A diagnostic LAMP assay for the destructive grapevine insect pest, phylloxera (Daktulosphaira vitifoliae).

Grape phylloxera (Daktulosphaira vitifoliae) is a destructive insect pest of grapevines that is highly invasive worldwide, despite strict biosecurity containment measures in place at farm and regional levels. Current phylloxera identification by visual inspection and laboratory-based molecular methods is time-consuming and costly. More rapid and cost-effective methods for identification of this pest would benefit industry, growers, and biosecurity services. Loop mediated isothermal amplification (LAMP) is a new portable technology available for rapid and accurate in-field molecular diagnostics. This study outlines the development of a new LAMP assay to enable the identification of phylloxera specimens. New LAMP primers were developed to specifically amplify phylloxera mitochondrial DNA (5'-COI), which we have shown is effective as a DNA barcode for identification of phylloxera, using LAMP technology. Positive LAMP reactions, containing phylloxera DNA, amplified in less than twelve minutes with an anneal derivative temperature of approximately 79 °C to 80 °C compared to a newly designed synthetic DNA (gBlock) fragment which had an anneal derivative temperature of 82 °C. No LAMP amplification was detected in any of the non-target species tested, i.e. no false-positive identification resulted for these species. We also successfully optimised a non-destructive DNA extraction procedure, HotSHOT "HS6", for use in the field on phylloxera adults, nymphs and eggs, to retain physical specimens. DNA extracted using this method was also suitable for species and genotype molecular identification methods, such as DNA barcoding, qPCR and microsatellite genotyping. The new LAMP assay provides a novel visual molecular tool for accurate diagnostics of phylloxera in the laboratory and field.

A LAMP assay for the detection of Bactrocera tryoni Queensland fruit fly (Diptera: Tephritidae).

LAMP assays are targeted molecular tests for the rapid detection of species in the laboratory and field. We developed a LAMP assay for an economically important fruit fly species, Queensland fruit fly, Bactrocera tryoni. This assay was assessed against a broad panel of target and non-target species and found to be specific, only amplifying the target species and closest relatives, in a portable real-time fluorometer (Genie III) in under 15 minutes with an anneal derivative temperature of 82.5 oC. The assay is sensitive to low levels of target DNA (>0.016 ng/µl), performing equally to the existing qPCR test. To enable retention of a physical voucher specimen, for potential morphological confirmation of LAMP results, a novel whole-specimen non-destructive DNA extraction method was developed, suitable for LAMP in the field. The stability of DNA extraction and LAMP reagents was tested under simulated and actual field conditions and shown to be robust. Our new assay now provides a portable molecular tool for the detection of this significant tephritid fruit fly pest species of biosecurity/quarantine concern. This has already proven invaluable for in-field diagnostics, providing real-time support influencing immediate actions, with negative results allowing the release of fruit produce, and positive results initiating fruit fly control measures.

Behavioral and Physiological Evidence for Palp Detection of the Male-Specific Attractant Cuelure in the Queensland Fruit Fly (Bactrocera tryoni).

The Queensland fruit fly, Bactrocera tryoni, is considered one of the worst horticultural pests in Australia attacking a large variety of fruit crops. To defeat pest insects, olfactory attractants have been developed and widely used in lure and kill strategies. Male B. tryoni are strongly attracted to the compound raspberry ketone and its synthetic analog, cuelure. Despite the strong behavioral response, a recent study failed to show any activation of antennal receptors to cuelure. Therefore, we hypothesized that cuelure may be detected by an accessory olfactory organ, the maxillary palp. Combining behavioral and physiological experiments we clearly demonstrate that male flies, but not female flies, primarily use the maxillary palps and not the antennae to detect and respond to cuelure. Furthermore, regardless of satiety status, male flies always preferred cuelure over a sugar rich source, unless the maxillary palps were excised.

Learning Influences Host Versus Nonhost Discrimination and Postalighting Searching Behavior in the Tephritid Fruit Fly Parasitoid Diachasmimorpha kraussii (Hymenoptera: Braconidae).

Compared with the extensive body of research on the olfactory behavior of parasitoids of leaf-feeding insects, less is known about the fine-tuning of olfactory behavior in parasitoids that use fruit-feeding insects as hosts. We investigated whether a tephritid fruit fly parasitoid, Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae), could discriminate between odors of fruits infested by larvae of a host species, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), compared to fruits infested by non-host larvae, Drosophila melanogaster (Diptera: Drosophilidae). Female wasps showed a significant preference for nectarines infested with B. tryoni, over uninfested fruits or fruits infested with D. melanogaster. When wasps were given prior experience of host or nonhost infested fruit, females demonstrated an increased ability to discriminate between host and nonhost related odors, but only when they were conditioned on host-infested (as opposed to nonhost infested) fruit. Insects provided with both host and nonhost stimuli showed no greater discriminatory learning compared to those provided with the rewarding stimuli alone. Previous experience also influenced postalighting behavior. Naïve females, and females with experience ovipositing at the top of fruit, oriented preferentially to the top of fruits upon alighting, while those with experience ovipositing at the base of fruits showed a significant difference in orientation, with 70% of wasps orientating preferentially toward the base. Similar learning-related changes were seen in search time and probing behavior. We discuss how pre- and post-alighting learning fine-tunes the behavioral responses of foraging wasps to their local environment.

Wolbachia infection alters olfactory-cued locomotion in Drosophila spp.

Wolbachia pipientis is an endosymbiotic bacterium present in diverse insect species. Although it is well studied for its dramatic effects on host reproductive biology, little is known about its effects on other aspects of host biology, despite its presence in a wide array of host tissues. This study examined the effects of three Wolbachia strains on two different Drosophila species, using a laboratory performance assay for insect locomotion in response to olfactory cues. The results demonstrate that Wolbachia infection can have significant effects on host responsiveness that vary with respect to the Wolbachia strain-host species combination. The wRi strain, native to Drosophila simulans, increases the basal activity level of the host insect as well as its responsiveness to food cues. In contrast, the wMel strain and the virulent wMelPop strain, native to Drosophila melanogaster, cause slight decreases in responsiveness to food cues but do not alter basal activity levels in the host. Surprisingly, the virulent wMelPop strain has very little impact on host responsiveness in D. simulans. This novel strain-host relationship was artificially created previously by transinfection. These findings have implications for understanding the evolution and spread of Wolbachia infections in wild populations and for Wolbachia-based vector-borne disease control strategies currently being developed.

How host plant variability influences the advantages to learning: a theoretical model for oviposition behaviour in Lepidoptera.

Learning can allow individuals to increase their fitness in particular environments. The advantage to learning depends on the predictability of the environment and the extent to which animals can adjust their behaviour. Earlier general models have investigated when environmental predictability might favour the evolution of learning in foraging animals. Here, we construct a theoretical model that predicts the advantages to learning using a specific biological example: oviposition in the Lepidoptera. Our model includes environmental and behavioural complexities relevant to host selection in these insects and tests whether the predictions of the general models still hold. Our results demonstrate how the advantage of learning is maximised when within-generation variability is minimised (the local environment consists mainly of a single host plant species) and between-generation variability is maximised (different host plant species are the most common in different generations). We discuss how our results: (a) can be applied to recent empirical work in different lepidopteran species and (b) predict an important role of learning in lepidopteran agricultural pests.

A general model for host plant selection in phytophagous insects.

We develop a general theoretical framework for exploring the host plant selection behaviour of herbivorous insects. This model can be used to address a number of questions, including the evolution of specialists, generalists, preference hierarchies, and learning. We use our model to: (i) demonstrate the consequences of the extent to which the reproductive success of a foraging female is limited by the rate at which they find host plants (host limitation) or the number of eggs they carry (egg limitation); (ii) emphasize the different consequences of variation in behaviour before and after landing on (locating) a host (termed pre- and post-alighting, respectively); (iii) show that, in contrast to previous predictions, learning can be favoured in post-alighting behaviour--in particular, individuals can be selected to concentrate oviposition on an abundant low-quality host, whilst ignoring a rare higher-quality host; (iv) emphasize the importance of interactions between mechanisms in favouring specialization or learning.