Differential pheromone profile as a contributor to premating isolation between two sympatric sibling fruit fly species.

Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) are sibling fruit fly species that are sympatric over much of their ranges. Premating isolation of these close relatives is thought to be maintained in part by allochrony-mating activity in B. tryoni peaks at dusk, whereas in B. neohumeralis, it peaks earlier in the day. To ascertain whether differences in pheromone composition may also contribute to premating isolation between them, this study used solid-phase microextraction and gas chromatography-mass spectrometry to characterize the rectal gland volatiles of a recently collected and a more domesticated strain of each species. These glands are typical production sites and reservoirs of pheromones in bactrocerans. A total of 120 peaks were detected and 50 were identified. Differences were found in the composition of the rectal gland emissions between the sexes, species, and recently collected versus domesticated strains of each species. The compositional variation included several presence/absence and many quantitative differences. Species and strain differences in males included several relatively small alcohols, esters, and aliphatic amides. Species and strain differences in females also included some of the amides but additionally involved many fatty acid esters and 3 spiroacetals. While the strain differences indicate there is also heritable variation in rectal gland emissions within each species, the species differences imply that compositional differences in pheromones emitted from rectal glands could contribute to the premating isolation between B. tryoni and B. neohumeralis. The changes during domestication could also have significant implications for the efficacy of Sterile Insect Technique control programs.

Assessment of the efficacy of Piper methysticum (Micrembryeae: Piperaceae) as a bioinsecticide, and/or spinosad combined with attractive plant volatiles for a novel lure and kill strategy against Bactrocera tryoni (Diptera: Tephritidae) in laboratory.

The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is a crop pest of global economic importance because of its wide range of hosts and its invasiveness capacities. To develop a novel integrated and sustainable crop protection, we have investigated the insecticidal properties of different varieties of kava (Piper methysticum [Frost]) extracted by two methods and the attractive effects of six plant volatiles identified from B. tryoni host plants to female, mated or not. We did not identify any significant insecticidal effect of the traditional Pacific kava plant at the tested concentrations. Among mated females, ethyl acetate compared to the no odor control elicited the highest attraction (87%, of which 60% for this odor), while ethyl butyrate was preferred compared with ethyl acetate in dual choice assays. Flies' preferences for specific odors depended on their mating status and the odor landscape they were confronted with. Combination with the commercial ingestion insecticide (Success 4: spinosad, 480 g/l, Dow AgroSciences, Valbonne, France) with the plant volatiles were tested to detect an increase in mortality related to the addition of an attractant. The 2-heptanone slightly showed a tend to increase the attractiveness of mated females within 4-6 h to the food bait, but the results were not statistically significant after 8 h. Further tests should be performed with other concentrations or mixtures of the identified host plant volatiles to develop a strong lure and kill strategy.

CRISPR/Cas9 Mutagenesis to Generate Novel Traits in Bactrocera tryoni for Sterile Insect Technique.

Sterile Insect Technique (SIT) is a biocontrol strategy that has been widely utilized to suppress or eradicate outbreak populations of insect pests such as tephritid fruit flies. As SIT is highly favored due to it being species-specific and environmentally friendly, there are constant efforts to improve the efficiency and efficacy of this method in particular at low pest densities; one of which is the use of genetically enhanced strains. Development of these desirable strains has been facilitated by the emergence of the CRISPR/Cas genome-editing technology that enables the rapid and precise genomic modification of non-model organisms. Here, we describe the manual microinjection of CRISPR/Cas9 reagents into tephritid pest Bactrocera tryoni (Queensland fruit fly) embryos to introduce ideal traits as well as the molecular methods used to detect successful mutagenesis.

Chemical Composition of the Rectal Gland and Volatiles Released by Female Queensland Fruit Fly, Bactrocera tryoni (Diptera: Tephritidae).

The composition of the rectal gland secretion and volatiles emitted by female Queensland fruit fly, Bactrocera tryoni was investigated. Esters were found to be the main compounds in the gland extracts and headspace, while amides were the minor compounds in the gland extracts and headspace. Ethyl dodecanoate, ethyl tetradecanoate, ethyl (Z9)-hexadecenoate and ethyl palmitate were the main esters in the gland extracts, while ethyl dodecanoate and ethyl tetradecanoate were the main esters in the headspace. Four amides (N-(3-methylbutyl)acetamide), N-(2-methylbutyl)propanamide, N-(3-methylbutyl)propanamide, and N-(3-methylbutyl)-2-methylpropanamide were found in the gland extracts and the headspace. Among the amides, N-(3-methylbutyl)acetamide and N-(3-methylbutyl)propanamide were the main amides in the gland extracts and the headspace. Traces of three spiroacetals were found both in the gland extracts and in the headspace. (E,E)-2,8-Dimethyl-1,7-dioxaspiro[5.5]undecane, (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane, (E,E)-2-propyl-8-methyl-1,7-dioxaspiro[5.5]undecane. All compounds found in the headspace were present in the extract of the rectal gland suggesting that the rectal gland is the main source of the headspace volatiles, whose function remains to be elucidated. This is the first comprehensive chemical analysis of the rectal gland secretions and volatiles of female B. tryoni, and further laboratory and field bioassays are required to determine the function of compounds identified in this study. Discovery of the same amides previously identified in the male rectal gland in the female rectal gland raises questions about the pheromonal role previously suggested for these compounds.

Screening mitochondrial DNA sequence variation as an alternative method for tracking established and outbreak populations of Queensland fruit fly at the species southern range limit.

Understanding the relationship between incursions of insect pests and established populations is critical to implementing effective control. Studies of genetic variation can provide powerful tools to examine potential invasion pathways and longevity of individual pest outbreaks. The major fruit fly pest in eastern Australia, Queensland fruit fly Bactrocera tryoni (Froggatt), has been subject to significant long-term quarantine and population reduction control measures in the major horticulture production areas of southeastern Australia, at the species southern range limit. Previous studies have employed microsatellite markers to estimate gene flow between populations across this region. In this study, we used an independent genetic marker, mitochondrial DNA (mtDNA) sequences, to screen genetic variation in established and adjacent outbreak populations in southeastern Australia. During the study period, favorable environmental conditions resulted in multiple outbreaks, which appeared genetically distinctive and relatively geographically localized, implying minimal dispersal between simultaneous outbreaks. Populations in established regions were found to occur over much larger areas. Screening mtDNA (female) lineages proved to be an effective alternative genetic tool to assist in understanding fruit fly population dynamics and provide another possible molecular method that could now be employed for better understanding of the ecology and evolution of this and other pest species.

Yeast: An Overlooked Component of Bactrocera tryoni (Diptera: Tephritidae) Larval Gut Microbiota.

Yeasts, often in hydrolyzed form, are key ingredients in the larval and adult diets of tephritid fruit fly colonies. However, very little is known about the presence or role of yeasts in the diets of tephritid fruit flies in nature. Previous studies have identified bacteria but not detected yeasts in the gut of Queensland fruit fly, Bactrocera tryoni (Froggatt), one of Australia's most economically damaging insect pests of horticultural crops and of significant biosecurity concern domestically and internationally. Here we demonstrate that cultivable yeasts are commonly found in the gut of B. tryoni larvae from fruit hosts. Analysis of the ITS1, 5.8S rRNA gene, and ITS2 sequences of randomly selected isolates identified yeasts and yeast-like fungi of the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Pichia, and Starmerella. The prevalence of these yeasts in fruits suggests that larvae consume the yeasts as part of their diet. This work highlights that yeasts should be considered in future tephritid larval gut microbiota studies. Understanding tephritid-microbial symbiont interactions will lead to improvements in artificial diets and the quality of mass-reared tephritids for the sterile insect technique.

Specialized Pheromone and Lure Application Technology as an Alternative Male Annihilation Technique to Manage Bactrocera tryoni (Diptera: Tephritidae).

The results of this study suggest that a novel male annihilation technique (specialized pheromone and lure application technology [SPLAT] incorporating cue-lure [CL] plus spinosad) is as effective as industry standard male annihilation controls, and is worth exploring further to manage Bactrocera tryoni (Froggatt) populations. Three lures were evaluated in a contact and feeding bioassay and a cage attractancy trial: 1) SPLAT-CL + spinosad; 2) SPLAT-CL without spinosad; and 3) wick-CL + malathion. In a field attraction trial, lures (1) and (3) were evaluated with a third treatment, caneite blocks-CL + malathion. Lures were weathered for 0, 1, 2, 4, or 8 wk, with an additional weathering treatment of 12 wk included in the field trial. In the contact and feeding bioassay, lures with SPLAT-CL + spinosad were >97% effective at 48 h for up to 2 wk weathering; however, wicks-CL + malathion killed B. tryoni within 2 h of exposure under all weathering periods. In the cage attractancy trial, SPLAT-CL + spinosad was as effective as, or performed better than, wicks-CL + malathion under all weathering treatments. The field study trap catches were similar for SPLAT-CL + spinosad and blocks-CL + malathion, and both had higher trap catches than wicks-CL + malathion at all weathering periods, except week 12. Overall, SPLAT-CL + spinosad compared favorably with current standard techniques for male annihilation and warrants further research. SPLAT-CL + spinosad may be a reduced-risk alternative for wicks-CL + malathion or blocks-CL + malathion for B. tryoni and other CL-responding fruit flies, such as Bactrocera cucurbitae Coquillett, because it contains a reduced-risk insecticide that poses a lower risk to humans and the environment and does not require labor-intensive handling and placement.

Field Evaluation of Melolure, a Formate Analogue of Cuelure, and Reassessment of Fruit Fly Species Trapped in Sydney, New South Wales, Australia.

In Australia, tephritids are usually attracted to either cuelure or methyl eugenol. Methyl eugenol is a very effective lure, but cuelure is less effective likely due to low volatility. A new formate analogue of cuelure, melolure, has increased volatility, resulting in improved efficacy with the melon fruit fly, Bactrocera cucurbitae Coquillett. We tested the efficacy of melolure with fruit fly species in Sydney as part of the National Exotic Fruit Fly Monitoring programme. This monitoring programme has 71 trap sites across Sydney, with each trap site comprising separate Lynfield traps containing either cuelure, methyl eugenol, or capilure lure. In 2008, an additional Lynfield trap with melolure plugs was added to seven sites. In 2009 and 2010, an additional Lynfield trap with melolure wicks was added to 11 trap sites and traps were monitored fortnightly for 2 yr. Capture rates for melolure traps were similar to cuelure traps for Dacus absonifacies (May) and Dacus aequalis (Coquillet), but melolure traps consistently caught fewer Bactrocera tryoni (Froggatt) than cuelure traps. However, trap sites with both a cuelure and melolure traps had increased capture rates for D. absonifacies and D. aequalis, and a marginally significant increase for B. tryoni. Melolure plugs were less effective than melolure wicks, but this effect may be related to lure concentration. The broader Bactrocera group species were attracted more to cuelure than melolure while the Dacus group species were attracted more to melolure than cuelure. There is no benefit in switching from cuelure to melolure to monitor B. tryoni, the most important fruit fly pest in Australia.