Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.</p

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.</p

New records and a description of a new species of fruit fly (Diptera: Tephritidae) from eastern Australia

The tropical fruit fly (Diptera: Tephritidae: Dacinae) fauna of Australia has been reasonably well studied using the male lures methyl eugenol and cue-lure. However, there has been little sampling of biodiverse Gondwanan rainforests of eastern Australia. Additionally, recently identified male lures have trapped new, or previously non-responsive species in northern Australia, south-east Asia and the Pacific. We wanted to determine if previously unknown species exist in these biodiverse and poorly sampled areas. Therefore, we conducted fruit fly trapping surveys using the male lures methyl eugenol, cue-lure, isoeugenol, methyl isoeugenol, dihydroeugenol, zingerone and protein baits. Trapping targeted previously known locations of rare species, and remnant Gondwanan forests of eastern Australia between 2017 and 2019. We recorded new distributions for “Bactrocera aurea” (May) and occurrences of rare species “Bactrocera mutabilis” (May) and “Bactrocera brunnea” (Perkins and May). This represented the first record of “Bactrocera brunnea” in over 25 years. Additionally, new lure responses were recorded for previously non-responsive “Bactrocera mutabilis”, and a single “Bactrocera phaleriae” (May); both trapped with isoeugenol. Bactrocera (Bactrocera) clarkei sp. n., is described from Couchy Creek Nature Reserve and the Border Ranges National Park, New South Wales

Towards a dated molecular phylogeny of the Tanypodinae (Chironomidae, Diptera)

A dated molecular phylogeny is proposed for the Tanypodinae, a diverse subfamily of Chironomidae (Diptera). We used molecular data from fragments of one ribosomal gene (28S), one nuclear protein-coding gene (CAD), and one mitochondrial protein-coding gene (COI), analysed using mixed model Bayesian and maximum likelihood inference methods. All proposed tribes were sampled, namely, Anatopyniini, Clinotanypodini, Coelopyniini, Fittkauimyiini, Macropelopiini, Natarsiini, Pentaneurini, Procladiini and Tanypodini. A multilocus dataset of 1938 characters was compiled from 123 individuals including outgroups. Monophyly was supported for all tribes although some relationships were not robust. Relationships between tribes and some genus groups are highly congruent with a morphology-based estimate. Relationships within tribe Pentaneurini mostly find weak support, yet previously hypothesised groupings and monophyly or lack thereof in well-sampled genera are revealed. The tempo of diversification of the family was deduced by divergence time analysis (BEAST). Origination of a subfamily stem group in the late Jurassic to early Cretaceous was inferred, with all tribes and many genera of Pentaneurini originating and diversifying in the Cretaceous. Some nodes are biogeographically informative. Gene sections supported the backbone, but more extensive sampling is needed to estimate shallower phylogenies and to better understand the tempo and diversification of Tanypodinae

Fruit Fly Larval Survival in Picked and Unpicked Tomato Fruit of Differing Ripeness and Associated Gene Expression Patterns

The larvae of frugivorous tephritid fruit flies feed within fruit and are global pests of horticulture. With the reduced use of pesticides, alternative control methods are needed, of which fruit resistance is one. In the current study, we explicitly tested for phenotypic evidence of induced fruit defences by running concurrent larval survival experiments with fruit on or off the plant, assuming that defence induction would be stopped or reduced by fruit picking. This was accompanied by RT-qPCR analysis of fruit defence and insect detoxification gene expression. Our fruit treatments were picking status (unpicked vs. picked) and ripening stage (colour break vs. fully ripe), our fruit fly was the polyphagous Bactrocera tryoni, and larval survival was assessed through destructive fruit sampling at 48 and 120 h, respectively. The gene expression study targeted larval and fruit tissue samples collected at 48 h and 120 h from picked and unpicked colour-break fruit. At 120 h in colour-break fruit, larval survival was significantly higher in the picked versus unpicked fruit. The gene expression patterns in larval and plant tissue were not affected by picking status, but many putative plant defence and insect detoxification genes were upregulated across the treatments. The larval survival results strongly infer an induced defence mechanism in colour-break tomato fruit that is stronger/faster in unpicked fruits; however, the gene expression patterns failed to provide the same clear-cut treatment effect. The lack of conformity between these results could be related to expression changes in unsampled candidate genes, or due to critical changes in gene expression that occurred during the unsampled periods.</p

A transcriptome-based analytical workflow for identifying loci for species diagnosis: A case study with Bactrocera fruit flies (Insecta: Tephritidae)

Development of novel molecular methods for accurate and economical identification of species has become critical both for pure biological research and a wide range of applied areas. The most widely used current molecular diagnostic tool, the mitochondrial cytochrome c oxidase subunit 1 gene (COI), the so-called ‘DNA barcode’, has been highly criticised and is known to be ineffective at distinguishing species in many groups. Alternative markers are needed to circumvent these issues and provide diagnosticians with a greater range of tools for making accurate identifications. To address this, we describe here a novel analytical workflow for diagnostic marker development that utilises near-genomic scale data to search for potential informative loci. The workflow takes advantage of orthologous gene databases, in combination with tests of phylogenetic resolution, and benchmarking of nucleotide variation against COI, to determine putative loci that might outperform COI. We use transcriptomes of 14 tephritid fruit flies, and especially the taxonomically complex genus Bactrocera, as a case study. Of 1646 orthologs searched, our workflow retained a total of five loci following our conservative filtering strategy. One locus, POP4, had strong potential as a novel diagnostic marker for Bactrocera fruit flies. POP4 discriminates most species in the training set of taxa, but like COI fails to separate the sibling species B. tryoni and B. neohumeralis. Further validation of this potential new marker against a broader taxonomic sample is ongoing. We advocate that this simple and efficient workflow is, with minor modification, customisable for diagnostic development in almost any taxonomic group

Opiine parasitoids (Hymenoptera Braconidae) and biological control of fruit flies (Diptera: Tephritidae) in Australia: Past, present and future

Opiine braconids are parasitoids of the immature stages of frugivorous tephritids. The female wasp lays her eggs into the eggs or larvae of the fruit fly host, where the immature wasp develops before emerging as a next-generation adult from the now dead host pupal case. In support of a new generation of Australian fruit fly parasitoid research, this paper comprehensively reviews what is known about the Australian fruit fly infesting opiines. Based on the most recent taxonomic revision 11 fruit fly infesting opiine species are documented to occur in Australia, but we consider as doubtful the record for Diachasmimorpha longicaudata and consider the record for Fopius illusorius to be tentative without further collections. We identify that the systematics and taxonomy of the Australian native fruit fly infesting opiines are in urgent need of further work. The history of fruit fly biological control in Australia is comprehensively reviewed, including the export of native Australian opiines for fruit fly control elsewhere in the world. Australia was actively involved in three major classical biological control programmes against fruit flies from the turn of the 1900s until the mid-1960s. Despite the introduction of 11 opiine species, plus numerous other natural enemies, only Fopius arisanus established in eastern Australia, where in South-east Queensland it can now cause between 30 and 40% mean parasitism. In addition to the exotic F. arisanus, the native species Diachasmimorpha kraussii and Diachasmimorpha tryoni also cause fruit fly parasitism in agriculturally important crops: both species have also been liberated widely outside of Australia for fruit fly control. Other Australian opiines have not been reared from flies infesting commercial crops and appear biologically restricted to the fruits and environs of Australian east-coast rainforests. The biology literature for D. tryoni and D. kraussii is comprehensively reviewed, while for F. arisanus, already reviewed elsewhere, key literature only is covered. Forward looking, we consider the potential for inoculative or inundative releases of opiines in areas where they do not currently occur to be good, while conservation biological control may help to increase the impacts of parasitoids in areas where they are already established.</p

A comprehensive phylogeny helps clarify the evolutionary history of host breadth and lure response in the Australian Dacini fruit flies (Diptera: Tephritidae)

The tribe Dacini (Diptera: Tephritidae) contains over 930 recognised species and has been widely studied due to the economic importance of some taxa, such as the Oriental fruit fly Bactrocera dorsalis. Despite the attention this group has received, very few phylogenetic reconstructions have comprehensively sampled taxa from a single biogeographic region, thereby limiting our capacity to address more targeted evolutionary questions. To study the evolution of diet breadth and male lure response, two key traits fundamental to understanding dacine diversity and the biology of pest taxa, we analysed 273 individuals representing 144 described species from Australia (80% continental coverage), the Pacific, and select close relatives from South-east Asia to estimate a dated molecular phylogenetic reconstruction of the Dacini. We utilised seven loci with a combined total of 4,332 nucleotides, to estimate both Bayesian and Maximum Likelihood phylogenies of the tribe. Consistent with other molecular phylogenies of the tribe, there was a high level of disagreement between the placement of species in the phylogeny and their current subgeneric and species-complex level taxonomies. The Australian fauna exhibit high levels of endemism, with radiations of both exclusively Australian clades, and clades that originate elsewhere (e.g. the Bactrocera dorsalis species group). Bidirectional movement of species has occurred between Papua New Guinea and Australia, with evidence for multiple incursions over evolutionary time. The Bactrocera aglaiae species group emerged sister to all other Bactrocera species examined. Divergence time estimates were ∼ 30 my younger than previously reported for this group, with the tribe diverging from its most recent common ancestor ∼ 43 mya. Ancestral trait reconstruction and tests for trait phylogenetic signal revealed a strong signal for the evolution of male lure response across the tree, with cue-lure/raspberry ketone lure response the ancestral trait. Methyl eugenol response has arisen on multiple, independent occasions. The evolution of host breadth exhibited a weaker signal; yet, basal groups were more likely to be host specialists. Both the evolution of lure response and host fruit use provide predictive information for the outbreak management of understudied pest fruit flies for which direct inference of these features may be lacking. Our results, which parallel those of earlier research into the closely-related African Dacus spp., demonstrate how geographically focussed taxon coverage allows Dacini phylogenetics to more explicitly test evolutionary hypotheses, thereby progressing our understanding of the evolution of this highly diverse and recently-radiated group of flies

Chronology in Late Antiquity: A lesson from the Palaestra

This article deals with the fundamental problem of working in Ostia : how to devise a respectable chronology for a city that was mostly cleared of soil in a few years, with the aid of railways and poor families, for the World Exhibition of 1942. In the absence of photographs and serious notes, we have little to fall back on. I offer a field methodology against despair, applied to the difficult case of the Palaestra. What we need to do is clean up large areas, establish phases between them, and undertake selective excavation of pottery-rich rubbish deposits. From the resultant Harris Matrix, a more nuanced history of Ostia can emerge, of late antique centuries, extending far beyond the « Hadrianic » fantasy of Mussolini. The scale of this exercise makes it possible to assess the reliability and relative utility of different dating methods. Most early methods now fail, but others remain. It seems that future chronology will depend on an uncomfortable cohabitation of old and new