species, with threats often changing between seasons. A comparison of pest outbreak reports in canola crops from previous literature to current reports from south-eastern Australia highlighted a decrease in the importance of red legged earth mite, while lucerne flea, balaustium and bryobia mites, and the “intangible” pests that include slugs, earwigs, and weevils are increasing in prominence. Pest reports for 2013 totaled 97 compared to 2014, 143 reports have been received up to July 25. Added to the usual establishment pests, outbreaks of green peach aphid and cutworms in 2014 highlight the difficulties growers had in establishing canola. Understanding intrinsic growth rates of pest populations’ is needed in order to better predict future pest outbreaks. A new integrated pest management approach is needed where the concept of rigid management strategies is aban...
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ABSTRACT In temperate ecosystems, invasive alien species pose a major threat to species diversity. The first step in understanding likely ecological consequences of invasive species is to record new distributions. Three invertebrate... more
ABSTRACT In temperate ecosystems, invasive alien species pose a major threat to species diversity. The first step in understanding likely ecological consequences of invasive species is to record new distributions. Three invertebrate species likely to affect Australian alpine and subalpine ecosystems are recorded for the first time above 1500 masl from the Victorian Alps: European Honey Bee Apis mellifera L., Grey Field Slug Deroceras reticulatum Muller, and European Wasp Vespula germanica (F.). Single records of other invasive alien species are mentioned but not explored. I present a simple online tool that is commonly used to model the distribution of species. Results show the value of extensive sampling, naturalist records and the need for reliable species distribution data. Building ecological models that can predict the likely consequences of invasive species is reliant on continual updating of historical species distribution records.
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Parthenogenetic species are assumed to represent evolutionary dead ends, yet parthenogenesis is common in some groups of invertebrates particularly in those found in relatively constant environments. This suggests that parthenogenetic... more
Parthenogenetic species are assumed to represent evolutionary dead ends, yet parthenogenesis is common in some groups of invertebrates particularly in those found in relatively constant environments. This suggests that parthenogenetic reproduction might be common in pest invertebrates from uniform agricultural environments. Based on the evaluations of two databases from North America and Italy, we found that parthenogenetic species comprised 45 per cent (North America) or 48 per cent (Italy) of pest species derived from genera where parthenogenesis occurred, compared with an overall incidence of 10 per cent or 16 per cent in these genera. In establishing these patterns, we included only genera containing at least some member species that reproduced by parthenogenesis. The high incidence of parthenogenesis in pest species is spread across different families and several insect orders. Parthenogenetic reproduction may be favoured in agricultural environments when particular clones have a high fitness across multiple generations. Increasing the complexity and variability of agricultural environments represents one way of potentially controlling parthenogenetic pest species.
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ABSTRACT Despite research efforts spanning many decades, invertebrate pest control in arable farming systems is still heavily reliant on broad-spectrum pesticides. Yet industry wants to implement Integrated Pest Management (IPM)... more
ABSTRACT Despite research efforts spanning many decades, invertebrate pest control in arable farming systems is still heavily reliant on broad-spectrum pesticides. Yet industry wants to implement Integrated Pest Management (IPM) principles. We suspect that the lack of implementation to date reflects the unpredictable nature of outbreaks of pests and unpredictable profit margins, creating an environment where growers prefer to follow a cautious chemical-based approach. Using southern Australia as a case study, we argue for a new approach where the concept of rigid management strategies is abandoned in favor of landscape changes, host plant resistance, ecological indicators, reliable predictors and emergency intervention strategies. This approach needs to be based on developing more stable crop environments that can limit fundamental niches available for exploitation by sporadic pest populations, and increasing crop resilience to resident herbivores. Fall-back strategies, including broad-spectrum chemical control, may still be required to protect productivity at times when outbreaks of pest populations take place. These are likely to be unavoidable, particularly given emerging climate challenges.
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ABSTRACT How alpine arthropods respond to climate warming is poorly understood. Empirical approaches to address this issue include experimental warming and characterizing changes in community composition across environmental gradients.... more
ABSTRACT How alpine arthropods respond to climate warming is poorly understood. Empirical approaches to address this issue include experimental warming and characterizing changes in community composition across environmental gradients. Here we compare these short- and longer-term approaches in understanding the likely effects of warming on arthropods from grassland-heathland vegetation in the Australian sub-alpine zone. Arthropod communities showed relatively small changes in composition in response to passive experimental warming in open topped chambers (OTCs) under the ITEX (International Tundra Experiment) protocol. Collembola, Katiannidae (Collembola), Australotomurus nr. barbatus (Collembola; Entomobryidae) and Saprophytic Coleoptera increased in abundance; however, none of these patterns were evident when considering a similar range of natural temperature variation (11.3 to 13.6°C) associated with elevation (1676 to 1891 m). Thus, experimental warming using OTCs was a poor predictor of likely changes along natural gradients. Responses to OTCs appear to be associated with thermal extremes and secondary effects such as increases in resources. These findings suggest caution is required when extending results from experimental warming to likely shifts in arthropod abundance along elevation gradients.