DOI: 10.1111/j.1570-7458.2008.00768.x Blackwell Publishing Ltd

Feeding on yeast hydrolysate enhances attraction to cue-lure in Queensland fruit flies, Bactrocera tryoni Christopher W. Weldon*, Diana Perez-Staples† & Phillip W. Taylor Centre for the Integrative Study of Animal Behaviour, Macquarie University, North Ryde, Sydney, NSW, 2109, Australia Accepted: 15 July 2008

Key words: dietary nitrogen, male attractant, phenylpropanoid, sterile insect technique, SIT, Q-fly, γ-irradiation, Tephritidae, Diptera

Abstract

Feeding on yeast hydrolysate (a source of nitrogen) has a strong influence on the physiology and behaviour of the Queensland fruit fly (Q-fly), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), affecting longevity, sexual maturation, oogenesis, and mating performance. In this study, we demonstrate that access to yeast hydrolysate also influences the development of attraction to cue-lure in Q-flies. We provided virgin Q-flies various periods of access to yeast hydrolysate (continuous, 48 h, 24 h, or deprived). Attraction of males to cue-lure was increased and occurred at an earlier age when they were fed yeast hydrolysate. Males given continuous access were strongly attracted to cue-lure at a younger age (8 days after emergence), but by 12 days after emergence attraction of males given access to yeast hydrolysate for 48 h did not differ from males given continuous access. Attraction by males deprived or given just 24 h access to yeast hydrolysate was always significantly lower than those of males with continuous access. Male attraction to cue-lure was highest in the early morning. While cue-lure is most often thought of as a male attractant, virgin female Q-flies were caught in cue-lure traps at dusk at ages when they are known to be sexually mature. We suggest that cue-lure or similar natural chemicals play a role in the Q-fly mating system. γ-Irradiation used to induce sterility had no significant effect on attraction to cue-lure by Q-flies.

Introduction Dietary sources of nitrogen have a strong influence on the physiology and behaviour of tephritid flies (Fletcher, 1987; Kaspi et al., 2000; Yuval et al., 2007). Access to yeast hydrolysate, a source of amino acids (Barry et al., 2003), has been found to increase longevity in fertile females of the Queensland fruit fly (Q-fly), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), as well as in irradiated flies of both sexes (Perez-Staples et al., 2007). Feeding on yeast hydrolysate by Q-flies also promotes ovarian development and oogenesis (Meats & Khoo, 1976; Meats et al., 2004), earlier mating ages, and increased mating propensity (Perez-Staples et al., 2007; Prabhu et al., 2008). Male Q-flies provided continuous access to yeast hydrolysate reach sexual maturity at a younger age, are more likely to *Correspondence: E-mail: [email protected] Present address: Laboratorio de Biotecnología y Ecología Aplicada (LABIOTECA), Universidad Veracruzana, Apartado Postal 250, CP 91090 Xalapa, Veracruz, Mexico.

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copulate, and have longer copulations than males fed sugar only (Perez-Staples et al., 2007). Furthermore, these benefits are evident even when males are allowed to feed on yeast hydrolysate for only 1 or 2 days after emerging (Perez-Staples et al., 2008). Given that feeding on yeast hydrolysate promotes maturation and sexual activity, it follows that it is also likely to promote responses to stimuli that are characteristic of sexually active, mature flies. Sexually mature male flies of the dacine genus Bactrocera are usually attracted to one of a number of chemicals belonging to the group of organic compounds referred to as phenylpropanoids (Fletcher, 1987). These ‘lures’, including cue-lure [4-(p-acetoxyphenyl)-2-butanone] and methyl eugenol (4-allyl-1,2-dimethoxy-benzene), are now used widely to monitor pest dacine populations. Q-flies and melon flies, Bactrocera cucurbitae Coquillett, are attracted to cue-lure (Drew, 1987; Meats & Hartland, 1999), whereas Oriental fruit flies, Bactrocera dorsalis Hendel, papaya fruit flies, Bactrocera papayae Drew & Hancock, and Philippines fruit flies, Bactrocera philippinensis (Drew & Hancock) are attracted to methyl eugenol (Shelly & Dewire, 1994; Shelly

© 2008 The Authors Entomologia Experimentalis et Applicata 129: 200–209, 2008 Journal compilation © 2008 The Netherlands Entomological Society

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et al., 1996; Hee & Tan, 1998). However, the biological function underpinning attraction to these lures is unresolved (Raghu, 2004). There is some evidence that feeding on these chemicals improves male sexual performance by increasing attractiveness of male pheromone, calling rate, and/or mating propensity in Bactrocera carambolae Drew & Hancock (Wee et al., 2007), B. cucurbitae (Shelly & Villalobos, 1995), B. dorsalis (Shelly & Dewire, 1994), B. papayae (Hee & Tan, 1998), and B. philippinensis (Shelly et al., 1996). This suggests that male attraction to phenylpropanoid compounds is related to sexual activity, and that attraction to lures can be explained by their similarity to compounds that play a role in the mating systems of these flies. Attraction to cue-lure by sexually mature male Q-flies is central to effective management of this important quarantine pest. Q-fly outbreaks in fruit production areas are combated using an area-wide integrated pest management programme that includes release of sterilised Q-flies (Meats et al., 2003). Recapture rates in cue-lure traps are often used as an indirect measure of sterile fly field performance (Meats, 1998a), but cue-lure-baited traps used to monitor wild and sterile Q-flies are notorious for their low and variable recapture rates (Meats et al., 2006). Given the importance of dietary intake of nitrogen for sexual maturation and the importance of sexual maturity for cue-lure attraction, inconsistencies in recapture rates may reflect nitrogen-deprivation rather than poor survival in the field. Current protocols for Q-fly sterile insect technique (SIT) specify release of adults that have been fed only sugar and water while housed in release boxes for 24 –48 h after emergence from the pupal phase (Meats et al., 2006). If released sterile flies are not able to locate adequate sources of dietary nitrogen in the field, they may not become sexually mature and may hence fail to be attracted to cue-lure-baited traps. Given the link between male sexual maturity and cue-lure attraction, and the reliance on cue-lure trap recaptures to assess field performance, it is important to understand how pre-release diet treatments affect trap recaptures before conducting field trials to assess field performance of Q-flies fed yeast hydrolysate. In the present study, we examined whether access to yeast hydrolysate influences attraction to cue-lure in male and female Q-flies. We provided yeast hydrolysate ad libitum for a number of periods after emergence that conform to those possible within current Q-fly SIT practice (48 h, 24 h, and deprivation). As a comparison, attraction to cue-lure by Q-flies given continuous access to yeast hydrolysate was also assessed. Based on the results of laboratory studies on sexual maturation of Q-flies given these same diet treatments (Perez-Staples et al., 2008), we

predicted that attraction to cue-lure by male Q-flies given continuous access to yeast hydrolysate would be much greater than males deprived of access to yeast hydrolysate, with attraction by males given 24-h and 48-h access being intermediate between these two extremes. To ensure that our findings are of the greatest possible applied value, we used both fertile and sterile Q-flies to establish whether γirradiation used to sterilize the flies affects the relationship between diet and attraction to cue-lure.

Materials and methods Source and treatment of flies

We obtained fertile and sterile Q-flies as pupae from a mass-rearing facility at Camden, New South Wales (Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries). Adults emerging from irradiated pupae produced by this facility are routinely released in SIT programmes to control Q-fly outbreaks throughout Australia. Batches of approximately 4 000 pupae were collected over four consecutive days at the facility and, consequently, cohorts of teneral adults emerged over a period of 4 days. We took two cohorts of flies emerging 2 days apart, which enabled (1) two replicates to be conducted under congruent conditions at a similar time, and (2) sufficient time to remove flies remaining in field cages from previous test days. Two batches of pupae were required to complete the experiment (two replicates per batch from the mass-rearing facility). Sterile flies were produced by exposure of hypoxic pupae to γ-radiation (70–75 Gy) from a cobalt-60 source before being sent by courier to the Centre for the Integrative Study of Animal Behaviour, Macquarie University, NSW, Australia (for details, see Collins et al., 2008a). We used the self-marking method of Steiner (1965) to dye flies with two fluorescent pigments (Astral Pink and Lunar Yellow, Fiesta®; FEX series, Swada, UK), which allowed fertile and sterile Q-flies to be discriminated. This procedure is used to dye sterile Q-flies released in SIT programmes in Australia. Following standard practice, we dusted pupae with fluorescent pigment (1 g pigment/10 000 pupae), which was taken into the ptilinal suture of adults emerging from their puparia. Pigment colours were rotated between replicates. Adult mortality is not affected by treatment with fluorescent pigments of different colour (Weldon, 2005). Fertile and sterile Q-flies were allowed to emerge into separate 5-l cages. On emergence, we divided flies into four groups of approximately equal size that were given access to yeast hydrolysate enzymatic (MP Biomedicals, Aurora, OH, USA) for one of four durations: (1) continuous, (2) 48 h, (3) 24 h, and (4) no access (deprived). Continuous access to a source of sugar (granulated sucrose) and water

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was also provided during the period of access to yeast hydrolysate and thereafter. At 2–3 days after emergence (DAE), we transferred 160–300 fertile and sterile male and female flies from each diet treatment to separate clean 5-l cages. This ensured that all flies tested were virgin, because laboratory-adapted, mass-reared flies given continuous access to yeast hydrolysate do not begin mating until 5 DAE (Meats et al., 2004). Adult flies were kept near a window in an out-building at ambient temperature to provide a natural light:dark cycle. Field cages

We undertook field cage bioassays in four field cages (3.6 × 3.6 × 2.2 m) with walls and roof of green 50% shade cloth, enclosing six large potted citrus plants. Field cages were located on the grounds of the Centre for the Integrative Study of Animal Behaviour, Macquarie University. It has recently been shown that field cages of white shade cloth provide optimal mating conditions for Q-flies (Collins et al., 2008b). However, green shade cloth cages were suitable for the current study, because we were not assessing mating behaviour. We placed white polypropylene cloth (Brattice J®; Rheem Australia, Sydney, Australia) on the ground in each field cage so that dead flies could be easily sighted. Field cages were furnished with water and feeding stations were hung from plant branches. Only sugar was provided at feeding stations for 48-h, 24-h, and deprived treatments; yeast hydrolysate and sugar were provided for the continuous protein treatment (despite providing flies with dishes of food, flies were never observed to feed from them). In each field cage, we suspended two Lynfield traps (Cowley et al., 1990) from plant branches 1–1.5 m above the ground. Lynfield traps in each field cage were furnished with a wick (four cotton dental rolls) baited with either (1) cue-lure (International Pheromone Systems, Wirral, UK) and malathion (Hy-MAL; Crop Care Australasia, QLD, Australia) (4.45 and 0.55 ml, respectively) (cue-lure trap), or (2) malathion only (0.55 ml) (null trap). Malathion [diethyl (dimethoxythiophosphorylthio) succinate] is an organophosphorus, cholinesterase-inhibiting insecticide that is used routinely in Q-fly monitoring traps and is not known to be attractive to insects (Vayssières et al., 2007). We recorded temperature, relative humidity, and light intensity in the shade in each field cage using data loggers (HOBO® H8; Onset Computer Corporation, Bourne, MA, USA) housed within small Stevenson screens suspended from plant branches. Observations

We tested development of attractiveness to cue-lure in Q-flies by releasing flies of each diet treatment, fertility,

and sex combination into field cages at 4, 8, 12, 16, and 20 DAE. On the afternoon of the day prior to a test day, we took flies from the 5-l cages in which they were housed, and transferred them to 1-l holding cages. There were usually 40 flies subsampled from each combination of diet treatment, fertility, and sex combination, although numbers varied somewhat due to occasional poor adult emergence and/or survival. Variation in availability of flies meant that some treatments could not be released for all ages in some replicates. All holding cages were furnished with a small plastic dish of sugar and a cotton dental roll soaked in water. We also provided a small dish of yeast hydrolysate for holding cages housing flies in the treatment with continuous access. On test days, we released flies from holding cages into field cages at least 15 min before astronomical sunrise (sunrisenset, version 2.2; National Mapping Division, Geoscience Australia, Belconnen, ACT, Australia). Each of the four field cages housed fertile and sterile male and female flies of one diet treatment. On the first full hour after sunrise, we removed captured flies from Lynfield traps and inspected the ground under traps for additional dead flies. We recorded sterility and sex of dead flies caught in and around traps. This was repeated each hour throughout the day until 1 h after astronomical sunset. Within a replicate, the same field cage was used for each test age of the same diet treatment. On the following day, we removed flies remaining in field cages. We completed four replicates: (1) 28 February–16 March 2007, (2) 2–18 March 2007, (3) 30 March–15 April 2007, and (4) 1–17 April 2007. Diet treatments were rotated among four field cages for each replicate. Data analysis

To improve analytical power, we collapsed hourly captures into four broad time periods: (1) early morning (sunrise– 9:00 hours), (2) late morning (10:00–12:00 hours), (3) early afternoon (13:00–15:00 hours), and (4) late afternoon (16:00 hours–sunset). Recapture data were not normally distributed (many zero counts), and could not be normalized by standard monotonic transformation. We estimated linear regression models for the total number of flies caught in or near traps, using bootstrapping (500 bootstrap samples) to estimate standard errors. The linear regression model of total number of flies caught in or near traps, including predictors of age, diet treatment, sex, sterility, time of day, and trap type, as well as covariates of field cage temperature, relative humidity, and light intensity in the shade, was very highly significant (χ2 = 582, d.f. = 167, P<0.001), and explained much of the variation in our data (R2 = 0.431). An a priori decision was made to test only the interactions between age, diet

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Table 1 Predictors of Queensland fruit fly attraction to cue-lure Effect

d.f.

χ2

P

Age Time of day Diet treatment Sex Age*time Age*treatment Age*sex Time*treatment Time*sex Treatment*sex Age*time*treatment Age*time*sex Age*treatment*sex Time*treatment*sex Age*time*treatment*sex Sterility Trap type Cage Temperature Relative humidity Light

4 3 3 1 12 12 4 9 3 3 36 12 12 9 36 1 1 3 1 1 1

141.71 88.41 39.78 214.11 133.14 31.69 151.26 48.39 181.57 33.24 63.65 132.24 29.69 54.72 60.58 0.01 186.87 11.00 0.65 3.78 4.32

<0.001 <0.001 <0.001 <0.001 <0.001 0.002 <0.001 <0.001 <0.001 <0.001 0.003 <0.001 0.003 <0.001 0.006 0.933 <0.001 0.012 0.419 0.052 0.038

treatment, sex, and time of day, as these were the effects of primary interest in this study. Post-hoc comparisons among variables were performed using contrasts to test simple effects. Contrasts are the only form of post-hoc comparison available to test interaction effects for this kind of model. All analyses were conducted using Stata Statistical Software: Release 10, 2007 (StataCorp, College Station, TX, USA).

Results There was a highly significant difference between cue-lure and null traps in number of flies caught per observation (Table 1), with flies being caught in cue-lure traps almost exclusively (Figure 1). Dead flies found on the ground under cue-lure traps accounted for 13.8 ± 3.3% of all flies captured across all replicates and treatments. No dead flies were found on the ground under null traps. Recaptures of sterile flies did not differ from those of fertile flies (Table 1, Figure 1). Trap captures were affected by the very highly significant interaction of fly age, diet treatment, sex, and time of day (Table 1), which limits interpretation of main effects, particularly in relation to time of day. The pattern of captures for males and females differed significantly with respect to time of day. Peak male response was in the early

morning and declined throughout the day (Figure 2). Contrasts indicated that females were caught significantly less than males in the early morning, while captures of males and females were not significantly different in the late afternoon. However, when accounting for diet treatment, at 16 and 20 DAE females given continuous access to yeast hydrolysate were caught more in the late afternoon than 48-h, 24-h, and deprived females (Figure 3). Trap captures were related with relative humidity and light intensity in the shade, but were not influenced by temperature (Table 1). These associations are likely due to correlation of high trap captures to lower temperatures and light levels recorded in the morning (Figure 4). Contrasts indicated that male captures in the early morning increased with fly age, but were also modulated by diet treatment. In the early morning by 8 DAE, males given continuous access to yeast hydrolysate were caught significantly more than 48-h and 24-h males, and deprived males were caught significantly less than 48-h and 24-h males (Figure 2). By 12 DAE, captures of 48-h males were not significantly different from that of continuous males in the early morning, but both were caught significantly more than 24-h and deprived males. Captures of 24-h and deprived males remained significantly less than 48-h and continuous males at 16 and 20 DAE.

Discussion Access to yeast hydrolysate increases Q-fly attraction to cue-lure. Males allowed to feed on yeast hydrolysate are attracted to the lure in greater numbers and at an earlier age compared to those deprived of yeast hydrolysate. The marked increase in attraction to cue-lure at 8 DAE in males given continuous access to yeast hydrolysate (Figure 1) corresponds with male sexual maturation on this diet. Laboratory studies show that mass-reared males begin mating at 5 DAE, with mean mating age of approximately 7 DAE (Meats et al., 2004). Delayed and low attraction to cue-lure by males given access to only sugar (Figure 1) corresponds with findings from recent studies that show low mating propensity (Perez-Staples et al., 2007; Prabhu et al., 2008), longer mating latencies, lower sperm transfer, and lower likelihood to inhibit female remating (PerezStaples et al., 2008) in males fed on sugar alone. When tested at 4 DAE, attraction to cue-lure by Q-flies fed yeast hydrolysate did not differ from deprived flies. This emphasizes that improved attraction to cue-lure by flies fed yeast hydrolysate is linked to improved rates of sexual maturation in fed flies, rather than a direct effect of yeast hydrolysate on attraction. Importantly for application of these results to the SIT, attraction to cue-lure by fertile and sterile males was

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Figure 1 Effect of diet treatment, age, and sterility on male and female Queensland fruit fly captures (mean + SE) in Lynfield traps with or without cue-lure. Males: (A) deprived of access to yeast hydrolysate, (B) 24-h access, (C) 48-h access, and (D) continuous access. Females: (E) deprived of access to yeast hydrolysate, (F) 24-h access, (G) 48-h access, and (H) continuous access.

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Figure 2 Diurnal attraction to cue-lure by male Queensland fruit flies of different ages [4, 8, 12, 16, and 20 days after emergence (DAE)] and given access to yeast hydrolysate over a range of durations. Diet treatments were (A) deprived of yeast hydrolysate, (B) 24-h access to yeast hydrolysate, (C) 48-h access, and (D) continuous access.

Figure 3 Diurnal attraction to cue-lure by female Queensland fruit flies of different ages [4, 8, 12, 16, and 20 days after emergence (DAE)] and given access to yeast hydrolysate over a range of durations. Diet treatments were (A) deprived of yeast hydrolysate, (B) 24-h access to yeast hydrolysate, (C) 48-h access, and (D) continuous access.

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Figure 4 (A) Temperature, (B) relative humidity, and (C) light intensity in the shade in four field cages used to test attraction of Queensland fruit flies to cue-lure.

increased by access to yeast hydrolysate for 24 or 48 h. At 8 DAE, attraction to cue-lure by males given access to yeast hydrolysate for 48 h was less than those given continuous access, but by 12 DAE attraction to cue-lure reached equally high levels. Access to yeast hydrolysate for 24 h led to higher levels of cue-lure attraction compared to males with no access, but attraction of 24-h males was always significantly less than attraction of males given continuous access. Sterile Q-flies used in SIT programmes are released as adults 24 –48 h after emergence (Meats et al., 2006). Inclusion of yeast hydrolysate in the diet provided to newly emerged sterile adults for a period of at least 24 h, but preferably 48 h, has the potential to improve trap recapture rates owing to faster rates of sexual development in male flies, and subsequent attraction to cue-lure. This would improve estimates of sterile male abundance in the field derived from trap recaptures. Furthermore, because male

attraction to cue-lure is linked to sexual maturity, feeding yeast hydrolysate to newly emerged sterile adults before release may improve sexual performance of males released in the field. A similar suggestion has been made for Mediterranean fruit flies, Ceratitis capitata Wiedemann (reviewed by Yuval et al., 2007). γ-Irradiation had no effect on attraction to cue-lure by Q-flies. This result was unexpected and differs from the outcome of studies on other tephritid species. Sterilization led to reduced attraction to food odours (protein hydrolysate) and food-based lures (BioLure®; Suterra LLC, Bend, OR, USA) in Caribbean fruit flies, Anastrepha suspensa (Loew), Mexican fruit flies, Anastrepha ludens (Loew), and C. capitata (Galun et al., 1985; Robacker, 1998). Few studies have attempted to define the physiological mechanisms underlying reduced response to food lures in these species, so it is difficult to speculate why γ-irradiation had no effect on response to cue-lure by Q-flies in our study. A recent study on Mediterranean fruit fly midgut proteolytic activity found no effect of sterilization (San Andres et al., 2007). The results from our study suggest that the ability of Q-flies to digest and absorb sources of nitrogen is unaffected by sterilization, leading to rates of sexual maturation (and attraction to cue-lure) similar to fertile (un-irradiated) flies. In addition, Perez-Staples et al. (2007) found no effect of irradiation on sexual maturation and activity of male or female Q-flies. Lynfield traps baited with cue-lure caught 86.2% of flies found dead in the field cage. The remainder of dead flies were found on the ground directly beneath the cue-lure traps, possibly after having initially entered the trap and escaping, or being affected by the insecticide before entering the trap. If Q-flies manage to escape from Lynfield traps before being killed by the insecticide, or are killed without having entered the trap, as our study suggests, this may lead to underestimation of an invasive population and failure to respond with timely control measures. However, effectiveness of current response thresholds, along with estimation of outbreak size, has been verified using trap capture data obtained using cue-lure traps (Meats, 1998a,b,c; Meats et al., 2003; Meats & Clift, 2005). This means that models estimating outbreak propagule size, probability of propagule extinction, and population size, already incorporate variability introduced by trap design. Nonetheless, redesign of cue-lure-baited traps used to monitor Q-fly populations should be a priority to enhance ability to detect and monitor lowdensity populations. We found that virgin female Q-flies are caught in cue-lure traps at ages when they are known to be sexually mature. In the laboratory, female Q-flies are sexually mature by as early as 5 DAE (at 25 °C), with mean mating

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age of 7 DAE when given unrestricted access to sugar and yeast hydrolysate (Meats et al., 2004; Perez-Staples et al., 2007; Prabhu et al., 2008). Our results are similar to those reported in other tephritids attracted to cue-lure and methyl eugenol. Unmated sexually mature females of the Australian species Bactrocera aquilonis (May) and Bactrocera opiliae Drew and Hardy are attracted to cue-lure and methyl eugenol, respectively (Fitt, 1981). Attraction of sexually mature virgin females to methyl eugenol has also been reported in wild tobacco fly, Bactrocera cacuminata (Hering) (Raghu & Clarke, 2003). It has been widely reported that female Q-flies are not attracted to, and are sometimes repelled by, cue-lure (Hill, 1986; Drew, 1987). Similar findings have also been reported for B. cucurbitae (Wong et al., 1991). However, the design of these studies may have precluded detection of female attraction to cue-lure. Drew (1987) housed male and female Q-flies together before testing their attraction to cue-lure, so females may have already mated. In other species where female response to male lures has been detected, attraction is not apparent in mated females (Fitt, 1981; Raghu & Clarke, 2003). Also, studies on attraction of Q-flies (Drew, 1987) and B. cucurbitae (Wong et al., 1991) did not include observations of attraction to cue-lure during the critical dusk mating period. Female Q-fly response to cue-lure may be linked to diurnal rhythms driving reproductive behaviour. Q-fly mating behaviour is restricted to the short period as light intensity falls at dusk (Myers, 1952; Tychsen, 1977; Drew & Lloyd, 1987). Males perch on leaves and commence rapid wing-vibration, or calling (Tychsen, 1977; Mankin et al., 2008), which is associated with release of a pheromone from rectal glands (Fletcher, 1968, 1969; Tan & Nishida, 1995). Sexually receptive females approach calling males, and copulation rapidly ensues. On an uncaged peach tree occupied by a large number of sexually mature males, virgin immature and mature females comprised around 90% of all visiting females (Drew & Lloyd, 1987). The association of responsiveness to cue-lure with female Q-fly reproductive maturity, and the temporal similarity of cue-lure response and reproductive behaviour at dusk, strongly implies a role for cue-lure, or similar chemicals, in the Q-fly mating system. Our results show that the peak period of attraction to cue-lure by male Q-flies is in the morning, and is out of phase with mating activity at dusk. Male Q-flies attracted to cue-lure in the morning are known to ingest the lure (Brieze-Stegeman et al., 1978). After feeding on cue-lure, or its hydrolysis product, raspberry ketone [4-(4-hydroxyphenyl)-2-butanone], a naturally occurring plant chemical, male Q-flies accumulate raspberry ketone in their rectal gland within 6 h of feeding (Tan & Nishida, 1995). In the

related B. carambolae, B. dorsalis, and B. papayae, males fed on methyl eugenol accumulate phenyl propanoid derivatives of that chemical [(E)-coniferyl alcohol, 2allyl-4,5-dimethoxyphenol, and 3,4-dimethoxycinnamyl alcohol)] in their rectal gland (Hee & Tan, 1998; Wee & Tan, 2005, 2007). These species and B. philippinensis also exhibit earlier precopulatory behaviour and improved sexual attractiveness when fed with methyl eugenol (Shelly & Dewire, 1994; Shelly et al., 1996; Hee & Tan, 1998; Wee & Tan, 2007; Wee et al., 2007). Similar sexual advantages have been found when male B. cucurbitae are fed cue-lure (Shelly & Villalobos, 1995). Although further study is required in this area of Q-fly reproductive biology, it seems likely that male response to cue-lure is driven by female preference for sex pheromones containing phenylpropanoid derivatives.

Acknowledgements We are very grateful to Alan Taylor (Macquarie University) and Roberto Munguía-Steyer (Monash University) for their statistical advice and programming assistance. We extend our thanks to John Prenter (Macquarie University) who helped to set up field cages and provided comments on earlier drafts of the manuscript. We thank Bernie Dominiak and Laura Jiang (NSW Department of Primary Industries) for providing Lynfield traps, baited wicks, mass-reared flies, and fluorescent pigments. Sterile flies were irradiated by Radiation and Dosimetry Services, Australian Nuclear Science and Technology Organisation. This project was facilitated by Horticulture Australia Limited (HAL) in partnership with Australian Citrus Growers and was funded by the Citrus levy (project code: CT05002). The Australian Government provides matched funding for all HAL R&D activities. Diana Perez-Staples was supported by a UNESCO-L’ORÉAL Co-sponsored Fellowship for Young Women in Life Sciences.

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