Metarhizium spp. isolates effective against Queensland fruit fly juvenile life stages in soil.

Queensland fruit fly, Bactrocera tryoni, Froggatt (Diptera: Tephritidae) is Australia's primary fruit fly pest species. Integrated Pest Management (IPM) has been adopted to sustainably manage this polyphagous species with a reduced reliance on chemical pesticides. At present, control measures are aimed at the adult stages of the fly, with no IPM tools available to target larvae once they exit the fruit and pupate in the soil. The use of entomopathogenic fungi may provide a biologically-based control method for these soil-dwelling life stages. The effectiveness of fungal isolates of Metarhizium and Beauveria species were screened under laboratory conditions against Queensland fruit fly. In bioassays, 16 isolates were screened for pathogenicity following exposure of third-instar larvae to inoculum-treated vermiculite used as a pupation substrate. The best performing Metarhizium sp. isolate achieved an average percentage mortality of 93%, whereas the best performing Beauveria isolate was less efficient, with an average mortality of 36%. Susceptibility to infection during different development stages was investigated using selected fungal isolates, with the aim of assessing all soil-dwelling life stages from third-instar larvae to final pupal stages and emerging adults. Overall, the third larval instar was the most susceptible stage, with average mortalities between 51-98% depending on the isolate tested. Moreover, adult mortality was significantly higher when exposed to inoculum during pupal eclosion, with mortalities between 56-76% observed within the first nine days post-emergence. The effect of temperature and inoculum concentration on insect mortality were assessed independently with candidate isolates to determine the optimum temperature range for fungal biological control activity and the rate required for application in field conditions. Metarhizium spp. are highly efficacious at killing Queensland fruit fly and have potential for use as biopesticides to target soil-dwelling and other life stages of B. tryoni.

The pest sap beetle Carpophilus (Myothorax) truncatus Murray, 1864 (Coleoptera: Nitidulidae)-a new synonymy and a related new species of Carpophilus.

Carpophilus truncatus Murray 1864, is a species of sap beetle which has been recorded from many countries worldwide, and has become recognised as an important pest of nuts. In this study, we present a re-description of C. truncatus including diagnostic photographic images of the adults and larvae, and demonstrate that Carpophilus jarijari Powell & Hamilton, 2019 is a junior subjective synonym of C. truncatus. Information about the species' distribution in Australia is updated. DNA barcode sequence data for C. truncatus is reviewed and augmented to enable differentiation from other morphologically similar Carpophilus species that are associated with nuts as hosts, including the cosmopolitan Carpophilus dimidiatus (Fabricius, 1792), for which C. truncatus has sometimes been misidentified. This analysis revealed that existing reference DNA sequences of "C. dimidiatus" consist of three highly genetically divergent lineages, representing three species: the cosmopolitan C. dimidiatus, the widespread C. truncatus, and a newly described species, Carpophilus imitatus sp. nov., known from south-eastern Asia and Australia.

Volatiles produced by symbiotic yeasts improve trap catches of Carpophilus davidsoni (Coleoptera: Nitidulidae): an important pest of stone fruits in Australia.

Carpophilus davidsoni (Dobson) is an important pest of Australian stone fruit. Current management practices for this beetle include the use of a trap that contains an attractant lure comprised of aggregation pheromones and a 'co-attractant' mixture of volatiles from fruit juice fermented using Baker's yeast, Saccharomyces cerevisiae (Hansen). We explored whether volatiles from yeasts Pichia kluyveri (Bedford) and Hanseniaspora guilliermondii (Pijper), which are closely associated with C. davidsoni in nature, might improve the effectiveness of the co-attractant. Field trials using live yeast cultures revealed that P. kluyveri trapped higher numbers of C. davidsoni compared to H. guilliermondii, and comparative GC-MS of volatile emissions of the two yeasts led to the selection of isoamyl acetate and 2-phenylethyl acetate for further investigation. In subsequent field trials, trap catches of C. davidsoni were significantly increased when 2-phenylethyl acetate was added to the co-attractant, compared to when isoamyl acetate was added, or both isoamyl acetate and 2-phenylethyl acetate. We also tested different concentrations of ethyl acetate in the co-attractant (the only ester in the original lure) and found contrasting results in cage bioassays and field trails. Our study demonstrates how exploring volatile emissions from microbes that are ecologically associated with insect pests can result in more potent lures for use in integrated pest management strategies. Results from laboratory bioassays screening volatile compounds should be treated with caution when making inferences regarding attraction under field conditions.

A Synthetic Blend of Fruit and Live Yeast Odours Shows Promise for Trapping Mated Female Queensland Fruit Fly, Bactrocera tryoni, in the Field.

Tephritid fruit flies are major horticultural pests of fruit and vegetable crops. Developing an odour lure that can attract mated female flies under field conditions has presented a major challenge to chemical ecologists around the globe. We have designed a new lure to attract female Queensland fruit fly, Bactrocera tryoni, based on the knowledge that this pest tephritid is attracted not only to odours from ripening fruits, but also to odours of symbiotic (gut-associated) yeasts on which the larvae feed. Initial field trials screened four volatile mixtures, each containing a base mix of three, short-chain "fruit ripening" esters and differing in the addition of long-chain "fruity" esters and / or alcohols produced by live yeasts. Results demonstrated that the lure formulated from short-chain esters and live yeast alcohols attracted the most female flies, with significantly higher catches than an odourless control trap, and a protein-baited trap. As electrophysiological studies exploring peripheral responses in tephritids to alcohols associated with fermentation have not been extensively investigated, we conducted this work on both the antennae and maxillary palps, and discovered that three yeast alcohols were detected by both sensory organs. Performance trials conducted in pome fruit and stone fruit orchards evaluated the six-component blend of short-chain ester and live yeast alcohol lure against a commercially available synthetic fruit-odour lure and a protein-based lure. In the apple orchard, the new lure caught significantly more female flies, and in particular mated females, compared to the commercially available lures. In the stone fruit orchard, while the new lure again caught the most mated female flies compared to the other lures, differences were not significant. The visual stimulus (trap design) used had a significant effect on capture, with a Ladd trap (red sphere on a yellow panel) being considerably more effective than the other traps. We discuss the implications of this study, including why the degree of effectiveness of the new lure might have differed among the test orchards (citrus, apple, stonefruit), and why yeast odours might increase attractiveness to "ripening fruit" volatile blends under field conditions.

Developing a non-destructive metabarcoding protocol for detection of pest insects in bulk trap catches.

Metabarcoding has the potential to revolutionise insect surveillance by providing high-throughput and cost-effective species identification of all specimens within mixed trap catches. Nevertheless, incorporation of metabarcoding into insect diagnostic laboratories will first require the development and evaluation of protocols that adhere to the specialised regulatory requirements of invasive species surveillance. In this study, we develop a multi-locus non-destructive metabarcoding protocol that allows sensitive detection of agricultural pests, and subsequent confirmation using traditional diagnostic techniques. We validate this protocol for the detection of tomato potato psyllid (Bactericera cockerelli) and Russian wheat aphid (Diuraphis noxia) within mock communities and field survey traps. We find that metabarcoding can reliably detect target insects within mixed community samples, including specimens that morphological identification did not initially detect, but sensitivity appears inversely related to community size and is impacted by primer biases, target loci, and sample indexing strategy. While our multi-locus approach allowed independent validation of target detection, lack of reference sequences for 18S and 12S restricted its usefulness for estimating diversity in field samples. The non-destructive DNA extraction proved invaluable for resolving inconsistencies between morphological and metabarcoding identification results, and post-extraction specimens were suitable for both morphological re-examination and DNA re-extraction for confirmatory barcoding.

Yeasts Influence Host Selection and Larval Fitness in Two Frugivorous Carpophilus Beetle Species.

We explored how gut-associated yeasts influence olfactory behaviour and resource use in two pest species of Carpophilus beetle that co-exist in Australian stone fruits. Molecular analysis of yeasts isolated from the gut of C. davidsoni (prefers ripe fruits) and C. hemipterus (prefers overripe and rotting fruits) revealed that the predominant species were Pichia kluyveri and Hanseniaspora guilliermondii. In olfactory attraction and oviposition trials, adult beetles preferred H. guilliermondii over P. kluyveri, and follow up GC-MS analysis revealed unambiguous differences between the odour profiles of these yeasts. In contrast to behavioural trials, larval feeding assays showed that fruit substrates inoculated with P. kluyveri yielded significantly faster development times, higher pupal mass, and a greater number of adult beetles, compared to H. guilliermondii - in other words, the lesser preferred yeast (by foraging adults) was more suitable for larval survival. Moreover, whilst larvae of both species survived to adulthood when fed solely on P. kluyveri (i.e. without a fruit substrate), only larvae of C. davidsoni could develop on H. guilliermondii; and only C. davidsoni reached adulthood feeding on a yeast-free fruit substrate. We discuss how these findings may relate to adaptations towards early colonising of fruits by C. davidsoni, enabling differences in resource use and potentially resource partitioning in the two beetles. More broadly, consideration of microbial interactions might help develop host selection theory. Our results could pave the way to more powerful attractants to mass-trap and monitor Carpophilus pests in fruit orchards.

Prospects and challenges of implementing DNA metabarcoding for high-throughput insect surveillance.

Trap-based surveillance strategies are widely used for monitoring of invasive insect species, aiming to detect newly arrived exotic taxa as well as track the population levels of established or endemic pests. Where these surveillance traps have low specificity and capture non-target endemic species in excess of the target pests, the need for extensive specimen sorting and identification creates a major diagnostic bottleneck. While the recent development of standardized molecular diagnostics has partly alleviated this requirement, the single specimen per reaction nature of these methods does not readily scale to the sheer number of insects trapped in surveillance programmes. Consequently, target lists are often restricted to a few high-priority pests, allowing unanticipated species to avoid detection and potentially establish populations. DNA metabarcoding has recently emerged as a method for conducting simultaneous, multi-species identification of complex mixed communities and may lend itself ideally to rapid diagnostics of bulk insect trap samples. Moreover, the high-throughput nature of recent sequencing platforms could enable the multiplexing of hundreds of diverse trap samples on a single flow cell, thereby providing the means to dramatically scale up insect surveillance in terms of both the quantity of traps that can be processed concurrently and number of pest species that can be targeted. In this review of the metabarcoding literature, we explore how DNA metabarcoding could be tailored to the detection of invasive insects in a surveillance context and highlight the unique technical and regulatory challenges that must be considered when implementing high-throughput sequencing technologies into sensitive diagnostic applications.

An improved culturing method for opiine fruit fly parasitoids and its application to parasitoid monitoring in the field.

Good culturing methods play an important role in the study of insect behavior and its application to pest management. Here, we describe and validate a new method for rearing the parasitoid wasp, Diachasmimorpha kraussii, which attacks some of the world's worst fruit fly pests and is an internationally used biological control agent. Our method differs from standard culturing approaches by presenting adult wasps with host-infested artificial media within a "culturing bag," which mimics a natural (fruit) oviposition substrate. In laboratory trials using wild collected D. kraussii, the culturing bag method was compared to the use of host-infested nectarines, and a commonly used laboratory method of presenting host-infested artificial media within Petri dishes. The culturing bag method proved to be a significant improvement on both methods, combining the advantages of high host survival in artificial media with parasitism levels that were the equivalent to those recorded using host-infested fruits. In our field study, culturing bags infested with the Queensland fruit fly, Bactrocera tryoni, and hung in a mixed peach and nectarine orchard proved to be effective "artificial fruits" attracting wild D. kraussii for oviposition. Significantly more adult wasps were reared from the culturing bags compared to field collected fruits. This was shown to be due to higher fruit fly larval density in the bags, as similar percentage parasitism rates were found between the culturing bags and ripe fruits. We discuss how this cheap, time-efficient method could be applied to collecting and monitoring wild D. kraussii populations in orchards, and assist in maintaining genetic variability in parasitoid laboratory cultures.

Two Gut-Associated Yeasts in a Tephritid Fruit Fly have Contrasting Effects on Adult Attraction and Larval Survival.

Yeast-insect interactions have been well characterized in drosophilid flies, but not in tephritid fruit flies, which include many highly polyphagous pest species that attack ripening fruits. Using the Queensland fruit fly (Bactrocera tryoni) as our model tephritid species, we identified yeast species present in the gut of wild-collected larvae and found two genera, Hanseniaspora and Pichia, were the dominant isolates. In behavioural trials using adult female B. tryoni, a fruit-agar substrate inoculated with Pichia kluyveri resulted in odour emissions that increased the attraction of flies, whereas inoculation with Hanseniaspora uvarum, produced odours that strongly deterred flies, and both yeasts led to decreased oviposition. Larval development trials showed that the fruit-agar substrate inoculated with the 'deterrent odour' yeast species, H. uvarum, resulted in significantly faster larval development and a greater number of adult flies, compared to a substrate inoculated with the 'attractive odour' yeast species, P. kluyveri, and a yeast free control substrate. GC-MS analysis of volatiles emitted by H. uvarum and P. kluyveri inoculated substrates revealed significant quantitative differences in ethyl-, isoamyl-, isobutyl-, and phenethyl- acetates, which may be responsible for the yeast-specific olfactory responses of adult flies. We discuss how our seemingly counterintuitive finding that female B. tryoni flies avoid a beneficial yeast fits well with our understanding of female choice of oviposition sites, and how the contrasting behavioural effects of H. uvarum and P. kluyveri raises interesting questions regarding the role of yeast-specific volatiles as cues to insect vectors. A better understanding of yeast-tephritid interactions could assist in the future management of tephritid fruit fly pests through the formulation of new "attract and kill" lures, and the development of probiotics for mass rearing of insects in sterile insect control programs.

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.

Bees at war: interspecific battles and nest usurpation in stingless bees.

We provide the first evidence for interspecific warfare in bees, a spectacular natural phenomenon that involves a series of aerial battles and leads to thousands of fatalities from both attacking and defending colonies. Molecular analysis of fights at a hive of the Australian stingless bee Tetragonula carbonaria revealed that the attack was launched by a related species, Tetragonula hockingsi, which has only recently extended its habitat into southeastern Queensland. Following a succession of attacks by the same T. hockingsi colony over a 4-month period, the defending T. carbonaria colony was defeated and the hive usurped, with the invading colony installing a new queen. We complemented our direct observations with a 5-year study of more than 260 Tetragonula hives and found interspecific hive changes, which were likely to be usurpation events, occurring in 46 hives over this period. We discuss how fighting swarms and hive usurpation fit with theoretical predictions on the evolution of fatal fighting and highlight the many unexplained features of these battles that warrant further study.

Understanding heliothine (Lepidoptera: Heliothinae) pests: what is a host plant?

Heliothine moths (Lepidoptera: Heliothinae) include some of the world's most devastating pest species. Whereas the majority of nonpest heliothinae specialize on a single plant family, genus, or species, pest species are highly polyphagous, with populations often escalating in size as they move from one crop species to another. Here, we examine the current literature on heliothine host-selection behavior with the aim of providing a knowledge base for research scientists and pest managers. We review the host relations of pest heliothines, with a particular focus on Helicoverpa armigera (Hübner), the most economically damaging of all heliothine species. We then consider the important question of what constitutes a host plant in these moths, and some of the problems that arise when trying to determine host plant status from empirical studies on host use. The top six host plant families in the two main Australian pest species (H. armigera and Helicoverpa punctigera Wallengren) are the same and the top three (Asteraceae, Fabaceae, and Malvaceae) are ranked the same (in terms of the number of host species on which eggs or larvae have been identified), suggesting that these species may use similar cues to identify their hosts. In contrast, for the two key pest heliothines in the Americas, the Fabaceae contains approximately 1/3 of hosts for both. For Helicoverpa zea (Boddie), the remaining hosts are more evenly distributed, with Solanaceae next, followed by Poaceae, Asteraceae, Malvaceae, and Rosaceae. For Heliothis virescens (F.), the next highest five families are Malvaceae, Asteraceae, Solanaceae, Convolvulaceae, and Scrophulariaceae. Again there is considerable overlap in host use at generic and even species level. H. armigera is the most widely distributed and recorded from 68 plant families worldwide, but only 14 families are recorded as a containing a host in all geographic areas. A few crop hosts are used throughout the range as expected, but in some cases there are anomalies, perhaps because host plant relation studies are not comparable. Studies on the attraction of heliothines to plant odors are examined in the context of our current understanding of insect olfaction, with the aim of better understanding the connection between odor perception and host choice. Finally, we discuss research into sustainable management of pest heliothines using knowledge of heliothine behavior and ecology. A coordinated international research effort is needed to advance our knowledge on host relations in widely distributed polyphagous species instead of the localized, piecemeal approaches to understanding these insects that has been the norm to date.

Increased plant volatile production affects oviposition, but not larval development, in the moth Helicoverpa armigera.

It is well established that herbivorous insects respond to changes in plant odour production, but little attention has been given to whether these responses relate to direct fitness costs of plant volatile production on insect growth and survival. Here, we use transgenic Nicotiana tabacum (tobacco) plants that produce relatively large amounts of the volatile (S)-linalool to study whether the responses of egg-laying herbivorous insects to linalool production relate directly to the growth and survival of offspring. In choice tests, fewer eggs were laid on transgenic plants compared with non-transformed controls, indicating that increased linalool emissions have a deterrent effect on Helicoverpa armigera oviposition. Larval survival and larval mass after feeding on transgenic leaves, however, was comparable to non-transformed controls. (S)-linalool, whether in volatile or sequestered form, does not appear to have a direct effect on offspring fitness in this moth. We discuss how the ecology of this polyphagous moth species may necessitate a high tolerance for certain volatiles and their related non-volatile compounds, and suggest that responses by adult female H. armigera moths towards increased linalool production may be context specific and relate to other indirect effects on fitness.

Insect odour perception: recognition of odour components by flower foraging moths.

Odours emitted by flowers are complex blends of volatile compounds. These odours are learnt by flower-visiting insect species, improving their recognition of rewarding flowers and thus foraging efficiency. We investigated the flexibility of floral odour learning by testing whether adult moths recognize single compounds common to flowers on which they forage. Dual choice preference tests on Helicoverpa armigera moths allowed free flying moths to forage on one of three flower species; Argyranthemum frutescens (federation daisy), Cajanus cajan (pigeonpea) or Nicotiana tabacum (tobacco). Results showed that, (i) a benzenoid (phenylacetaldehyde) and a monoterpene (linalool) were subsequently recognized after visits to flowers that emitted these volatile constituents, (ii) in a preference test, other monoterpenes in the flowers' odour did not affect the moths' ability to recognize the monoterpene linalool and (iii) relative preferences for two volatiles changed after foraging experience on a single flower species that emitted both volatiles. The importance of using free flying insects and real flowers to understand the mechanisms involved in floral odour learning in nature are discussed in the context of our findings.

Learning, odour preference and flower foraging in moths.

Floral volatiles play a major role in plant-insect communication. We examined the influence of two volatiles, phenylacetaldehyde and alpha-pinene, on the innate and learnt foraging behaviour of the moth Helicoverpa armigera. In dual-choice wind tunnel tests, adult moths flew upwind towards both volatiles, with a preference for phenylacetaldehyde. When exposure to either of these volatiles was paired with a feeding stimulus (sucrose), all moths preferred the learnt odour in the preference test. This change in preference was not seen when moths were exposed to the odour without a feeding stimulus. The learnt preference for the odour was reduced when moths were left unfed for 24 h before the preference test. We tested whether moths could discriminate between flowers that differed in a single volatile component. Moths were trained to feed on flowers that were odour-enhanced using either phenylacetaldehyde or alpha-pinene. Choice tests were then carried out in an outdoor flight cage, using flowers enhanced with either volatile. Moths showed a significant preference for the flower type on which they were trained. Moths that were conditioned on flowers that were not odour-enhanced showed no preference for either of the odour-enhanced flower types. The results imply that moths may be discriminating among odour profiles of individual flowers from the same species. We discuss this behaviour within the context of nectar foraging in moths and odour signalling by flowering plants.