Tephritid Integrative Taxonomy: Where We Are Now, with a Focus on the Resolution of Three Tropical Fruit Fly Species Complexes.

Accurate species delimitation underpins good taxonomy. Formalization of integrative taxonomy in the past decade has provided a framework for using multidisciplinary data to make species delimitation hypotheses more rigorous. We address the current state of integrative taxonomy by using as a case study an international project targeted at resolving three important tephritid species complexes: Bactrocera dorsalis complex, Anastrepha fraterculus complex, and Ceratitis FAR (C. fasciventris, C. anonae, C. rosa) complex. The integrative taxonomic approach has helped deliver significant advances in resolving these complexes: It has been used to identify some taxa as belonging to the same biological species as well as to confirm hidden cryptic diversity under a single taxonomic name. Nevertheless, the general application of integrative taxonomy has not been without issue, revealing challenges that must be considered when undertaking an integrative taxonomy project. Scrutiny of this international case study provides a unique opportunity to document lessons learned for the benefit of not only tephritid taxonomists, but also the wider taxonomic community.

Prion propagation and toxicity in vivo occur in two distinct mechanistic phases.

Mammalian prions cause fatal neurodegenerative conditions including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. Prion infections are typically associated with remarkably prolonged but highly consistent incubation periods followed by a rapid clinical phase. The relationship between prion propagation, generation of neurotoxic species and clinical onset has remained obscure. Prion incubation periods in experimental animals are known to vary inversely with expression level of cellular prion protein. Here we demonstrate that prion propagation in brain proceeds via two distinct phases: a clinically silent exponential phase not rate-limited by prion protein concentration which rapidly reaches a maximal prion titre, followed by a distinct switch to a plateau phase. The latter determines time to clinical onset in a manner inversely proportional to prion protein concentration. These findings demonstrate an uncoupling of infectivity and toxicity. We suggest that prions themselves are not neurotoxic but catalyse the formation of such species from PrP(C). Production of neurotoxic species is triggered when prion propagation saturates, leading to a switch from autocatalytic production of infectivity (phase 1) to a toxic (phase 2) pathway.

Do Fruit Ripening Volatiles Enable Resource Specialism in Polyphagous Fruit Flies?

Frugivorous tephritid fruit flies have lineages with high levels of host generalism. These insects use olfaction to locate fruits, but how they are able to recognize the odors of so many different host species is poorly understood. We used a series of behavioral experiments to investigate the role of fruit ripening volatiles as host cues in the Queensland fruit fly, Bactrocera tryoni (Froggatt), a polyphagous pest in Australia. Odors of mature guava (Psidium guajava) attracted female and male flies more strongly than three other ripening stages and guava pulp. We analyzed volatiles from guava odor and selected eleven compounds, all of which elicited an electrophysiological response in the antenna of female flies. Three of these, ethyl acetate, ethyl butyrate, and ethyl propionate, were released at the highest rates from the most attractive ripening stage. In behavioral trials, these three esters were not attractive individually, whereas a combination was necessary and sufficient in attracting female flies. The three-component blend was as attractive as the entire 11-component blend, which without these key volatiles was not attractive. Moreover, injecting low ranking hosts (squash and cucumber) with the three volatiles increased attraction in ovipositing female flies. These fruit flies are classed as generalists, but like many polyphagous insects they could be regarded as resource specialists, preferring specific plant reproductive stages with predictable odor cues. Exploring olfaction from this perspective could improve our understanding of host choice in polyphagous insects, and the selection of volatiles to be used as attractants in insect pest management.

N-terminal domain of prion protein directs its oligomeric association.

The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar ß-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined ß-sheet-rich oligomer, containing ∼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23-90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91-231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein.

Sexual selection in true fruit flies (Diptera: Tephritidae): transcriptome and experimental evidences for phytochemicals increasing male competitive ability.

In male tephritid fruit flies of the genus Bactrocera, feeding on secondary plant compounds (sensu lato male lures = methyl eugenol, raspberry ketone and zingerone) increases male mating success. Ingested male lures alter the male pheromonal blend, normally making it more attractive to females and this is considered the primary mechanism for the enhanced mating success. However, the male lures raspberry ketone and zingerone are known, across a diverse range of other organisms, to be involved in increasing energy metabolism. If this also occurs in Bactrocera, then this may represent an additional benefit to males as courtship is metabolically expensive and lure feeding may increase a fly's short-term energy. We tested this hypothesis by performing comparative RNA-seq analysis between zingerone-fed and unfed males of Bactrocera tryoni. We also carried out behavioural assays with zingerone- and cuelure-fed males to test whether they became more active. RNA-seq analysis revealed, in zingerone-fed flies, up-regulation of 3183 genes with homologues transcripts to those known to regulate intermale aggression, pheromone synthesis, mating and accessory gland proteins, along with significant enrichment of several energy metabolic pathways and gene ontology terms. Behavioural assays show significant increases in locomotor activity, weight reduction and successful mating after mounting; all direct/indirect measures of increased activity. These results suggest that feeding on lures leads to complex physiological changes, which result in more competitive males. These results do not negate the pheromone effect, but do strongly suggest that the phytochemical-induced sexual selection is governed by both female preference and male competitive mechanisms.

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin.

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.

Pharmacological chaperone for the structured domain of human prion protein.

In prion diseases, the misfolded protein aggregates are derived from cellular prion protein (PrP(C)). Numerous ligands have been reported to bind to human PrP(C) (huPrP), but none to the structured region with the affinity required for a pharmacological chaperone. Using equilibrium dialysis, we screened molecules previously suggested to interact with PrP to discriminate between those which did not interact with PrP, behaved as nonspecific polyionic aggregates or formed a genuine interaction. Those that bind could potentially act as pharmacological chaperones. Here we report that a cationic tetrapyrrole [Fe(III)-TMPyP], which displays potent antiprion activity, binds to the structured region of huPrP. Using a battery of biophysical techniques, we demonstrate that Fe(III)-TMPyP forms a 11 complex via the structured C terminus of huPrP with a K(d) of 4.5 ± 2 µM, which is in the range of its IC(50) for curing prion-infected cells of 1.6 ± 0.4 µM and the concentration required to inhibit protein-misfolding cyclic amplification. Therefore, this molecule tests the hypothesis that stabilization of huPrP(C), as a principle, could be used in the treatment of human prion disease. The identification of a binding site with a defined 3D structure opens up the possibility of designing small molecules that stabilize huPrP and prevent its conversion into the disease-associated form.

Divergent east-west lineages in an Australian fruit fly, (Bactrocera jarvisi), associated with the Carpentaria Basin divide.

Bactrocera jarvisi is an endemic Australian fruit fly species (Diptera: Tephritidae). It occurs commonly across tropical and subtropical coastal Australia, from far-northern Western Australia, across the 'Top End' of the Northern Territory, and then down the Queensland east coast. Across this range, its distribution crosses several well documented biogeographic barriers. In order to better understand factors leading to the divergence of Australian fruit fly lineages, we carried out a population genetic study of B. jarvisi from across its range using genome-wide SNP analysis, utilising adult specimens gained from trapping and fruit rearing. Populations from the Northern Territory (NT) and Western Australia were genetically similar to each other, but divergent from the genetically uniform east-coast (= Queensland, QLD) population. Phylogenetic analysis demonstrated that the NT population derived from the QLD population. We infer a role for the Carpentaria Basin as a biogeographic barrier restricting east-west gene flow. The QLD populations were largely panmictic and recognised east-coast biogeographic barriers play no part in north-south population structuring. While the NT and QLD populations were genetically distinct, there was evidence for the historically recent translocation of flies from each region to the other. Flies reared from different host fruits collected in the same location showed no genetic divergence. While a role for the Carpentaria Basin as a barrier to gene flow for Australian fruit flies agrees with existing work on the related B. tryoni, the reason(s) for population panmixia for B. jarvisi (and B. tryoni) over the entire Queensland east coast, a linear north-south distance of >2000km, remains unknown.

Prion Propagation is Dependent on Key Amino Acids in Charge Cluster 2 within the Prion Protein.

To dissect the N-terminal residues within the cellular prion protein (PrPC) that are critical for efficient prion propagation, we generated a library of point, double, or triple alanine replacements within residues 23-111 of PrP, stably expressed them in cells silenced for endogenous mouse PrPC and challenged the reconstituted cells with four common but biologically diverse mouse prion strains. Amino acids (aa) 105-111 of Charge Cluster 2 (CC2), which is disordered in PrPC, were found to be required for propagation of all four prion strains; other residues had no effect or exhibited strain-specific effects. Replacements in CC2, including aa105-111, dominantly inhibited prion propagation in the presence of endogenous wild type PrPC whilst other changes were not inhibitory. Single alanine replacements within aa105-111 identified leucine 108 and valine 111 or the cluster of lysine 105, threonine 106 and asparagine 107 as critical for prion propagation. These residues mediate specific ordering of unstructured CC2 into ß-sheets in the infectious prion fibrils from Rocky Mountain Laboratory (RML) and ME7 mouse prion strains.

Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wing-shape data.

BACKGROUND: Bactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status. RESULTS: Geometric morphometric results generated from 15 landmarks for wings of 169 flies revealed significant differences in wing shape between almost all sites following canonical variate analysis. For the combined data set there was a greater isolation-by-distance (IBD) effect under a 'non-Euclidean' scenario which used geographical distances within a biogeographical 'Sundaland context' (r(2) = 0.772, P < 0.0001) as compared to a 'Euclidean' scenario for which direct geographic distances between sample sites was used (r(2) = 0.217, P < 0.01). COI sequence data were obtained for 156 individuals and yielded 83 unique haplotypes with no correlation to current taxonomic designations via a minimum spanning network. beast analysis provided a root age and location of 540kya in northern Thailand, with migration of B. dorsalis s.l. into Malaysia 470kya and Sumatra 270kya. Two migration events into the Philippines are inferred. Sequence data revealed a weak but significant IBD effect under the 'non-Euclidean' scenario (r(2) = 0.110, P < 0.05), with no historical migration evident between Taiwan and the Philippines. Results are consistent with those expected at the intra-specific level. CONCLUSIONS: Bactrocera dorsalis s.s., B. papayae and B. philippinensis likely represent one species structured around the South China Sea, having migrated from northern Thailand into the southeast Asian archipelago and across into the Philippines. No migration is apparent between the Philippines and Taiwan. This information has implications for quarantine, trade and pest management.

The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil.

Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection.

A molecular phylogeny for the Tribe Dacini (Diptera: Tephritidae): systematic and biogeographic implications.

With well over 700 species, the Tribe Dacini is one of the most species-rich clades within the dipteran family Tephritidae, the true fruit flies. Nearly all Dacini belong to one of two very large genera, Dacus Fabricius and Bactrocera Macquart. The distribution of the genera overlap in or around the Indian subcontinent, but the greatest diversity of Dacus is in Africa and the greatest diversity of Bactrocera is in south-east Asia and the Pacific. The monophyly of these two genera has not been rigorously established, with previous phylogenies only including a small number of species and always heavily biased to one genus over the other. Moreover, the subgeneric taxonomy within both genera is complex and the monophyly of many subgenera has not been explicitly tested. Previous hypotheses about the biogeography of the Dacini based on morphological reviews and current distributions of taxa have invoked an out-of-India hypothesis; however this has not been tested in a phylogenetic framework. We attempted to resolve these issues with a dated, molecular phylogeny of 125 Dacini species generated using 16S, COI, COII and white eye genes. The phylogeny shows that Bactrocera is not monophyletic, but rather consists of two major clades: Bactrocera s.s. and the 'Zeugodacus group of subgenera' (a recognised, but informal taxonomic grouping of 15 Bactrocera subgenera). This 'Zeugodacus' clade is the sister group to Dacus, not Bactrocera and, based on current distributions, split from Dacus before that genus moved into Africa. We recommend that taxonomic consideration be given to raising Zeugodacus to genus level. Supportive of predictions following from the out-of-India hypothesis, the first common ancestor of the Dacini arose in the mid-Cretaceous approximately 80mya. Major divergence events occurred during the Indian rafting period and diversification of Bactrocera apparently did not begin until after India docked with Eurasia (50-35mya). In contrast, diversification in Dacus, at approximately 65mya, apparently began much earlier than predicted by the out-of-India hypothesis, suggesting that, if the Dacini arose on the Indian plate, then ancestral Dacus may have left the plate in the mid to late Cretaceous via the well documented India-Madagascar-Africa migration route. We conclude that the phylogeny does not disprove the predictions of an out-of-India hypothesis for the Dacini, although modification of the original hypothesis is required.

Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS.

hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN-NAD+-phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN-NAD+-glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.

Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces.

Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental advance was the cell culture-based scrapie cell assay, allowing prion titres to be estimated more precisely and an order of magnitude faster than by animal bioassays. Here we describe a bioassay method that exploits the marked binding affinity of prions to steel surfaces. Using steel wires as a concentrating and sensitization tool and combining with an adapted scrapie cell endpoint assay we can achieve, for mouse prions, a sensitivity 100x higher than that achieved in standard mouse bioassays. The rapidity and sensitivity of this assay offers a major advance over small animal bioassay in many aspects of prion research. In addition, its specific application in assay of metal-bound prions allows evaluation of novel prion decontamination methods.

Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.

The motor protein SecA drives the transport of polypeptides through the ubiquitous protein channel SecYEG. Changes in protein-nucleotide binding energy during the hydrolytic cycle of SecA must be harnessed to drive large conformational changes resulting in channel opening and vectorial substrate polypeptide transport. Here, we elucidate the ATP hydrolysis cycle of SecA from Escherichia coli by transient and steady-state methods. The basal ATPase activity of SecA is very slow with the release of ADP being some 600-fold slower than hydrolysis. Upon binding to SecYEG the release of ADP is stimulated but remains rate-limiting. ADP release is fastest in the fully coupled system when a substrate protein is being translocated; in this case hydrolysis and ADP release occur at approximately the same rate. The data imply that ADP dissociation from SecA is accompanied by a structural rearrangement that is strongly coupled to the protein interface and protein translocation through SecYEG.

Folding kinetics of the human prion protein probed by temperature jump.

Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (approximately 3,000 s(-1)), with the extrapolated folding rate constant at approximately 20 degrees C in physiological conditions reaching 20,000 s(-1). By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant phi-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix-helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrP(C) conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state that is not compact (m value analysis gives a beta(t) value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible effect on stability but still led to formation of aberrant conformations during folding implies an easily perturbed folding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal dominant condition, 20 of the 33 amino acid replacements occur in the helix-2/3 sequence.

Competition: A Missing Component of Fruit Fly (Diptera: Tephritidae) Risk Assessment and Planning.

Tephritid fruit flies are internationally significant pests of horticulture. Because they are also highly invasive and of major quarantine concern, significant effort is placed in developing full or partial pest risk assessments (PRAs) for fruit flies, while large investments can be made for their control. Competition between fruit fly species, driven by the need to access and utilise fruit for larval development, has long been recognised by researchers as a fundamental component of fruit fly biology, but is entirely absent from the fruit fly PRA literature and appears not be considered in major initiative planning. First presenting a summary of the research data which documents fruit fly competition, this paper then identifies four major effects of fruit fly competition that could impact a PRA or large-scale initiative: (i) numerical reduction of an existing fruit fly pest species following competitive displacement by an invasive fruit fly; (ii) displacement of a less competitive fruit fly pest species in space, time or host; (iii) ecological resistance to fruit fly invasion in regions already with competitively dominant fruit fly species; and (iv) lesser-pest fruit fly resurgence following control of a competitively superior species. From these four major topics, six more detailed issues are identified, with each of these illustrated by hypothetical, but realistic biosecurity scenarios from Australia/New Zealand and Europe. The scenarios identify that the effects of fruit fly competition might both positively or negatively affect the predicted impacts of an invasive fruit fly or targeted fruit fly control initiative. Competition as a modifier of fruit fly risk needs to be recognised by policy makers and incorporated into fruit fly PRAs and major investment initiatives.

The Fallacy of Year-Round Breeding in Polyphagous Tropical Fruit Flies (Diptera: Tephritidae): Evidence for a Seasonal Reproductive Arrestment in Bactrocera Species.

The genus Bactrocera (Diptera: Tephritidae) is endemic to the monsoonal rainforests of South-east Asia and the western Pacific where the larvae breed in ripe, fleshy fruits. While most Bactrocera remain rainforest restricted, species such as Bactrocera dorsalis, Bactrocera zonata and Bactrocera tryoni are internationally significant pests of horticulture, being both highly invasive and highly polyphagous. Almost universally in the literature it is assumed that Bactrocera breed continuously if temperature and hosts are not limiting. However, despite that, these flies show distinct seasonality. If discussed, seasonality is generally attributed to the fruiting of a particular breeding host (almost invariably mango or guava), but the question appears not to have been asked why flies do not breed at other times of the year despite other hosts being available. Focusing initially on B. tryoni, for which more literature is available, we demonstrate that the seasonality exhibited by that species is closely correlated with the seasons of its endemic rainforest environment as recognised by traditional Aboriginal owners. Evidence suggests the presence of a seasonal reproductive arrest which helps the fly survive the first two-thirds of the dry season, when ripe fruits are scarce, followed by a rapid increase in breeding at the end of the dry season as humidity and the availability of ripe fruit increases. This seasonal phenology continues to be expressed in human-modified landscapes and, while suppressed, it also partially expresses in long-term cultures. We subsequently demonstrate that B. dorsalis, across both its endemic and invasive ranges, shows a very similar seasonality although reversed in the northern hemisphere. While high variability in the timing of B. dorsalis population peaks is exhibited across sites, a four-month period when flies are rare in traps (Dec-Mar) is highly consistent, as is the fact that nearly all sites only have one, generally very sharp, population peak per year. While literature to support or deny a reproductive arrest in B. dorsalis is not available, available data is clear that continuous breeding does not occur in this species and that there are seasonal differences in reproductive investment. Throughout the paper we reinforce the point that our argument for a complex reproductive physiology in Bactrocera is based on inductive reasoning and requires specific, hypothesis-testing experiments to confirm or deny, but we do believe there is ample evidence to prioritise such research. If it is found that species in the genus undergo a true reproductive diapause then there are very significant implications for within-field management, market access, and biosecurity risk planning which are discussed. Arguably the most important of these is that insects in diapause have greater stress resistance and cold tolerance, which could explain how tropical Bactrocera species have managed to successfully invade cool temperate regions.

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.

Aversive responses of Queensland fruit flies towards larval-infested fruits are modified by fruit quality and prior experience.

For frugivorous fruit flies, the decision whether to accept or reject a host fruit for oviposition is influenced by a variety of fruit quality factors. Additionally, ovipositing flies may be influenced by the presence of eggs or larvae already within the host fruit. Species of the genus Bactrocera have been shown to avoid ovipositing into larval-infested fruits. However, the observed oviposition aversion in Bactrocera is variable, with some studies showing that deterrence to infested fruits may not always occur, but what may influence such variation is unknown. Using the polyphagous fruit fly Bactrocera tryoni (Froggatt), we tested if the quality of host fruit for offspring survival was a factor in influencing a female fly's decision whether to oviposit or not into larval-infested fruits. In both small cages and field cages, ovipositing B. tryoni did not discriminate between infested and non-infested high-quality fruits. However, when given a choice between poor-quality infested and non-infested fruits, significantly more flies selected and oviposited in non-infested fruits. For example, B. tryoni did not discriminate between infested and non-infested guava (a fruit in which there is high offspring survival), but more flies selected and oviposited on non-infested than on infested green apples (a fruit in which there is poor offspring survival). Small cage experiments also showed that prior oviposition experience on a larval-infested host negated the previously observed aversive response for that particular infested host fruit. The results are discussed in the light of a long recognised, but often ignored fact that herbivore host choice is about the sum of both the positive and negative cues received from the host.