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.

Cuelure but not zingerone make the sex pheromone of male Bactrocera tryoni (Tephritidae: Diptera) more attractive to females

In tephritid fruit flies of the genus Bactrocera Macquart, a group of plant derived compounds (sensu amplo ‘male lures’) enhance the mating success of males that have consumed them. For flies responding to the male lure methyl eugenol, this is due to the accumulation of chemicals derived from the male lure in the male rectal gland (site of pheromone synthesis) and the subsequent release of an attractive pheromone. Cuelure, raspberry ketone and zingerone are a second, related group of male lures to which many Bactrocera species respond. Raspberry ketone and cuelure are both known to accumulate in the rectal gland of males as raspberry ketone, but it is not known if the emitted male pheromone is subsequently altered in complexity or is more attractive to females. Using Bactrocera tryoni as our test insect, and cuelure and zingerone as our test chemicals, we assess: (i) lure accumulation in the rectal gland; (ii) if the lures are released exclusively in association with the male pheromone; and (iii) if the pheromone of lure-fed males is more attractive to females than the pheromone of lure-unfed males. As previously documented, we found cuelure was stored in its hydroxyl form of raspberry ketone, while zingerone was stored largely in an unaltered state. Small but consistent amounts of raspberry ketone and β-(4-hydroxy-3-methoxyphenyl)-propionic acid were also detected in zingerone-fed flies. Males released the ingested lures or their analogues, along with endogenous pheromone chemicals, only during the dusk courtship period. More females responded to squashed rectal glands extracted from flies fed on cuelure than to glands from control flies, while more females responded to the pheromone of calling cuelure-fed males than to control males. The response to zingerone treatments in both cases was not different from the control. The results show that male B. tryoni release ingested lures as part of their pheromone blend and, at least for cuelure, this attracts more females.

Bactrocera frauenfeldi (Diptera: Tephritidae), an invasive fruit fly in Australia that may have reached the extent of its spread due to environmental variables

Bactrocera frauenfeldi (Schiner), the ‘mango fruit fly’, is a horticultural pest originating from the Papua New Guinea region. It was first detected in Australia on Cape York Peninsula in north Queensland in 1974 and had spread to Cairns by 1994 and Townsville by 1997. Bactrocera frauenfeldi has not been recorded further south since then despite its invasive potential, an absence of any controls and an abundance of hosts in southern areas. Analysis of cue-lure trapping data from 1997 to 2012 in relation to environmental variables shows that the distribution of B. frauenfeldi in Queensland correlates to locations with a minimum temperature for the coldest month >13.2°C, annual temperature range <19.3°C, mean temperature of the driest quarter >20.2°C, precipitation of the wettest month >268 mm, precipitation of the wettest quarter >697 mm, temperature seasonality <30.9°C (i.e. lower temperature variability) and areas with higher human population per square kilometre. Annual temperature range was the most important variable in predicting this species' distribution. Predictive distribution maps based on an uncorrelated subset of these variables reasonably reflected the current distribution of this species in northern Australia and predicted other areas in the world potentially at risk from invasion by this species. This analysis shows that the distribution of B. frauenfeldi in Australia is correlated to certain environmental variables that have most likely limited this species' spread southward in Queensland. This is of importance to Australian horticulture in demonstrating that B. frauenfeldi is unlikely to establish in horticultural production areas further south than Townsville.

The host specificity and climatic suitability of the gall flyCecidochares connexa(Diptera: Tephritidae), a potential biological control agent for Chromolaena odorata(Asteraceae) in Australia

The gall fly Cecidochares connexa (Diptera: Tephritidae) is a potential biological control agent for Chromolaena odorata in Australia. Its host specificity was determined against 18 species in the tribe Eupatorieae (Family Asteraceae) in which C. odorata belongs, in quarantine in Brisbane, Australia. Oviposition occurred and flies developed on only C. odorata and Praxelis clematidea, both of which are in the subtribe Praxelinae. P. clematidea is considered a weed outside tropical America. In both multiple-species-minus-C. odorata choice tests and single-species no-choice tests, the mean number of galls/plant was significantly greater on C. odorata (48 and 41, respectively) than on P. clematidea (2 and 9, respectively). There were also significantly more adults emerging from C. odorata (mean 129 and 169, respectively) in the two types of tests than from P. clematidea (1 and 8, respectively). Paired choice, multiple generation (continuation) and time dependent tests further clarified the extent that C. connexa could develop on P. clematidea. In these tests, the mean number of galls formed and the mean number of emerging adults were consistently less for P. clematidea than C. odorata and populations of C. connexa could not be maintained on P. clematidea. Galls were not seen on any other plant species tested. This study supports the results of host specificity testing conducted in seven other countries and confirms that C. connexa poses little risk to other plant species in Australia. C. connexa has been released in 10 countries and an application seeking approval to release in Australia has been submitted to the Australian Government.

Low-dose methyl bromide fumigation as a quarantine disinfestation treatment for nectarines against Queensland fruit fly (Diptera:Tephritidae)

White nectarines (Prunus persica var. nucipersica) were fumigated with methyl bromide (MB) at a nominal treatment dose of 18 g m-3 at 18°C for 5 h and 30 min as a quarantine disinfestation treatment against Bactrocera tryoni, the Queensland fruit fly. Three large scale trials were conducted against each of the four immature lifestages, eggs and first, second and third instars. There were no survivors from the estimated 43,614 eggs, 41,873 first instars, 41,345 second instars and 33,549 third instars treated, thereby resulting in an efficacy of GROTERDAN99.99% mortality at the 95% confidence level for each lifestage. Of the 12 trials reported herein, the highest concentration of MB, sampled from the chamber headspace analysed by gas chromatography, was 18.7 g m-3. The maximum chamber temperature from 5 min readings was 19.7°C and the maximum fruit core temperature was 19.5°C. The treatment time for all trials was exactly 5.5 h. Thus the recommended treatment dose to disinfest nectarines from B. tryoni is 19.0 g m-3 MB at 20.0°C for 5.5 h. Fruit quality trials were conducted on white nectarines at three combinations of treatment parameters: 15 g m-3 MB at 19°C for 5.25 h; 18 g m-3 MB at 19°C for 5.5 h and 21 g m-3 MB at 19°C for 5.5 h. The fruit were stored at 0, 4 and 8 days at 4°C and 8 days at 4°C followed by 4 d at 22°C. They were then were assessed for skin colour, flesh colour, skin defects, flesh defects, fruit weight loss, flesh firmness, total soluble solids, titratable acidity and rots. There was no significant difference between untreated control and MB treated fruits in any of the parameters measured. Thus the treatments did not have adverse effects on fruit quality.

A new species of gall midge (Diptera: Cecidomyiidae) feeding on ornamental Cordyline fruticosa(Asparagaceae) in Australia

Larvae of an undescribed gall midge were found feeding on leaves and stems within leaf sheaths and between leaf blades of potted plants of Cordyline fruticosa (Asparagaceae) in a production nursery in Queensland. The following varieties of the host plant were infested: Apple Blossom', Glauca', Kilauea', Negra', Pink Diamond, 'Purple Prince' and Willy's Gold'. The new species, Dasineura cordylineaeKolesik sp. nov., is described and its cytochrome oxidase unit I mitochondrial gene segment is sequenced. The new species is the first known gall midge feeding on a plant species of the genus Cordyline. Orange larvae induce oval shallow swellings on the leaf and stem tissue, which becomes necrotised during the later stage of larval feeding. Necrotic areas remain visible to the end of leaves' lives and decrease the market value of the plants. In the production nursery investigated, the lesions caused by the gall midge provided an entry for a fungal infection by Fusarium sp. inflicting further injury to plants. Larvae of the new species were preyed on by larvae of Gaurax sp. (Diptera: Chloropidae). This is the first worldwide record of Chloropidae preying on Cecidomyiidae.

The host specificity and climatic suitability of the gall fly Cecidochares connexa (Diptera: Tephritidae), a potential biological control agent for Chromolaena odorata (Asteraceae) in Australia

Host specificity tests on Gynaikothrips ficorum (Marchal) and Gynaikothrips uzeli (Zimmerman) (Thysanoptera: Phlaeothripidae) have shown that under experimental conditions, G. ficorum will induce leaf galls on both Ficus benjamina L. and Ficus microcarpa L. f. (Rosales: Moraceae), but G. uzeli will induce galls only on F. benjamina. A further interesting aspect of the results is that gall induction by G. uzeli on F. benjamina appears to have been suppressed in the presence of F. microcarpa plants in the same cage. Liothrips takahashii (Moulton) (Thysanoptera: Phlaeothripidae), an inquiline in the galls of these Gynaikothrips, is reported for the first time from Australia, mainland China, Malaysia, Costa Rica, and western USA.

Protein feeding of Queensland fruit fly Bactrocera tryoni and cucumber fly Zeugodacus cucumis (Diptera: Tephritidae) on non-host vegetation: effect of plant species and bait height

Perimeter-baiting of non-crop vegetation using toxic protein baits was developed overseas as a technique for control of melon fly, Zeugodacus (Zeugodacus) cucurbitae (Coquillett) (formerly Bactrocera (Zeugodacus) cucurbitae), and evidence suggests that this technique may also be effective in Australia for control of local fruit fly species in vegetable crops. Using field cage trials and laboratory reared flies, primary data were generated to support this approach by testing fruit flies' feeding response to protein when applied to eight plant species (forage sorghum, grain sorghum, sweet corn, sugarcane, eggplant, cassava, lilly pilly and orange jessamine) and applied at three heights (1, 1.5 and 2 m). When compared across the plants, Queensland fruit fly, Bactrocera tryoni (Froggatt), most commonly fed on protein bait applied to sugarcane and cassava, whereas more cucumber fly, Zeugodacus (Austrodacus) cucumis (French) (formerly Bactrocera (Austrodacus) cucumis), fed on bait applied to sweet corn and forage sorghum. When protein bait was applied at different heights, B. tryoni responded most to bait placed in the upper part of the plants (2 m), whereas Z. cucumis preferred bait placed lower on the plants (1 and 1.5 m). These results have implications for optimal placement of protein bait for best practice control of fruit flies in vegetable crops and suggest that the two species exhibit different foraging behaviours.

Soybean Stem Fly, Melanagromyza sojae (Diptera: Agromyzidae), in the New World: detection of high genetic diversity from soybean fields in Brazil

Soybean Stem Fly (SSF), Melanagromyza sojae (Zehntner), belongs to the family Agromyzidae and is highly polyphagous, attacking many plant species of the family Fabaceae, including soybean and other beans. SSF is regarded as one of the most important pests in soybean fields of Asia (e.g., China, India), North East Africa (e.g., Egypt), parts of Russia, and South East Asia. Despite reports of Agromyzidae flies infesting soybean fields in Rio Grande do Sul State (Brazil) in 1983 and 2009 and periodic interceptions of SSF since the 1940s by the USA quarantine authorities, SSF has not been officially reported to have successfully established in the North and South Americas. In South America, M. sojae was recently confirmed using morphology and its complete mitochondrial DNA (mtDNA) was characterized. In the present study, we surveyed the genetic diversity of M. sojae, collected directly from soybean host plants, using partial mtDNA cytochrome oxidase I (COI) gene, and provide evidence of multiple (>10) maternal lineages in SSF populations in South America, potentially representing multiple incursion events. However, a single incursion involving multiple-female founders could not be ruled out. We identified a haplotype that was common in the fields of two Brazilian states and the individuals collected from Australia in 2013. The implications of SSF incursions in southern Brazil are discussed in relation to the current soybean agricultural practices, highlighting an urgent need for better understanding of SSF population movements in the New World, which is necessary for developing effective management options for this significant soybean pest. © FUNPEC-RP.

‘Ladd traps’ as a visual trap for male and female Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae)

Bactrocera tryoni (Froggatt) is Australia's major horticultural insect pest, yet monitoring females remains logistically difficult. We trialled the ‘Ladd trap’ as a potential female surveillance or monitoring tool. This trap design is used to trap and monitor fruit flies in countries other (e.g. USA) than Australia. The Ladd trap consists of a flat yellow panel (a traditional ‘sticky trap’), with a three dimensional red sphere (= a fruit mimic) attached in the middle. We confirmed, in field-cage trials, that the combination of yellow panel and red sphere was more attractive to B. tryoni than the two components in isolation. In a second set of field-cage trials, we showed that it was the red-yellow contrast, rather than the three dimensional effect, which was responsible for the trap's effectiveness, with B. tryoni equally attracted to a Ladd trap as to a two-dimensional yellow panel with a circular red centre. The sex ratio of catches was approximately even in the field-cage trials. In field trials, we tested the traditional red-sphere Ladd trap against traps for which the sphere was painted blue, black or yellow. The colour of sphere did not significantly influence trap efficiency in these trials, despite the fact the yellow-panel/yellow-sphere presented no colour contrast to the flies. In 6 weeks of field trials, over 1500 flies were caught, almost exactly two-thirds of them being females. Overall, flies were more likely to be caught on the yellow panel than the sphere; but, for the commercial Ladd trap, proportionally more females were caught on the red sphere versus the yellow panel than would be predicted based on relative surface area of each component, a result also seen the field-cage trial. We determined that no modification of the trap was more effective than the commercially available Ladd trap and so consider that product suitable for more extensive field testing as a B. tryoni research and monitoring tool.

Composition of chemical attractants affects trap catches of the Australian sheep blowfly, lucilia cuprina, and other blowflies

Numbers of Lucilia cuprina (Australian sheep blowfly), Chrysomya spp., and Calliphora spp. blowflies caught on sticky traps baited with various synthetic attractants or a standard liver/sodium sulfide attractant in western Queensland were recorded. Numbers of each genus collected were influenced by the composition of the chemical attractants. Attractant mixtures based on 2-mercaptoethanol, indole, butanoic/pentanoic acid, and a sodium sulfide solution gave 5- to 20-fold higher L. cuprina catches than the liver standard. These blends attracted similar numbers of Chrysomya spp. (0.85–2.7× ) and fewer Calliphora spp. (0.02–0.2× ) compared to the liver standard. These synthetic attractants were more effective and selective for L. cuprina than the standard liver/sodium sulfide attractant, and they can be packaged in controlled-release dispensers to generate constant, prolonged release of the attractant.

Identification and preliminary evaluation of viriditoxin, a metabolite of Paecilomyces varioti, as an insecticide for sheep blowfly, Lucilia cuprina (Wied.)

Solvent extracts of cultures of the fungus Paecilomyces varioti are toxic to sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae). Different components of the culture extracts were isolated and bioassayed with L. cuprina. The component with most toxicity was purified and identified from its proton magnetic resonance spectrum as viriditoxin, a known antibiotic metabolite of the fungus. The insecticidal properties of viriditoxin were then evaluated. Mean LCso values for first instar larvae of organophosphate susceptible and resistant strains of L. cuprina were 7.5 and 8.4 ppm respectively. Pilot implant trials in sheep demonstrated that the compound provided protection for 9-17 weeks against both strains of L. cuprina. No adverse effects on the trial sheep were detected.

Do parasitic flies attack mites? Evidence in Baltic amber

We provide the first evidence of a small-headed fly planidium (first instar larva; Diptera: Acroceridae) associated with a whirligig mite (Acari: Acariformes: Prostigmata: Anystina: Anystidae) in Baltic amber. This fossil is surprising as parasitic nematodes are the only metazoans known to successfully attack acariform mites, and Acroceridae are believed to be host-restricted parasitoids of spiders. The fossil corroborates a previously published, but widely dismissed, paper that first reported parasitism of parasitengone mites by acrocerid planidia. The possible natural history implications of this find are discussed.

Synthesis, Binding and Bioactivity of [gamma]-Methylene [gamma]-Lactam Ecdysone Receptor Ligands: Advantages of QSAR Models for Flexible Receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or [`]attractors'. We describe the synthesis, in vitro binding and selected in vivo toxicity data for [gamma]-methylene [gamma]-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognized by a single conformation of the EcR binding pocket.