Richard Glatz

G-Protein-Coupled Receptors in Drug Discovery: Nanosizing Using Cell-Free Technologies and Molecular Biology Approaches

Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a genericmolecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, highthroughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formatswill limit the development of higher density GPCRassay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.

A new extant family of primitive moths from Kangaroo Island, Australia, and its significance for understanding early Lepidoptera evolution

We report the first discovery since the 1970s of a new extant family (Aenigmatineidae fam.n.) of homoneurous moths, based on the small Aenigmatinea glatzella sp.n. from Kangaroo Island off southern Australia. It exhibits a combination of extraordinary anatomical characters, and, unlike most homoneurous moths, its larva is a conifer‐feeder (stem mining in Callitris, Cupressaceae). While the adults mouthparts are strongly regressed, evidence from other morphological characters and from a Bayesian analysis of 25 genetic loci convincingly places the taxon among Glossata (‘tongue moths’). An unexpected tongue moth clade including Acanthopteroctetidae and Neopseustidae, suggested with low support in recent molecular analyses, remarkably becomes strongly supported when Aenigmatinea is included in the molecular analysis; the new taxon becomes subordinated in that clade (as sister group to Neopseustidae) and the clade itself appears as the sister group of all Heteroneura, representing the vast majority of all Lepidoptera. Including Aenigmatinea into the analysis thereby strengthens the surprising indication of non‐monophyly of Myoglossata, and the new phylogeny requires an additional number of ad hoc assumptions of convergence/character reversals in early Lepidoptera evolution.

Identification of an in Vitro Interaction between an Insect Immune Suppressor Protein (CrV2) and Gα Proteins

The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to Gα subunits (but not the Gβγ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nm was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or Gα proteins also reduced the TR-FRET signal. The activation state of the Gα subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila Gαo compared with rat Gαi1. In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species.

Host stage preference of Dolichogenidea tasmanica (Cameron, 1912) (Hymenoptera: Braconidae), a parasitoid of Epiphyas postvittana (Walker, 1863) (Lepidoptera: Tortricidae)

Epiphyas postvittana (Walker, 1863), or light brown apple moth (LBAM), is a highly polyphagous native Australian species and a major insect pest in Australian vineyards. Dolichogenidea tasmanica (Cameron, 1912) is the most abundant parasitoid of the larval stages of LBAM. The prime objective of our study was to determine the parasitism success of D. tasmanica on different larval stages of E. postvittana. Additionally, we aimed to find out if larval head capsule width could be used to determine the instar number by examining the variability in head capsule size of each instar of LBAM and the effect of rearing temperature on larval head capsule size. To determine which of the first three larval stages of LBAM parasitised is most effectively by D. tasmanica, choice and no‐choice tests were conducted in a wind tunnel. Head capsule widths varied with rearing temperature, especially in the final fifth or sixth instar. We showed that the ranges of head capsule widths overlaps between successive instars three through six, which makes it impossible to distinguish these instars of LBAM using head capsule size alone. We showed that first, second and third instars of LBAM are parasitised by D. tasmanica. No‐choice and choice tests revealed that there are significant differences in parasitism among the three susceptible instars, with the highest percentage of parasitism found in second instars in no‐choice and choice tests (65.1% and 65.8%, respectively) compared with first instars (61.1% and 45.1%) and third instars (27.3% and 37.5%). Wasps developed faster in third instars, but the wasp females size was not significantly different among the instars attacked. These data confirm that D. tasmanica is capable of parasitising early larval instars, which is important for minimising damage through biological control.

GPCR Expression Using Baculovirus-Infected Sf 9 Cells

Expression of proteins in insect cells using recombinant baculoviruses has gained wide use in the G protein-coupled receptor (GPCR) community. This expression system produces high yields of functional receptor, is able to perform post-translational modifications, and is readily adaptable to large-scale culture. Here, we describe the generic methods for expressing a GPCR using baculovirus-infected insect cells, including the maintenance of insect cell culture. Data are presented for polyhedrin promoter-driven expression of a C-terminal 6 x histidine-tagged mammalian M(2) muscarinic receptor in Sf9 cells. Results demonstrate that expressed receptor could be detected and quantified using radiolabeled ligand binding, that expression was maximal at approximately 72 h post-infection, and that expression levels could be altered by addition of various ligands to cultures of infected insect cells.

Strategic national approach for improving the conservation management of insects and allied invertebrates in Australia: Conservation strategy for Australian insects

Despite progress in recent decades, the conservation management of insects and allied invertebrates in Australia is challenging and remains a formidable task against a background of poor taxonomic and biological knowledge, limited resources (funds and scientific expertise) and a relatively low level of community engagement, education and awareness. In this review, we propose a new, strategic national approach for the conservation of insects and allied invertebrates in Australia to complement and build on existing actions and increase awareness with the general public and government. A review of all species listed under relevant State and Territory Acts, national legislation (EPBC Act) and on international lists (IUCN Red List) indicated that of the 285 species currently listed under these conservation schedules, 10 (3%) are considered extinct, 204 (72%) threatened (Critically Endangered, Endangered or Vulnerable) and 71 (25%) are classified as other (Threatened, Near Threatened, Rare or Least Concern). Comparison of the geographic ranges of listed species in relation to bioregions (IBRA regions) shows a striking discordance in spatial representation across the Australian landscape, reflecting an ad hoc approach to threatened species conservation and the concentration of invertebrate biologists in urban centres of temperate coastal Australia. There is a positive relationship between the number of threatened species and extent of protection according to the National Reserve System within each IBRA region, exemplifying the anomaly in spatial representativeness of listed species. To overcome these shortfalls, we propose a novel educational, regional approach based on selecting, for each of the 89 IBRA regions, a relatively small set of ‘flagship taxa’ (threatened species and/or ‘iconic’ species of high scientific/social value), which are then promoted and/or nominated for listing by the scientific community. Such species could be adopted by local community groups whereby a community‐based regional approach would ensure spatial representativeness of insect conservation across the entire Australian continent. This novel approach may ultimately provide a better strategy for the conservation management of habitats and threatened ecological communities, reducing extinction risk of threatened species and addressing key threatening processes. Members of the Australian entomological community are strongly encouraged to nominate candidate taxa as flagship species for wider promotion and/or listing nationally under the EPBC Act.

Developmental Penalties Associated with Inducible Tolerance in Helicoverpa armigera to Insecticidal Toxins from Bacillus thuringiensis

ABSTRACT Exposure of insect larvae to sublethal concentrations of crystal toxins from the soil bacterium Bacillus thuringiensis (Bt toxins) causes the induction of immune and metabolic responses that can be transmitted to offspring by epigenetic inheritance mechanisms. Given that the elevated immune status carries significant developmental penalties, we wanted to establish the relationships between immune induction, tolerance to the toxin and developmental penalties. A laboratory culture of Helicoverpa armigera was induced by a sublethal bacterial suspension containing crystal toxin Cry1Ac in one generation and maintained in the presence of toxin, acquiring significant levels of tolerance to the toxin within 12 generations of continuous exposure. Comparing tolerant and susceptible insects, we show that the induction of larval immune response and the coincident alteration of development-related metabolic activities by elicitors in the larval gut (larval induction) differs from the elevated immune status transmitted by epigenetic mechanisms (embryonic induction). Because the damaging effects of larval induction processes are higher compared to embryonic induction, it is likely that overall developmental penalties depend on the relative contribution of the two induction processes. When insects are kept with the same amount of toxin in the diet for subsequent generations, the embryonic induction process increases its contribution compared to the larval induction, resulting in reduced overall developmental penalty, while tolerance to the toxin is maintained.

Molecular Engineering of G Protein-Coupled Receptors and G Proteins for Cell-Free Biosensing

The ability to express and purify modified recombinant proteins, so they retain their biological function in a cell-free format, has provided a basis for development of molecular biosensors. Here we utilize recombinant G Protein-coupled receptors (GPCRs) and their G proteins for cell-free detection of various binding partners. Fusion peptides were used to improve surface-attachment and fluorescent-labelling capabilities. A novel homogeneous fluorescence resonance energy transfer (FRET)-based assay was developed to detect rearrangements in the G protein heterotrimer. By using this heterotrimeric ‘molecular switch’, we are developing a generic technology such that multiple GPCRs could be assayed for ligand-mediated activation while tethered to surfaces or in solution, with increased throughput compared to current assay platforms.

Host discrimination by the solitary endoparasitoid Dolichogenidea tasmanica (Hymenopotera: Braconidae)

Successful parasitism of a host partly depends on a females assessment of its quality, including whether the host has already been parasitised or not. We conducted experiments to elucidate host discrimination by Dolichogenidea tasmanica (Hymenoptera: Braconidae). It is the most commonly collected parasitoid of light brown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae). To assess the rate of superparasitism avoidance by D. tasmanica, female wasps were given choices between (1) unparasitised hosts versus freshly self-parasitised hosts, (2) unparasitised hosts versus hosts at 24 h post-self-parasitisation and (3) freshly self-parasitised hosts versus hosts freshly parasitised by a conspecific female. Results confirm that host discrimination occurs in D. tasmanica. Females avoid laying eggs in hosts that have been parasitised by themselves or conspecifics, even though the frequency of first encounter with either an unparasitised or a parasitised host was the same for all choices. Thus, it appears that females are not able to discriminate the host parasitisation status prior to contacting a host, but host acceptance is not random. Host discrimination is time-dependent, with greater avoidance of superparasitism after 24 h. The ability of female D. tasmanica to distinguish healthy from parasitised hosts suggests that it could be an effective biological control agent in regulation of host populations. It should also ensure production efficiency in parasitoid mass-rearing.

Curious case of the Kangaroo Island honeybee Apis mellifera Linnaeus, 1758 (Hymenoptera: Apidae) sanctuary

Humans have had a long association with the honeybee Apis mellifera Linnaeus, 1758, which has been exploited for production of honey and for the crop pollination services it provides. This association facilitated movement of this species to such a degree that it is now virtually ubiquitous in all areas with flowering plants and available water. On Kangaroo Island (KI), a ‘sanctuary’ was created for the Ligurian bee subspecies A. mellifera ligustica, which is exotic to Australia and the entire New World. The Ligurian Bee Act was enacted in 1885 on the basis of perceived genetic purity and isolation of KI honeybee populations, and was updated in 1931 and 1997. It supports biosecurity protocols preventing importation of bees, bee‐keeping equipment and bee‐related products such as honey and wax. This represents a rare example of legislative protection for an invertebrate in Australia. This legislation and the apparent isolation of KI bees from mainland bees in the time since its enactment have led to the popular assertion that KI honeybee populations represent the last ‘pure’ genetic population of A. mellifera ligustica. However, historical accounts of bee introductions to KI show that A. mellifera mellifera‐like bees were present on KI prior to the introduction of A. mellifera ligustica, and that multiple A. mellifera ligustica introductions to KI occurred using bees of mixed heritage. Indeed, DNA sequence analyses of KI honeybees clearly indicate that while there is limited genetic diversity (supporting the assertions of limited introductions and recent geographic/genetic isolation), they are in fact hybrids and share more similarity with the A. mellifera mellifera subspecies. Therefore, the relevant state legislation should be updated to remove any mention of Ligurian or other honeybee strains. However, the biosecurity protocols relating to KI should continue due to the low incidence of some honeybee diseases and the threat posed by Varroa mite.