A review of 16 years of quality control parameters at a mass‐rearing facility producing Queensland fruit fly,Bactrocera tryoni

From 1996 to 2012, the mass‐rearing facility at Camden (NSW, Australia) has been producing Queensland fruit flies, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). During this time, the facility has regularly recorded fly quality parameters, creating a unique data set that provides an invaluable opportunity to evaluate the interrelationships among standard quality control (QC) parameters and test for redundant QC variables. Here, we conducted an exploratory data analysis to reveal relationships among the QC parameters. We found that pupal weight, adult lifespan, and longevity under nutritional stress (i.e., survival duration without food or water) had distinct monthly trends, suggesting that these QC parameters are sensitive to seasonal conditions. Furthermore, emergence percentage, flight ability, and adult lifespan were adversely affected by the dyeing/handling/irradiation process associated with sterile insect releases. Using a multivariate approach and controlling for monthly and yearly patterns, we showed that pupal weight and egg hatch are consistently negatively related and that percentage male and emergence rates are consistently negatively related. These results suggest that these correlation pairs measure similar quality information and hence one QC variable from each pair could be dropped. Flight ability was not strongly correlated with any of the QC variables, suggesting that this QC variable remains a useful QC metric. Finally, the longevity under nutritional stress QC appears to be fairly insensitive to QCs and we suggest that it should be replaced by the standard mortality under stress test.     

Precocious Mexican fruit fly methoprene‐fed males inhibit female receptivity and perform sexually as mature males

The sterile insect technique (SIT) has been used successfully for the control of fruit flies. The efficiency of this technique can be significantly reduced when sterile released insects are exposed to adverse conditions and predators, as a great number of sterile insects die before reaching sexual maturity and thus fail to mate with wild females. Treatments with juvenile hormone (JH) analogues such as methoprene (M) significantly reduce the time to reach sexual maturity by sterile Anastrepha ludens (Loew) (Diptera: Tephritidae) males. In this study, we compared the sexual performance of non‐treated sexually mature males with young males that had been sexually accelerated with M. Furthermore, we compared the ability of M‐fed males in inhibiting female remating compared with sexually mature males. Results showed that at 5 days M‐fed males had lower mating success than mature males; however, 6‐day‐old (0.1%) M‐fed males had the same amount of matings as mature 13‐day‐old males. Young 5‐ to 10‐day‐old M‐fed males also had similar number of matings as mature non‐treated 12‐ to 17‐day‐old males. There were no differences in copula duration between treatments. Moreover, there were no differences between the fertility, fecundity or refractory period of females mated with either young male fed M or normal sexually mature males. These results indicated that young males that were sexually accelerated with M have the same sexual performance as non‐treated sexually mature males. Implications of using M as a pre‐release treatment for A. ludens controlled through SIT are discussed.     

Potential for pre-release diet supplements to increase the sexual performance and longevity of male Queensland fruit flies

1 Recent studies have shown that continuous access to a protein source (yeast hydrolysate) can greatly enhance the sexual performance of male Queensland fruit flies ( Bactrocera tryoni ; 'Q-flies'). However, in Sterile Insect Technique programmes used to eradicate or suppress wild populations, mass-reared Q-flies are typically fed only sucrose and water for up to 2 days before release.
2 We investigated whether adding a protein source to the diet of male Q-flies for a 24- or 48-h window after emergence and then removing it is sufficient to enhance mating probability, latency to mate, copula duration, probability of sperm storage, number of sperm stored, female remating tendency and longevity of male Q-flies.
3 Protein-fed males were more likely to mate than males fed only sucrose, especially when young. Protein-fed males also had shorter mating latencies and longer copulations than protein-deprived males.
4 Females mated by protein-fed males were more likely to store sperm, stored more sperm and were less likely to remate than were females mated by protein-deprived males. Females were also less likely to remate if their first mate had been large.
5 Overall, providing male Q-flies access to a protein source for a 24- or 48-h window early on in their adult life was sufficient to greatly enhance all assessed measures of performance. Although 24-h access was sufficient for a notable enhancement, further benefits were evident in males provided 48-h access.
6 The results are discussed in terms of the practical implications for Sterile Insect Technique programs used to eradicate or suppress wild Q-fly populations.     

Dispersal of mass‐reared sterile,laboratory‐domesticated and wild male Queensland fruit flies

Queensland fruit flies, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) (‘Q‐flies’) were released as sexually immature adults from a point within an orchard. Marked male Q‐flies were recaptured in the trap furthest from the release point (1087 m) by 2 weeks after release, although 98.25 ± 1.04% of recaptured males were trapped <500 m from the release point. Comparison of gamma‐irradiated (sterile), laboratory‐adapted and wild male Q‐flies indicated that dispersal distance was not significantly affected by fly type. There was no significant correlation between temperature and mean dispersal distance, but total recaptures were significantly negatively correlated with increasing daily maximum, minimum and average temperature.     

Yeast hydrolysate supplement increases starvation vulnerability of Queensland fruit fly

Post‐teneral diets containing yeast hydrolysate are reported to increase longevity, reproductive development and sexual performance of Queensland fruit fly (‘Q‐fly’) Bactrocera tryoni Froggatt (Diptera: Tephritidae). Consequently, diets including yeast hydrolysate are recommended for sterile Q‐flies before release in sterile insect technique (SIT) programmes. However, in some tephritids, diets including yeast hydrolysate are associated with an increased vulnerability to starvation. In the present study, the effects of yeast hydrolysate supplementation before release are considered with respect to the longevity of released Q‐fly when food becomes scarce. Experiments are carried out in three settings of varying resemblance to field conditions: 5‐L laboratory cages, 107‐L outdoor cages and 14 140‐L field cages containing potted citrus trees. In all experimental settings, compared with flies that received only sucrose, male and female Q‐flies that are provided with yeast hydrolysate during the first 2 days of adult life have a significantly shorter survival when subsequently deprived of food. Yeast supplementation appears to commit Q‐flies to a developmental trajectory that renders them more vulnerable to starvation. The practical significance of these findings for SIT depends on how often the releases are carried out under conditions in which Q‐flies experience extreme food shortages in the field.     

Methoprene-induced matings of young Queensland fruit fly males are effective at inducing sexual inhibition in females

Pre-release dietary treatment with methoprene, a juvenile hormone analogue, decreases the age at which male Queensland fruit flies mature and hence may decrease the post-release delay until released sterile flies participate in sterile insect technique (SIT) programmes. However, if matings of young methoprene-treated males are not effective at inducing sexual inhibition in their mates, then this treatment may not enhance SIT. The present study investigates efficacy of matings of methoprene-treated males at inducing sexual inhibition in their mates. Methoprene incorporated into a diet of sugar and yeast hydrolysate (w/w 3:1) for 48 hr after emergence resulted in significantly increased male mating propensity when flies were <10 days of age, but not when older, and longer copulations. Copula latency did not vary with methoprene treatment but did decrease with age. The matings of young methoprene-treated males were effective at inducing sexual inhibition in their mates, matching the efficacy of untreated mature males. Regardless of treatment, females had reduced tendency to remate if their first mate was 15 days of age than if their first mate was younger (6, 8 days) or older (20, 25, 30 days). Females mated by methoprene-treated males that did remate tended to remate later in the day than females mated by untreated males. Also, second copula durations of females first mated by a 6- to 10-day-old male were shorter if the male was methoprene treated. These patterns in remating females may indicate greater efficacy of the initial mating of methoprene-treated males. Overall, we find that the additional matings of young methoprene-treated male Queensland fruit flies are effective at inducing sexual inhibition in their mates. This finding supports the incorporation of methoprene into pre-release diet for SIT.     

Sexual development of wild and mass‐reared male Queensland fruit flies in response to natural food sources

Diet has a profound influence on the fitness of adult tephritid flies. Mass‐reared flies are provided yeast hydrolysate as a rich source of nutrition that supports rapid sexual development and mating success. In contrast, wild tephritid flies often live in environments where food may be hard to find, and these are the conditions that sexually immature mass‐reared sterile males encounter when released into the field during sterile insect technique campaigns. The effect of natural food sources (bat guano, bird droppings, citrus pollen, and wheat pollen) on the sexual development of adult mass‐reared fertile, mass‐reared sterile, and wild male Queensland fruit flies, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), was determined by measuring ejaculatory apodeme size. Inclusion of yeast hydrolysate in the adult diet was associated with faster growth of the ejaculatory apodeme in comparison with all other diets. Effects of diet were far less pronounced in mass‐reared males, which may indicate reduced nutritional requirements, whereas the ejaculatory apodeme of wild males fed on natural sources of food or sucrose alone did not increase in size over the first 20 days of adult life.     

Effects of radiation on the fertility of the Ethiopian fruit fly,Dacus ciliatus

The Ethiopian fruit fly, Dacus ciliatus (Loew) (Diptera: Tephritidae), is a significant pest of cucurbit crops in Asia and Africa and is currently controlled with insecticides. The sterilizing effect of gamma radiation on D. ciliatus adults was investigated to assess the suitability of sterile insect technique (SIT) for use as an alternative, non‐chemical strategy for the control of this pest. Late pupae (48 h before emergence) were irradiated with 60, 80, 100, 120, and 140 Gy of gamma rays emitted by a ⁶⁰Co source. Following emergence, the biological characteristics of the experimental cohorts (including all possible male‐female combinations of irradiated and untreated flies) were recorded. No significant negative effects of irradiation on pupal eclosion or the ability of newly emerged flies to fly were observed. Samples of eggs at reproductive fly‐ages (12‐, 15‐, and 17‐day‐old pairs) were collected and their hatch rates were assessed. At 60 Gy, females were completely sterilized, whereas complete sterilization of the males was observed only at 140 Gy (a small amount of fertility persisted even at 120 Gy). In addition to the above experiments, three fruit infestation trials were conducted with zucchini [Cucurbita pepo L. (Cucurbitaceae)] as the plant host and the pupae produced in those trials were collected and recorded. We observed significant (ca. 10%) infestation following treatment with up to 120 Gy and zero progeny only at 140 Gy, mirroring the egg‐hatch results. Our findings support the feasibility of SIT for the control of D. ciliatus.     

Remating inhibition in female Queensland fruit flies: effects and correlates of sperm storage

Reproductive success of male insects commonly hinges both on their ability to secure copulations with many mates and also on their ability to inseminate and inhibit subsequent sexual receptivity of their mates to rival males. We here present the first investigation of sperm storage in Queensland fruit flies (Tephritidae: Bactrocera tryoni; a.k.a. 'Q-flies') and address the question of whether remating inhibition in females is directly influenced by or correlated with number of sperm stored from their first mates. We used irradiation to disrupt spermatogenesis and thereby experimentally reduce the number of sperm stored by some male's mates while leaving other aspects of male sexual performance (mating probability, latency until copulating, copula duration) unaffected. Females that mated with irradiated rather than normal males were less likely to store any sperm at all (50% vs. 89%) and, if some sperm were stored, the number was greatly reduced (median 11 vs. 120). Despite the considerable differences in sperm storage, females mated by normal males and irradiated males were similarly likely to remate at the next opportunity, indicating (1) number of sperm stored does not directly drive female remating inhibition and (2) factors actually responsible for remating inhibition are similarly expressed in normal and irradiated males. While overall levels of remating were similar for mates of normal and irradiated males, factors responsible for female remating inhibition were positively associated with presence and number of sperm stored by mates of normal but not irradiated males. We suggest seminal fluids as the most likely factor responsible for remating inhibition in female Q-flies, as these are likely to be transported in proportion to number of sperm in normal males, be uninfluenced by irradiation, and be transported without systematic relation to sperm number in irradiated males.     

Seminal fluids mediate sexual inhibition and short copula duration in mated female Queensland fruit flies

Molecules in male seminal fluid transferred to female insects during mating can have potent effects on their subsequent sexual and reproductive behaviour. Like many other tephritids, female Queensland fruit flies (Bactrocera tryoni) typically have diminished sexual receptivity after their first mating. Also, copulations of females that do remate tend to be shorter than those of virgins. We here find that virgin females injected with small doses (0.1, 0.2 or 0.5 male equivalents) of extracts from the male reproductive tract accessory tissues, which consist of male accessory glands, ejaculatory apodeme and ejaculatory duct (AG/EA/ED), have diminished receptivity and short copula duration very similar to naturally mated females. In contrast, virgin females injected with saline or with high doses of AG/EA/ED (1 or 2 male equivalents) that likely exceed the range of natural variation retain the higher levels of sexual receptivity and longer copulations of un-injected virgins. We conclude that reduced sexual receptivity and shorter copulations of mated female Q-flies are mediated by products in the male seminal fluid derived from the male reproductive tract accessory tissues.     

Effects of irradiation dose rate on quality and sterility of Queensland fruit flies, Bactrocera tryoni (Froggatt)

Queensland fruit fly (Bactrocera tryoni; Q‐fly) pupae are routinely irradiated to induce reproductive sterility in adults released in a sterile insect technique programme. Although there have been some studies of how total dose influences fly quality, dose rate has not been considered. In the present study, pupae were irradiated at a target dose range of 70–75 Gy at dose rates of approximately 5, 7, 26, 57 and 80 Gy/min and were then subjected to routine IAEA/FAO/USDA quality control tests including emergence, flight ability, mortality under stress and sterility induction. No significant effects of dose rate were found on emergence or flight ability. Sterility induction was also found to be independent of dose rate, a result conforming to a ‘one‐hit’ ionizing event hypothesis. Flies irradiated at higher dose rates suffered increased mortality under stress. This appears to stem from an increased tendency to over‐shoot the target dose when irradiating at high dose rates. We recommend that, to reduce potential error in total target dose, the lowest practical dose rate be used when irradiating Q‐fly pupae for use in the sterile insect technique.     

Transfer and fate of male ejaculate in female Queensland fruit flies

Abstract Insect seminal fluid commonly comprises a complex cocktail of proteins and other biochemical components that migrate away from the female reproductive tract to sites elsewhere in the female body and elicit changes in female reproductive behaviour. The transfer of male seminal fluid molecules to reproductive and somatic tissues of the female Queensland fruit fly (‘Q‐fly’) Bactrocera tryoni is examined and some putative target sites identified. Male Q‐flies are fed a diet containing radiolabelled (³⁵S) amino acids, which are incorporated into male accessory gland products. Radioactivity diminishes within the accessory glands and increases in all assessed parts of the female body during copulation, indicating the transfer of these products into the female soma via the reproductive tract. There are significant changes in the absolute and proportional radioactivity profiles among female tissues over the next 22 h, with substantial reductions in the thorax and increases in the head. This is consistent with accumulation of behaviour‐modifying male products at binding sites in the female head. Parallels can be drawn between the data in the present study and seminal fluid distribution profiles and receptor binding documented in other insects.     

Control of copula duration and sperm storage by female Queensland fruit flies

Copula duration and sperm storage patterns can directly or indirectly affect fitness of male and female insects. Although both sexes have an interest in the outcome, research has tended to focus on males. To investigate female influences, we compared copula duration and sperm storage of Queensland fruit fly females that were intact, or had been incapacitated through decapitation or abdomen isolation. We found that copulations were far longer when females had been incapacitated, indicating that constraints imposed on copula duration by intact females had been relaxed. Repeatability of copula duration for males was very low regardless of female treatment, and this is also consistent with strong female influence. Number of sperm in the spermathecae was not influenced by female treatment, suggesting that female abdominal ganglia control the transport of sperm to these long-term storage organs. However, more sperm were found in the ventral receptacles of incapacitated females compared to intact females. Overall, results implicate cephalic ganglia in regulation of copula duration and short-term sperm storage in the ventral receptacle and abdominal ganglia in regulation of long-term sperm storage in the spermathecae.     

Effects of field cage colour and supplementary shade on environmental conditions and mating behaviour of Queensland fruit flies,Bactrocera tryoni

The mating performance field cage test is a required periodic quality‐control assessment for factory‐reared fruit flies used for the sterile insect technique. The FAO/IAEA/USDA guidelines for assessing fly quality state that if during tests a large proportion of flies call and mate on cage walls, away from host trees, then environmental conditions within the cage need to be adjusted and tests repeated. Here we test effects of cage design, specifically mesh colour (green, white) and addition of supplementary shade, on the mating behaviour of Queensland fruit fly (Q‐fly), Bactrocera tryoni Froggatt (Diptera: Tephritidae). Observations were made over a 4‐h period at dusk when these flies mate. Changes in environmental conditions in each cage over the dusk period varied with cage design. We recorded the highest proportion of matings taking place on trees as opposed to cage walls (>90%) in the unshaded white cage, the shaded white and un‐shaded green cages being intermediate (ca. 70%), and the shaded green cage had the least (ca. 40%). The effects of field cage colour and supplementary shade on mating behaviour are discussed. We recommend that Q‐fly field cage tests should be conducted in cages with a light coloured mesh, and that supplementary shading should only be applied if there is a need to adjust temperature and light within the cage.     

Activity patterns of Queensland fruit flies (Bactrocera tryoni) are affected by both mass‐rearing and sterilization

Mass‐reared sterile tephritid flies released in sterile insect technique (SIT) programmes exhibit behaviour, physiology and longevity that often differ from their wild counterparts. In the present study, video recordings of flies in laboratory cages are used to determine whether the sequential processes of mass‐rearing and sterilization (using gamma radiation) that are integral to SIT affect general activity patterns of male and female Queensland fruit flies Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) (‘Q‐flies'). Compared with wild flies, mass‐reared flies exhibit a marked reduction in overall activity, and further reduction is found after sterilization. In terms of the frequency of activities, both fertile and sterile mass‐reared Q‐flies fly less often and exhibit more bouts of inactivity and grooming than wild Q‐flies. In addition, in terms of the duration of activities, fertile and sterile mass‐reared Q‐flies spend less time flying and more time walking, grooming and being inactive than wild Q‐flies. Although fertile and sterile mass‐reared flies are similar in other regards, sterile mass‐reared flies spend more time being inactive than fertile mass‐reared flies. These findings raise new questions about how changes in behaviour and activity levels may influence the performance of mass‐reared sterile Q‐flies in the field, as well as the physiological and metabolic processes that are involved. The frequency and duration of inactivity could provide a simple but powerful and biologically relevant test for quality in mass‐rearing and SIT programs.     

Effects of post-teneral diet on foraging success of sterile male Mediterranean fruit flies

Post‐teneral diets containing protein have been shown to enhance the copulatory success of sterile male Mediterranean fruit flies, Ceratitis capitata (Wied.) (Diptera: Tephritidae). However, ingesting protein was also found to negatively affect male survival, in particular when males faced starvation following release in the field. Accordingly, the objective of the present study was to determine the effects of various post‐teneral diets, presented to sterile males prior to release, on their subsequent ability to forage for carbohydrates and protein in the field. Using mark‐release recapture and analytic biochemical methods, we found that both protein‐fed and protein‐deprived males foraged successfully for protein and sugar in a field enclosure when these resources were available. We conclude that protein‐fed sterile males are able to exploit sources of nutrition in the release environment, and their inability to overcome starvation is not a concern for control operations using the sterile insect technique.     

Temperature effects on “overwintering” phenology of a polyphagous,tropical fruit fly (Tephritidae) at the subtropical/temperate interface

Around the world, several pest tephritids are extending their ranges from warm tropical or Mediterranean climates into cooler temperate regions. The ability to tolerate climatic diversity is uncommon among insects, and understanding the population phenology drivers of such species across different parts of their range will be critical for their management. Here, we determined the role of temperature versus fruit availability on the population phenology of Queensland fruit fly, Bactrocera tryoni. Using a field site located at the subtropical/temperate interface, with host fruits continuously available, we monitored the development times and abundance of B. tryoni, a species which has invaded temperate Australia from the tropics. From fruit samples held at ambient and controlled conditions, the abundance of emerging flies was highly variable among collection dates, but the variance did not reflect the observed changes in temperature. For most samples, the survival rate of flies in a field site was lower than predicted by a day‐degree population model fitted with mean daily field temperatures. The development time of the immature stage in the field was prolonged, presumably due to cooler ambient conditions, but the fitted day‐degree population model consistently over‐predicted estimated development times. Our results indicate that at the subtropical/temperate interface, the decline in B. tryoni populations during winter is only partly driven by temperature and host availability. We classify B. tryoni as a climate generalist, which likely employs physiological as well as behavioural mechanisms to achieve broad climatic tolerance ranges.     

Artificial selection,pre‐release diet,and gut symbiont inoculation effects on sterile male longevity for area‐wide fruit‐fly management

Longevity is an important life‐history trait for successful and cost‐effective application of the sterile insect technique. Furthermore, it has been shown that females of some species – e.g., Anastrepha ludens (Loew) (Diptera: Tephritidae) – preferentially copulate with ‘old’, sexually experienced males, rather than younger and inexperienced males. Long‐lived sterile males may therefore have greater opportunity to find and mate with wild females than short‐lived males, and be more effective in inducing sterility into wild populations. We explored the feasibility of increasing sterile male lifespan through selection of long‐lived strains and provision of pre‐release diets with added protein, and inoculated with bacterial symbionts recovered from cultures of the gut of wild Anastrepha obliqua (Macquart). Artificial selection for long‐lived A. ludens resulted in a sharp drop of fecundity levels for F1 females. Nevertheless, the cross of long‐lived males with laboratory females produced a female F1 progeny with fecundity levels comparable to those of females in the established colony. However, the male progeny of long‐lived males*laboratory females did not survive in higher proportions than laboratory males. Provision of sugar to A. obliqua adults resulted in increased survival in comparison to adults provided only with water, whereas the addition of protein to sugar‐only diets had no additional effect on longevity. Non‐irradiated males lived longer than irradiated males, and supplying a generic probiotic diet produced no noticeable effect in restoring irradiated male longevity of A. obliqua. We discuss the need to evaluate the time to reach sexual maturity and survival under stress for long‐lived strains, and the inclusion of low amounts of protein and specific beneficial bacteria in pre‐release diets to increase sterile male performance and longevity in the field.