Bacterial Communities Are Less Diverse in a Strepsipteran Endoparasitoid than in Its Fruit Fly Hosts and Dominated by Wolbachia.

Microbiomes play vital roles in insect fitness and health and can be influenced by interactions between insects and their parasites. Many studies investigate the microbiome of free-living insects, whereas microbiomes of endoparasitoids and their interactions with parasitised insects are less explored. Due to their development in the constrained environment within a host, endoparasitoids are expected to have less diverse yet distinct microbiomes. We used high-throughput 16S rRNA gene amplicon sequencing to characterise the bacterial communities of Dipterophagus daci (Strepsiptera) and seven of its tephritid fruit fly host species. Bacterial communities of D. daci were less diverse and contained fewer taxa relative to the bacterial communities of the tephritid hosts. The strepsipteran's microbiome was dominated by Pseudomonadota (formerly Proteobacteria) (> 96%), mainly attributed to the presence of Wolbachia, with few other bacterial community members, indicative of an overall less diverse microbiome in D. daci. In contrast, a dominance of Wolbachia was not found in flies parasitised by early stages of D. daci nor unparasitised flies. Yet, early stages of D. daci parasitisation resulted in structural changes in the bacterial communities of parasitised flies. Furthermore, parasitisation with early stages of D. daci with Wolbachia was associated with a change in the relative abundance of some bacterial taxa relative to parasitisation with early stages of D. daci lacking Wolbachia. Our study is a first comprehensive characterisation of bacterial communities in a Strepsiptera species together with the more diverse bacterial communities of its hosts and reveals effects of concealed stages of parasitisation on host bacterial communities.

Host-endoparasitoid-endosymbiont relationships: concealed Strepsiptera provide new twist to Wolbachia in Australian tephritid fruit flies.

Wolbachia are widespread endosymbionts that affect arthropod reproduction and fitness. Mostly maternally inherited, Wolbachia are occasionally transferred horizontally. Previously, two Wolbachia strains were reported at low prevalence and titres across seven Australian tephritid species, possibly indicative of frequent horizontal transfer. Here, we performed whole-genome sequencing of field-caught Wolbachia-positive flies. Unexpectedly, we found complete mitogenomes of an endoparasitic strepsipteran, Dipterophagus daci, suggesting that Wolbachia in the flies are linked to concealed parasitization. We performed the first genetic characterization of D. daci and detected D. daci in Wolbachia-positive flies not visibly parasitized, and most but not all Wolbachia-negative flies were D. daci-negative, presumably reflecting polymorphism for the Wolbachia infections in D. daci. We dissected D. daci from stylopized flies and confirmed that Wolbachia infects D. daci, but also found Wolbachia in stylopized fly tissues, likely somatic, horizontally transferred, non-heritable infections. Furthermore, no Wolbachia cif and wmk genes were detected and very low mitogenomic variation in D. daci across its distribution. Therefore, Wolbachia may influence host fitness without reproductive manipulation. Our study of 13 tephritid species highlights that concealed early stages of strepsipteran parasitization led to the previous incorrect assignment of Wolbachia co-infections to tephritid species, obscuring ecological studies of this common endosymbiont and its horizontal transmission by parasitoids.

Wolbachia pseudogenes and low prevalence infections in tropical but not temperate Australian tephritid fruit flies: manifestations of lateral gene transfer and endosymbiont spillover?

BACKGROUND: Maternally inherited Wolbachia bacteria infect many insect species. They can also be transferred horizontally into uninfected host lineages. A Wolbachia spillover from an infected source population must occur prior to the establishment of heritable infections, but this spillover may be transient. In a previous study of tephritid fruit fly species of tropical Australia we detected a high incidence of identical Wolbachia strains in several species as well as Wolbachia pseudogenes. Here, we have investigated this further by analysing field specimens of 24 species collected along a 3,000 km climate gradient of eastern Australia. RESULTS: Wolbachia sequences were detected in individuals of nine of the 24 (37 %) species. Seven (29 %) species displayed four distinct Wolbachia strains based on characterisation of full multi locus sequencing (MLST) profiles; the strains occurred as single and double infections in a small number of individuals (2-17 %). For the two remaining species all individuals had incomplete MLST profiles and Wolbachia pseudogenes that may be indicative of lateral gene transfer into host genomes. The detection of Wolbachia was restricted to northern Australia, including in five species that only occur in the tropics. Within the more widely distributed Bactrocera tryoni and Bactrocera neohumeralis, Wolbachia also only occurred in the north, and was not linked to any particular mitochondrial haplotypes. CONCLUSIONS: The presence of Wolbachia pseudogenes at high prevalence in two species in absence of complete MLST profiles may represent footprints of historic infections that have been lost. The detection of identical low prevalence strains in a small number of individuals of seven species may question their role as reproductive manipulator and their vertical inheritance. Instead, the findings may be indicative of transient infections that result from spillover events from a yet unknown source. These spillover events appear to be restricted to northern Australia, without proliferation in host lineages further south. Our study highlights that tropical fruit fly communities contain Wolbachia pseudogenes and may be exposed to frequent horizontal Wolbachia transfer. It also emphasises that global estimates of Wolbachia frequencies may need to consider lateral gene transfer and Wolbachia spillover that may be regionally restricted, transient and not inherited.

Loop-mediated isothermal amplification (LAMP) assays for detection of the New Guinea fruit fly Bactrocera trivialis (Drew) (Diptera: Tephritidae).

The cue-lure-responding New Guinea fruit fly, Bactrocera trivialis, poses a biosecurity risk to neighbouring countries, e.g., Australia. In trapping programs, lure caught flies are usually morphologically discriminated from non-target species; however, DNA barcoding can be used to confirm similar species where morphology is inconclusive, e.g., Bactrocera breviaculeus and B. rufofuscula. This can take days-and a laboratory-to resolve. A quicker, simpler, molecular diagnostic assay would facilitate a more rapid detection and potential incursion response. We developed LAMP assays targeting cytochrome c oxidase subunit I (COI) and Eukaryotic Translation Initiation Factor 3 Subunit L (EIF3L); both assays detected B. trivialis within 25 min. The BtrivCOI and BtrivEIF3L assay anneal derivatives were 82.7 ± 0.8 °C and 83.3 ± 1.3 °C, respectively, detecting down to 1 × 101 copies/µL and 1 × 103 copies/µL, respectively. Each assay amplified some non-targets from our test panel; however notably, BtrivCOI eliminated all morphologically similar non-targets, and combined, the assays eliminated all non-targets. Double-stranded DNA gBlocks were developed as positive controls; anneal derivatives for the COI and EIF3L gBlocks were 84.1 ± 0.7 °C and 85.8 ± 0.2 °C, respectively. We recommend the BtrivCOI assay for confirmation of suspect cue-lure-trapped B. trivialis, with BtrivEIF3L used for secondary confirmation when required.

Comparative Trap Catches of Male Bactrocera, Dacus, and Zeugodacus Fruit Flies (Diptera: Tephritidae) With Four Floral Phenylbutanoid Lures (Anisyl Acetone, Cue-Lure, Raspberry Ketone, and Zingerone) in Queensland, Australia.

The male fruit fly attractants, cue-lure (CL) and raspberry ketone (RK), are important in pest management. These volatile phenylbutanoids occur in daciniphilous Bulbophyllum Thouar (Orchidaceae: Asparagales) orchids, along with zingerone (ZN) and anisyl acetone (AA). While these four compounds attract a similar range of species, their relative attractiveness to multiple species is unknown. We field tested these compounds in two fruit fly speciose locations in north Queensland, Australia (Lockhart and Cairns) for 8 wk. Of 16 species trapped in significant numbers, 14 were trapped with CL and RK, all in significantly greater numbers with CL traps than RK traps (at least in higher population locations). This included the pest species Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) (CL catches ca. 5× > RK), Bactrocera neohumeralis (Hardy) (Diptera: Tephritidae) and Bactrocera bryoniae (Tryon) (Diptera: Tephritidae) (CL catches ca. 3× > RK), and Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) (in Cairns-CL catches ca. 1.6× > RK). Seven species were trapped with AA, and all were also caught in CL and RK traps in significantly greater numbers, with the exception of B. frauenfeldi. For this species, catches were not statistically different with CL, RK, and AA in Lockhart, and RK and AA in Cairns. Seven species were trapped with ZN, two at this lure only, and the remainder also with CL or RK but in significantly greater numbers. This is the first quantitative comparison of the relative attractiveness of CL, RK, AA, and ZN against multiple species, and supports the long-held but untested assumption that CL is broadly more attractive lure than RK.

Impacts of climate change on high priority fruit fly species in Australia.

Tephritid fruit flies are among the most destructive horticultural pests posing risks to Australia's multi-billion-dollar horticulture industry. Currently, there are 11 pest fruit fly species of economic concern in Australia. Of these, nine are native to this continent (Bactrocera aquilonis, B. bryoniae, B. halfordiae, B. jarvisi, B. kraussi, B. musae, B. neohumeralis, B. tryoni and Zeugodacus cucumis), while B. frauenfeldi and Ceratitis capitata are introduced. To varying degrees these species are costly to Australia's horticulture through in-farm management, monitoring to demonstrate pest freedom, quarantine and trade restrictions, and crop losses. Here, we used a common species distribution model, Maxent, to assess climate suitability for these 11 species under baseline (1960-1990) and future climate scenarios for Australia. Projections indicate that the Wet Tropics is likely to be vulnerable to all 11 species until at least 2070, with the east coast of Australia also likely to remain vulnerable to multiple species. While the Cape York Peninsula and Northern Territory are projected to have suitable climate for numerous species, extrapolation to novel climates in these areas decreases confidence in model projections. The climate suitability of major horticulture areas currently in eastern Queensland, southern-central New South Wales and southern Victoria to these pests may increase as climate changes. By highlighting areas at risk of pest range expansion in the future our study may guide Australia's horticulture industry in developing effective monitoring and management strategies.

Isoeugenol, a More Attractive Male Lure for the Cue-Lure-Responsive Pest Fruit Fly Bactrocera curvipennis (Diptera: Tephritidae: Dacinae), and New Records of Species Responding to Zingerone in New Caledonia.

Bactrocera curvipennis (Froggatt) is a polyphagous pest fruit fly endemic to New Caledonia that is weakly attracted to the male lure cue-lure (CL). Effective male lures are important for the monitoring and management of numerous pest species of Dacinae fruit flies. However, if a species is weakly responsive to these lures its detection and control is difficult. Recently in Oceania and Asia, more attractive male lures (isoeugenol, methyl-isoeugenol, dihydroeugenol, and zingerone) were identified for several weakly CL- and methyl eugenol (ME)-responsive species. To determine if these lures may be more attractive to B. curvipennis, we field tested them in comparison to CL and ME in New Caledonia. Bactrocera curvipennis catch with isoeugenol-baited traps (mean 20.3 ± 3.0) was 15 times greater than with CL (1.3 ± 0.8) and catch with dihydroeugenol (5.7 ± 1.6) was four times greater than with CL. This is the first record of B. curvipennis responding to these lures. It was also the only species that responded to isoeugenol in this study. Bactrocera fulvifacies (Perkins) (Diptera: Tephritidae), a rarely encountered species 'nonresponsive' to male lures, was attracted to zingerone with its trap catch (2,574 flies) approaching that of Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) at CL (2,724 flies). Another nonresponsive species, Dacus aneuvittatus (Drew) (Diptera: Tephritidae), was also trapped by zingerone-baited traps. This is the first record of these species responding to a male lure. The significantly greater response of B. curvipennis to isoeugenol would make it a considerably more effective attractant for use in surveillance and control programs.

Systematic Modification of Zingerone Reveals Structural Requirements for Attraction of Jarvis's Fruit Fly.

Tephritid fruit flies are amongst the most significant horticultural pests globally and male chemical lures are important for monitoring and control. Zingerone has emerged as a unique male fruit fly lure that can attract dacine fruit flies that are weakly or non-responsive to methyl eugenol and cuelure. However, the key features of zingerone that mediate this attraction are unknown. As Jarvis's fruit fly, Bactrocera jarvisi (Tryon), is strongly attracted to zingerone, we evaluated the response of B. jarvisi to 37 zingerone analogues in a series of field trials to elucidate the functional groups involved in attraction. The most attractive analogues were alkoxy derivatives, with isopropoxy being the most attractive, followed by ethoxy and trifluoromethoxy analogues. All of the phenolic esters tested were also attractive with the response typically decreasing with increasing size of the ester. Results indicate that the carbonyl group, methoxy group, and phenol of zingerone are key sites for the attraction of B. jarvisi and identify some constraints on the range of structural modifications that can be made to zingerone without compromising attraction. These findings are important for future work in developing and optimising novel male chemical lures for fruit flies.

Combining Cue-Lure and Methyl Eugenol in Traps Significantly Decreases Catches of Most Bactrocera, Zeugodacus and Dacus Species (Diptera: Tephritidae: Dacinae) in Australia and Papua New Guinea.

Male fruit fly attractants, cue-lure (CL) and methyl eugenol (ME), are important in the monitoring and control of pest fruit fly species. Species respond to CL or ME but not both, and there are conflicting reports on whether combining CL (or its hydroxy analogue raspberry ketone) and ME decreases their attractiveness to different species. Fruit fly monitoring programs expend significant effort using separate CL and ME traps and avoiding lure cross-contamination, and combining the two lures in one trap would create substantial savings. To determine if combining lures has an inhibitory effect on trap catch, CL and ME wicks placed in the same Steiner trap were field tested in comparison to CL alone and ME alone in Australia and Papua New Guinea (PNG). In Australia, 24 out of 27 species trapped were significantly more attracted to CL or ME alone than the combination ME/CL lure, including the pests Bactrocera bryoniae (Tryon), B. frauenfeldi (Schiner), B. kraussi (Hardy), B. neohumeralis (Hardy), B. tryoni (Froggatt) (CL-responsive), and B. musae (Tryon) (ME-responsive). In PNG, 13 out of 16 species trapped were significantly more attracted to CL or ME alone than the ME/CL combination, including the pests B. bryoniae, B. frauenfeldi, B. neohumeralis, B. trivialis (Drew), Zeugodacus cucurbitae (Coquillett) (CL-responsive) and B. dorsalis (Hendel), B. musae, and B. umbrosa (Fabricius) (ME-responsive). This study shows that combining CL and ME in the one trap in equal parts significantly reduces catches of most species of Dacini fruit flies in Australia and PNG.

Methyl-isoeugenol, a Highly Attractive Male Lure for the Cucurbit Flower Pest Zeugodacus diversus (Coquillett) (syn. Bactrocera diversa) (Diptera: Tephritidae: Dacinae).

Effective male fruit fly attractants, such as cue lure (CL) and methyl eugenol (ME), are important in the monitoring and management of pest species through lure and kill techniques of trapping and male annihilation. However, some species are only weakly responsive to these lures, making their detection and control difficult. Zeugodacus diversus (Coquillett), a pest of cucurbit flowers in Asia, is weakly attracted to ME. Recently in Australia and Papua New Guinea, the eugenol analogues isoeugenol, methyl-isoeugenol, and dihydroeugenol were found to be effective attractants for species with a weak response to ME and CL, as well as several nonresponsive species. Additionally, studies from the early 1900s indicated that Z. diversus was attracted to isoeugenol. To determine if these eugenol analogues may be more effective attractants for Z. diversus, we field tested them in Bangladesh in comparison to ME, as well as CL and zingerone. Z. diversus was significantly more attracted to all three eugenol analogues than ME, with it most attracted to methyl-isoeugenol. Its attraction to methyl-isoeugenol was 49 times greater than its attraction to ME (respective means 23.58 flies/trap/day (FTD) and 0.48 FTD). Z. diversus was also consistently trapped at methyl-isoeugenol at all trap clearances including when populations were low, whereas it was only trapped at ME at 6 out of the 13 clearances. This study demonstrates that methyl-isoeugenol is a highly attractive lure for Z. diversus and would be a valuable inclusion as an attractant in monitoring and male annihilation programs.

Field Trapping Bactrocera latifrons (Diptera: Tephritidae) with Select Eugenol Analogs That Have Been Found to Attract Other 'Non-Responsive' Fruit Fly Species.

Bactrocera latifrons (Hendel) (Diptera: Tephritidae) is a pest fruit fly species native to Oriental Asia which has invaded and established in Hawaii and Tanzania and has been recovered in detection trapping in California. It is largely non-responsive to the male lures cuelure and methyl eugenol. Alpha-ionol + cade oil is a moderately effective male B. latifrons attractant, but is not as attractive as cuelure or methyl eugenol are to other fruit fly species. An improved attractant is therefore desired. With the recent success in finding other non-responsive fruit fly species attracted to isoeugenol, methyl-isoeugenol, or dihydroeugenol in Australia and other countries, we wanted to assess whether B. latifrons might also respond to these “eugenol analogs.” Working with wild B. latifrons populations in Hawaii, we assessed the relative catch of B. latifrons in traps baited with the eugenol analogs with catch in traps baited with alpha-ionol, alpha-ionol + cade oil, or alpha-ionol + eugenol. Catch was significantly higher in traps baited with alpha-ionol + cade oil relative to traps with any of the other baits. There was, though, some male B. latifrons catch in traps baited with dihydroeugenol or isoeugenol but none in traps baited with methyl-isoeugenol.

Author Correction: Potential impacts of climate change on habitat suitability for the Queensland fruit fly.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Potential impacts of climate change on habitat suitability for the Queensland fruit fly.

Anthropogenic climate change is a major factor driving shifts in the distributions of pests and invasive species. The Queensland fruit fly, Bactrocera tryoni Froggatt (Qfly), is the most economically damaging insect pest of Australia's horticultural industry, and its management is a key priority for plant protection and biosecurity. Identifying the extent to which climate change may alter the distribution of suitable habitat for Qfly is important for the development and continuation of effective monitoring programs, phytosanitary measures, and management strategies. We used Maxent, a species distribution model, to map suitable habitat for Qfly under current climate, and six climate scenarios for 2030, 2050 and 2070. Our results highlight that south-western Australia, northern regions of the Northern Territory, eastern Queensland, and much of south-eastern Australia are currently suitable for Qfly. This includes southern Victoria and eastern Tasmania, which are currently free of breeding populations. There is substantial agreement across future climate scenarios that most areas currently suitable will remain so until at least 2070. Our projections provide an initial estimate of the potential exposure of Australia's horticultural industry to Qfly as climate changes, highlighting the need for long-term vigilance across southern Australia to prevent further range expansion of this species.