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Queensland fruit flies Bactrocera tryoni (Froggatt) have a long adult maturation phase which, together with high mortality rates, can substantially reduce number of released flies that survive to mature and contribute to sterile insect technique (SIT) programmes. This constraint on SIT can potentially be addressed by incorporating methoprene, a juvenile hormone analogue, into an adult diet of sugar and yeast hydrolysate for 2 days after emergence. Methoprene treatments have been found to accelerate sexual development of male Queensland fruit fly, resulting in increased mating propensity of 5–7-day-old males in no-choice laboratory trials. Before considering deployment of methoprene as a pre-release treatment in SIT, it is necessary to demonstrate mating competitiveness and compatibility of methoprene-treated flies under field-like conditions. In the present study, we assessed whether methoprene treatment increases ability of sterile males (5 and 7 days old) to compete with mature (wild or laboratory) males for matings with mature (wild or laboratory) females in field cages. We also investigated mating compatibility to test for sexual isolation between sterile flies and mature (wild or laboratory) fertile flies. In mating competitiveness tests, methoprene-treated males of either age outperformed mature wild or laboratory males for matings with mature wild or laboratory females, respectively. Untreated 5- and 7-day-old males were less competitive than mature wild or laboratory mature males and hence had lower relative sterility indexes. Methoprene-treated males mated earlier in the evening and continued mating for longer than untreated sterile males and mature wild or laboratory males. In mating compatibility trials, methoprene-treated males mated equally with methoprene-treated females and mature females, whereas methoprene-treated females tended to mate more often with mature males than with methoprene-treated males. However, untreated flies of both sexes exhibited substantial sexual isolation. Pairings that comprised methoprene-treated males and mature females had shorter mating latency and longer copulations than other pairings. Unlike males, methoprene-treated females did not exhibit changes in mating latency or duration. Overall, the present study supports the use of pre-release dietary methoprene treatment in Queensland fruit fly SIT.

Sterile insect technique (SIT) is an environmentally benign pest management technique that involves releasing millions of sterile insects to suppress reproduction of pest populations. Many fruit flies, including Queensland fruit fly (Bactrocera tryoni Froggatt, ‘Q-fly’), have long adult maturation periods such that pre-maturation mortality can greatly reduce abundance of sexually active sterile males and impede SIT efficacy. Q-fly is the most difficult and costly challenge to market access for Australia’s horticulture industries, and has been targeted for intensive use of SIT program. We here demonstrate potential of pre-release caffeine supplements as a novel means to accelerate sexual maturation in male Q-fly. In mating trials, analytical caffeine was very effective at accelerating sexual maturation, while no positive effects of caffeine-containing instant coffee or guarana supplements were detected. In parallel, development of testes and ejaculatory apodemes was accelerated in males provided analytical caffeine but not instant coffee or guarana. High doses of guarana and instant coffee reduced longevity while even the highest doses of analytical caffeine did not affect longevity. Pre-release caffeine supplements promote sexual maturation in Q-flies, and similar benefits are expected in other fruit flies having long adult maturation periods.

Larval diets used for artificial rearing can have a significant effect on insect biology. The Queensland fruit fly (aka ‘Qfly’), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is one of the greatest challenges for fruit growers in Australia. The sterile insect technique (SIT) is being developed to manage outbreaks in regions that remain free of Qfly and to reduce populations in regions where this species is endemic. Factory scale rearing is essential for SIT; however, artificial larval diets are known to affect the microbiome of Qfly, which may then affect fly performance. In this study, high-throughput Illumina sequencing was used to assess the Qfly microbiome in colonies reared, for five generations from nature, on two common artificial diets (carrot and gel). At generation five (G5), the microbiome was assessed in larvae, pupae, adult males and adult females and standard fly quality control parameters were assessed together with additional performance measures of mating propensity and survival under nutritional stress. At the genus level, bacterial communities were significantly different between the colonies reared on the two larval diets. However, communities converged at Phyla to family taxonomic levels. Interestingly, pathogenic bacterial genera (e.g., Morganella, Citrobacter, Providencia, Burkholderia) were highly abundant in all developmental stages of Qfly reared on the gel diet, when compared to the carrot diet. Despite abundance of these commonly pathogenic genera, a greater percentage of egg hatching, heavier pupal weight and a higher percentage of fliers were found in the Qfly reared on the gel diet. Mating propensity and survival under nutritional stress was similar for adult Qfly that had been reared on the two larval diets. Overall, our findings demonstrate that the artificial larval diet strongly influences the microbiome and quality control measures of Qfly, with likely downstream effects on performance of flies released in SIT programs.

Juvenile hormone is an important regulator of sexual development in insects, and application of methoprene, a juvenile hormone analogue, together with access to a protein-rich diet, has been found to accelerate sexual maturation of several tephritid fruit fly species including Queensland fruit fly Bactrocera tryoni (‘Q-fly’). Such accelerated development is a potentially valuable means to increase participation of released males in sterile insect technique programs. However, there is a risk that benefits of accelerated maturation might be countered by increased vulnerability to starvation and desiccation. The present study investigates this possibility. After emergence, flies were treated with three levels of methoprene (0, 0.05%, and 0.5%) incorporated into a diet of sugar and yeast hydrolysate for two days after emergence. Survival of groups and individual flies was assessed under conditions of food stress, food and water stress, and ad libitum access to diet, and survival of individual flies was also assessed under desiccation stress. Most flies provided ad libitum access to diet were still alive at 7 days, whereas all stressed flies died within 4 days. Desiccation stressed flies had the shortest survival followed by food and water stress, and then food stress. Methoprene supplements increased susceptibility of flies to each stress. Flies subjected to food and water stress had the least lipid reserves at death, whereas flies subjected to desiccation stress retained the least water reserves. To investigate mechanisms that might underlie reduced survival under stress; we also quantified activity level of flies that were subjected to food and water stress and desiccation stress. Activity level was greater for flies provided methoprene, but did not vary with stress type or sex, suggesting that increased vulnerability of flies to stress is related to elevated metabolism associated with elevated activity. Deleterious effects of methoprene supplements on stress tolerance indicate a need for careful consideration of the conditions that will be encountered by flies in the field before deploying methoprene as a pre-release treatment in Q-fly sterile insect technique programs.

Methoprene supplements added to diets of yeast hydrolysate and sugar promote early expression of sexual behaviour and mating in male Queensland fruit fly (Bactrocera tryoni; 'Q-fly') and show promise as a pre-release treatment for sterile insect technique programs. Currently it is not known whether the early mating behaviour of methoprene-treated male Q-flies is only behavioural or is coupled with accelerated development of reproductive organs. Accordingly, the present study investigates whether incorporation of methoprene into diets of yeast hydrolysate and sugar (1:3) or sugar alone, accelerate development of testes, ejaculatory apodeme, and accessory glands in male Q-flies and ovaries in females. All organs increased in size as the flies aged and matured, and development rate of all organs was far greater when the flies were provided yeast hydrolysate in addition to sugar. Incorporation of methoprene into diets containing yeast hydrolysate was found to strongly accelerate development of testes and ejaculatory apodeme, but not accessory glands, in males. In the absence of yeast hydrolysate, methoprene treatment had only a modest effect on male organ development. In contrast to males, development of ovaries in female Q-flies did not respond to dietary methoprene supplements, regardless of whether they were fed yeast hydrolysate and sugar or sugar alone. These findings of diet-dependent effects of methoprene supplements on reproductive organs are a close match to previous studies investigating effects of methoprene supplements on mating behaviour. Overall, methoprene supplements substantially enhance the positive effects of protein rich adult diet on the early expression of sexual behaviour and accelerate development of reproductive organs in male, but not female, Q-flies. Methoprene supplements added to pre-release diets of yeast hydrolysate and sugar show promise as a means of accelerating reproductive development of Q-flies released in sterile insect technique programs, and may also bias operational sex ratio in favour of males.

Sterile insect technique (SIT) is an environmentally benign pest management technique that relies on released sterile male insects mating with, and curtailing reproduction of, wild females. However, for species with high mortality rates and long adult maturation phases, a large proportion of the released insects can die before maturing and so fail to contribute to SIT. To counter this problem, inclusion of yeast hydrolysate in pre-release diets and treatment of pupae or adults with methoprene, a juvenile hormone analogue, have been investigated as means of accelerating development of some fruit flies, including Queensland fruit fly, Bactrocera tryoni (Froggatt) (‘Q-fly’). Methoprene has most often been administered topically in acetone solution, which is toxic, flammable, and impractical for operational settings. As a practical alternative, we incorporated methoprene (0, 0.05, 0.1, and 0.5%) into Q-fly adult diet of sugar only or sugar mixed with yeast hydrolysate for 2 days, and then provided sugar only for the rest of the trial period. Mating performance of males and females was tested from 4 to 30 days of age. Flies provided sugar mixed with yeast hydrolysate had increased mating propensity in comparison with flies that were provided sugar only. At all ages and for both diets, all methoprene doses increased male mating probability. Methoprene treatment did not affect copula latency of males that received yeast hydrolysate, but males that received only sugar mated earlier if they had received 0.05% methoprene. Methoprene treatment of males was also associated with longer copulations, which may affect fertility of females that later remate. Females differed from males in that methoprene treatment did not significantly affect mating probability or latency, but resembled males in that methoprene treatment resulted in longer copulations. Sex differences in response to methoprene may lead to male-biased operational sex ratio when bisex Q-fly strains are used in SIT. Yeast hydrolysate increased longevity of both males and females, but methoprene treatment did not affect longevity. Overall, findings of the present study indicate that Q-fly sexual maturation can be accelerated, and SIT might hence be enhanced, by incorporation of methoprene and yeast hydrolysate in pre-release diet.

Queensland fruit flies Bactrocera tryoni (‘Q-fly’) have long adult prereproductive development periods, which can present challenges for sterile insect technique (SIT) programs. Holding the sterile flies in release facilities is expensive for control programs. Alternatively, releases of sexually immature males can lead to substantial mortality of sterile males before they mature. Recent studies have reported effectiveness of dietary supplementation with a mosquito larvicide (NOMOZ) that contains S-methoprene, a juvenile hormone analogue, for accelerating sexual development of fertile Q-fly males. However, it is not known whether effects on sterile flies are comparable to effects on fertile flies, or whether effects of methoprene-containing larvicide are comparable to effects of analytical standard methoprene such has been used in most studies. Here we address both knowledge gaps, investigating the effects of analytical standard methoprene and NOMOZ mixed with food and provided for 48 h following emergence on sexual development and longevity of fertile and sterile Q-flies. Compared with controls, fertile and sterile male Q-flies that were provided diets supplemented with methoprene from either source exhibited substantially accelerated sexual development by 2–3 d and longer mating duration. Unlike males, females did not respond to methoprene treatment. Although fertile and sterile flies were generally similar in sexual development and response to methoprene treatment, sterile flies of both sexes tended to have shorter copula duration than fertile flies. Neither methoprene supplements nor sterilization affected longevity of flies. The present study confirms effectiveness of dietary methoprene supplements in accelerating sexual development of both fertile and sterile male (but not female) Q-flies, and also confirms that low-cost mosquito larvicides that contain methoprene can achieve effects similar to those for high-cost analytical grade methoprene as prerelease supplements for Q-fly SIT.

Tephritid fruit flies are amongst the most damaging insect pests of horticulture globally. Some of the key fruit fly species are managed using the sterile insect technique (SIT), whereby millions of sterile males are released to suppress reproduction of pest populations. Male annihilation technique (MAT), whereby sex specific lures are used to attract and kill males, is often used to reduce wild male numbers before SIT programs commence, providing released sterile males an increased numerical advantage. Overall program efficacy might be improved if MAT could be deployed simultaneously with SIT, continuously depleting fertile males from pest populations and replacing them with sterile males. However, such 'male replacement' requires a means of suppressing attraction of released sterile males to lures used in MAT. Previous studies have found that exposure of some fruit flies to lure compounds as mature adults can suppress subsequent response to those lures, raising the possibility of pre-release treatments. However, this approach requires holding flies until after maturation for treatment and then release. The present study takes a novel approach of exposing immature adult male Queensland fruit flies (Bactrocera tryoni, or 'Qfly') to raspberry ketone (RK) mixed in food, forcing these flies to ingest RK at ages far younger than they would naturally. After feeding on RK-supplemented food for two days after emergence, male Qflies exhibited a reduction in attraction to cuelure traps that lasted more than 20 days. This approach to RK exposure is compatible with current practises, in which Qflies are released as immature adults, and also yields advantages of accelerated reproductive development and increased mating propensity at young ages.