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Keywords:

  • Diet;
  • longevity;
  • sexual behaviour;
  • sterile insect technique;
  • tephritidae

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Abstract Adult diet is an important determinant of sexual activity in many tephritid fruit flies. Whether availability of protein (hydrolysed yeast) in addition to sucrose influences sexual activity or longevity of male and female Queensland fruit flies (Bactrocera tryoni Froggatt, ‘Q-flies’), and whether irradiation of flies as pupae modifies their dietary needs, is investigated. Previous studies on groups of flies suggest that protein is required for sexual maturation of females but not males. By contrast, this study of individual flies demonstrates that protein in the adult diet provides a massive boost to sexual activity of both males and females. Mating probability increases with age from 4-14 days as the flies began to mature. However, mating probability reaches much higher levels when the flies are provided with protein. Although males and females mate at similar rates when provided with protein, females suffer a greater reduction in mating probability than males when deprived of protein. In addition to increased mating probability, access to dietary protein is also associated with reduced latency from onset of dusk until copulation. Furthermore, young male flies with access to dietary protein have longer copula duration than males fed only sucrose. Irradiation of flies as pupae has no apparent effect on mating probability, the latency to copulate or copula duration. However, when deprived of protein, sterile flies (especially males) suffer a greater reduction in longevity compared with fertile flies. Overall, access to dietary protein increases longevity for both males and females, although females live longer than males on both diets. These findings suggest that prerelease provision of dietary protein has the potential to greatly enhance the efficacy of Q-flies used in the sterile insect technique.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Diet is a powerful influence on sexual development, sexual performance, and longevity in adults of many tephritid flies. In particular, access to protein is key in many species including Mediterranean fruit flies (Ceratitis capitata; Taylor & Yuval, 1999; Kaspi et al., 2000), apple maggot-flies (Rhagoletis pomonella; Webster & Stoffolano, 1978; Webster et al., 1979), melon flies (Bactrocera cucurbitae; McInnis et al., 2004; Shelly et al., 2005) and various Anastrepha species, such as Anastrepha obliqua, Anastrepha serpentina, and Anastrepha striata, but not Anastrepha ludens (Jácome et al., 1995, 1999; Aluja et al., 2001b). As well as being a vital component of foraging for wild tephritid populations, protein acquisition is also an important consideration for flies released in sterile insect technique (SIT) programmes. If released sterile flies fail to acquire sufficient protein in nature, they may have delayed sexual maturation, diminished sexual performance, and shortened lifespan. Whether released flies reliably acquire adequate nutrition, and whether prerelease provisioning of protein can improve performance, remain the topics of ongoing debate (Kaspi & Yuval, 2000; Aluja et al., 2001b; Shelly et al., 2002; Shelly & Kennelly, 2003; Maor et al., 2004).

The Queensland fruit fly (Bactrocera tryoni, or ‘Q-fly’) is one of Australia’s most economically important insects, infesting a wide array of commercial fruits crops (Drew & Hooper, 1983; Sutherst et al., 2000). Post-teneral nutrition, and especially protein acquisition, appears to be key to Q-fly reproductive performance and longevity. For adults in nature, bacteria gleaned from leaf surfaces are implicated as the key source of protein (Courtice & Drew, 1983; Drew et al., 1983; Drew & Lloyd, 1987; Vijaysegaran et al., 1997). Under laboratory conditions, and in mass-reared cultures, protein is usually provided in the form of hydrolysed yeast (Monro & Osborn, 1967; Meats et al., 2004). Drew et al. (1983) found that although mixed sex groups of Q-flies fed just sucrose and water have very low fecundity, low fertility, and short lifespan, improvements in each of these measures can be achieved through protein-rich supplements of hydrolysed yeast or bacteria cultures. Drew (1987) later reported that, although groups of flies produce fertile offspring regardless of whether the males had received protein, they fail to produce eggs if females are denied protein.

These studies indicate that protein is important for Q-fly populations and that there are sex differences in dependence on protein for production of fertile offspring. However, despite the clear trends in these studies of group fecundity and fertility, the effects of protein on individual mating performance remain to be investigated. Furthermore, there has not been any investigation of whether sterilization of flies for use in SIT has any influence on their protein requirements for mating. The present study builds upon the earlier work of Drew and colleagues by investigating the importance of access to a protein source for the mating performance and longevity of male and female Q-flies, and also considers whether fertile and sterile flies have different requirements.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

General methods

Fertile and sterile (irradiated, 73 Gy at approximately 65% pupal development) Q-flies were obtained as pupae from the Fruit Fly Production Facility located at New South Wales Department of Primary Industries’ Elizabeth Macarthur Agricultural Institute. Adults emerging from irradiated pupae from this facility are routinely released in a sterile release program to control Q-fly populations around the Tri-State Fruit Fly Exclusion Zone that covers large areas of New South Wales, Victoria and South Australia, as well as occasional outbreaks elsewhere (Sutherst et al., 2000). The adult flies emerged in a laboratory at Macquarie University (Sydney, Australia) and were initially housed in 5-L cages containing approximately 200 flies. All cages were supplied with water-soaked cotton wool and a dish of dry granular sucrose as food. Half of the cages were also supplied with a dish of dry hydrolysed yeast enzymatic (MP Biomedicals, Aurora, OH) as a source of dietary protein. All cages were maintained at 24–26 °C and 65–75% RH. An LD 12 : 12 h artificial photoperiod was maintained, although flies also experienced an artificial dawn and dusk in which the lights ramped up and down through 1 h. Adult flies were separated according to sex within 3 days after emerging by aspirator. No calling, courting, or mating was observed in cages prior to separating the sexes. Experiments were carried out for flies from 4 days of age until they reached the peak of their sexual activity at 14 days. A second series of experiments was carried out at 11, 20 and 30 days to observe sexual activity when flies were older.

Experiment 1: Sexual activity 4–14 days

For each of the eight combinations of diet, sex, and irradiation treatment, 11 cages containing ten flies each were set up. A sample of flies from these cages was tested for sexual activity at each day of age from 4–14 days. For each combination of diet, sex, and irradiation treatment, six flies were tested on each day against 10–14-day-old fertile sugar and protein-fed males or females. All flies used in each treatment on any day were taken from the same cage (i.e. each source cage contained four additional flies in case some died).

Between 3–5 h before artificial dusk began in the laboratory, each tested fly was transferred to a 1-L clear plastic cage. Next, a 10–14-day-old protein-fed fertile fly of the opposite sex was introduced to the cage. The two flies were left to interact and were observed for mating from 1 h before the laboratory light-phase ended until all sexual activity had ended. The times when copulation began and ended (observed using dim red light from a torch with a red filter covering the lens) were noted. Three replicates of this experiment were carried out using flies from batches obtained at least 6 weeks apart.

The day after the mating experiments, all flies were anaesthetized using CO2 and were then frozen (−19 °C) in individual vials. The right wing of all flies (mated and unmated, female and male) was removed and mounted onto double-sided adhesive tape along one side of a microscope slide. A strip of paper marked with the identity of each fly was mounted to the other side next to the wings. A second microscope slide was then pressed onto the tape holding the wings and labels to protect them from dust. In this way, the wings and labels were firmly secured and held flat between two glass microscope slides. Each wing was then photographed using a digital camera (Progres C10, 3 megapixels; Jenoptik, Germany) through the phototube of a stereomicroscope (Olympus SZX12, Japan) at × 20 magnification. The camera transferred the digital images directly to a computer hard-drive. The images were then measured using Image Tools, version 2.25 (a shareware program available from the University of Texas, San Antonio, Texas, U.S.A.). As a measure of fly size, wing length (mm) was measured from the intersection of the anal and median band to the margin of the costal band and the R4 + 5 vein.

Probability of mating was assessed using multiple logistic regression, with significance tested using likelihood ratio tests. Copula latency and duration were analysed by analysis of covariance (ancova). In all models, first-order interactions were considered initially. Nonsignificant interactions were excluded from the analysis, and models re-run. Effects of nominal predictors in ancova on continuous outcomes are presented as least square means (LSM, back-transformed if dependent variables have been transformed for analysis), correcting for other factors in the model.

Experiment 2: Sexual activity 11–30 days

These experiments were identical to those investigating sexual activity at 4–14 days, except that flies were tested at 11, 20, and 30 days of age. The analysis differed from experiment 1 only in that the age effect was treated as continuous in experiment 1 and as nominal in experiment 2 (with only three time points). Three replicates of this experiment were carried out using batches of flies obtained at least 1 week apart.

Experiment 3: Effects of protein on longevity

These experiments were set up on the day that flies emerged as adults. For each of the diet, sex and irradiation treatments, three 1-L cages were set up with 10–15 flies. All cages were provided with water-soaked cotton wicks and sucrose in dishes on the floor of the cage. Sucrose + protein flies had a second dish in their cages, containing hydrolysed yeast. Survival of flies was checked daily, and dead flies were removed from the cages. Two replicates of this experiment were carried out using batches of flies obtained 1 week apart.

Longevity was assessed using Cox’s proportional hazards survival regression with significance tested using likelihood ratio tests. When flies died of un-natural causes or escaped, these cases were included as censored data (see Hosmer & Lemeshow, 2003).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mating probability

Experiment 1: 0–14 days Diet had potent effects on mating probability, although the strength of this effect varied both with the age and sex of the tested fly (Table 1, Fig. 1). Flies on the sucrose + protein diet showed a steep increase in mating probability over the 14-day testing period (b= 0.48 ± 0.03, = 792, G1= 293.516, P < 0.001). Although flies on the sucrose only diet also showed a significant increase in mating probability over the tested period, the slope was shallower than observed for the sucrose + protein diet (b= 0.21 ± 0.05, n= 792, G1= 22.085, P < 0.001). Accordingly, the difference in sexual activity of flies on the two diets increased over the testing period (Fig. 1).

image

Figure 1. Effects of access to dietary protein on the percentage of flies mating each day of age from 4–14 days in experiment 1 (all replicates combined). White bars are sucrose only; dark bars are sucrose + protein.

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Table 1.  Predictors of mating probability.
Sourced.f.GP
Days 4–14
 Replicate212.6600.002
 Age1163.726< 0.001
 Fertility10.2270.633
 Sex13.9950.046
 Size10.0810.777
 Diet1373.741< 0.001
 Diet × Age118.799< 0.001
 Diet × Sex14.9630.026
Days 11–30
 Replicate20.9030.637
 Age20.4770.788
 Fertility10.2110.646
 Sex11.0070.316
 Diet1178.962< 0.001
 Size13.8070.051
 Diet × Sex111.787< 0.001

Although neither sex showed high mating probability on the sucrose only diet, males performed better than females on this diet (Fig. 1). For flies on the sucrose + protein diet, there was no significant difference in mating probability of the two sexes (n= 792, G1= 0.127, P= 0.722), whereas for flies on the sucrose only diet males were significantly more likely to mate than were females (n= 792, G1= 7.558, P= 0.006).

Experiment 2: 11–30 days Diet also influenced mating probability over the longer time frame of experiment 2 and, as in experiment 1, this effect varied between the sexes (Table 1, Fig. 2). Again, although mating probability of males and females was similarly high when on the sucrose + protein diet (n= 239, G1= 0.351, P= 0.553), more males than females mated when on the sucrose only diet (but still to a far lower level than when on the sucrose + protein diet) (n= 244, G1= 21.788, P < 0.001).

image

Figure 2. Effects of access to dietary protein on the percentage of flies mating at 11, 20 and 30 days of age in experiment 2 (all replicates combined). White bars are sucrose only; dark bars are sucrose + protein.

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Copula latency

Experiment 1: 0–14 days Latency from the beginning of artificial dusk until initiation of copula (log-transformed, Table 2) decreased as the flies aged (b=−0.035 ± 0.012) and was similar for sterile and fertile flies. Diet had a significant effect on copula latency in males but not in females; Tukey’s HSD test (α= 0.05) revealed no significant differences in copula latency among females on either diet and males on the sucrose only diet, but that males on the sucrose + protein diet had significantly shorter copula latency than any other group (back-transformed LSMs: female sucrose + protein = 23.8 min, female sucrose only = 26.6 min, male sucrose + protein = 17.7 min, male sucrose only = 29.3 min). Males and females also differed in the relationship between size and copula latency. Large males tended to mate later than small males (b= 0.654 ± 0.292, n= 251, F1,244= 5.017, P= 0.026), but size was not related to copula latency in females (b=−0.146 ± 0.242, n= 230, F1,223 = 0.366, P= 0.546).

Table 2.  Predictors of copula latency (log-transformed).
Sourced.f.FP
Days 4–14
 Replicate2353.409< 0.001
 Age18.2940.004
 Fertility11.8050.180
 Sex10.9760.324
 Size12.5060.114
 Diet111.508< 0.001
 Sex × Size17.3260.007
 Diet × Sex14.1030.043
Days 11–30
 Replicate26.2640.002
 Age24.4960.012
 Fertility10.0280.868
 Sex11.9340.165
 Diet138.716< 0.001
 Size10.1530.696

Experiment 2: 11–30 days Latency from the beginning of artificial dusk until initiation of copula (log-transformed, Table 2) was similar for sterile and fertile flies and for males and females. The latency to copulate increased significantly as the flies aged (back-transformed LSMs: 11 days = 8.1 min, 20 days = 9.5 min, 30 days = 11.9 min) and was significantly shorter for flies on the sucrose + protein diet than for flies on the sucrose only diet (back-transformed LSMs: 7.0 min and 13.5 min, respectively). Size had no effect on the latency to copulate (Table 2).

Copula duration

Experiment 1: 0–14 days Copula duration (log-transformed, Table 3) was similar for sterile and fertile flies, increased as flies aged (b= 0.031 ± 0.014), and also varied significantly with diet for males but not for females. Tukey’s HSD test (α= 0.05) revealed no significant differences in copula duration among females on either diet and males on the sucrose + protein only diet, but that males on the sucrose only diet had significantly shorter copula duration than any other group (back-transformed LSMs: female protein + sucrose = 95.4 min, female sucrose only = 68.4 min, male sucrose + protein = 86.6 min, male sucrose only = 34.0 min).

Table 3.  Predictors of copula duration (log-transformed).
Sourced.f.FP
Days 4–14
 Replicate20.8960.409
 Age15.0730.025
 Fertility10.9490.331
 Sex113.563< 0.001
 Size11.6650.198
 Diet142.842< 0.001
 Diet × Sex19.8170.002
Days 11–30
 Replicate22.6140.075
 Age22.5090.083
 Fertility11.2150.271
 Sex12.0670.152
 Diet121.484< 0.001
 Size13.5190.062

Experiment 2: 11-30 days The duration of copulation (log-transformed, Table 3) did not vary significantly with age, fertility, sex or size. However, flies fed sucrose + protein had significantly longer copulations than flies fed sucrose only (back-transformed LSMs: protein + sucrose = 80.6 min, female sucrose only = 49.9 min).

Effects of protein on longevity

Proportional hazards survival analysis revealed significant effects of sterility, diet and sex on longevity (Table 4, Fig. 3) (median longevity for fertile flies: female protein + sucrose = 87 days, female sucrose only = 59 days, male sucrose + protein = 41 days, male sucrose only = 38 days; median longevity for sterile flies: female protein + sucrose = 81 days, female sucrose only = 27 days, male sucrose + protein = 45 days, male sucrose only = 23 days). There was a significant interaction between diet and sterility; although sterile and fertile flies had similar longevity when on the sucrose + protein diet, sterile flies had reduced longevity compared with fertile flies when on the sucrose only diet. There was also a significant interaction between diet and sex; females lived longer than males on both diets, but gained a much greater increase in longevity on the sucrose + protein diet (median longevity of flies on sucrose + protein divided by median longevity of flies on sucrose only: fertile males = 1.08, fertile females = 1.47, sterile males = 1.96, sterile females = 3.00).

image

Figure 3. Effects of access to dietary protein on survival of Fertile and Sterile Queensland fruit flies (all replicates combined). Solid bars are sucrose + protein; broken lines are sucrose only.

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Table 4.  Predictors of longevity.
Sourced.f.GP
Replicate11.0020.317
Fertility14.0160.045
Sex148.611< 0.001
Diet153.480< 0.001
Diet × Fertility116.738< 0.001
Diet × Sex110.969< 0.001

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Access to dietary protein has a profound effect on sexual activity of Queensland fruit flies. In the present study, virgin male and female Q-flies fed both sucrose and yeast hydrolysate (a protein source) increase rapidly in mating probability to a peak of 80–90% within 12 days of emerging and maintain high levels of mating for at least 30 days. By stark contrast, males and females fed only sucrose show consistently much lower mating probability, at no time matching the performance of protein-fed conspecifics. As suggested by the earlier work of Drew (1987) on fecundity and fertility of groups of flies, females were more sensitive to the absence of protein, showing a relatively greater reduction in mating probability when on the sucrose only diet. This difference is likely related to sex differences in the nutritional demands on development of reproductive tissues. Female Q-flies need at least 0.1 mg of protein per day to develop their ovaries (Drew, 1987; Meats & Leighton, 2004). Similarly, protein intake also influences the rate of ovarian development in A. obliqua and A. ludens (Aluja et al., 2001a).

During the first 2 weeks of adulthood mating probability increases with age more steeply for flies fed on sucrose + protein compared with flies fed on sucrose only (i.e. significant age-diet interaction). Consequently, the differences in mating probability between these treatments increase as the flies age. However, once the flies on the two diet treatments have reached their peak of mating probability at 10–12 days, the difference between the treatments remains quite stable until the maximum age tested of 30 days.

In addition to effects on mating tendency, access to protein also influences the latency to copulate; of the males that mated, those receiving protein tend to mate sooner after the onset of dusk. Similarly, protein-fed C. capitata tend to mate earlier than protein-deprived conspecifics (Blay & Yuval, 1997; Kaspi et al., 2000). This result may indicate greater overall vigour, and courtship effort of protein-fed males. In other tephritids such as C. capitata, A. striata, and A. serpentina, males fed on sucrose and protein call more frequently than flies fed on sucrose only (Kaspi et al., 2000; Aluja et al., 2001b). Latency to copulate decreases as flies age from 4–14 days, whereas it increases again at later ages. At this earlier age, larger males take longer to mate. Perhaps as males and females approach full sexual maturity, males invest more effort in courtship. Indeed, in C. capitata, calling incidence increases with age (Papadopoulos et al., 1998). Females may also be more eager to accept a young mate. Older flies that have long since reached sexual maturity may invest less effort in calling and courtship, hence taking longer to secure a mate.

Young males on the sucrose-only diet tend to have shorter copulations than those receiving both sucrose and protein. When the flies are older, both males and females on the sucrose only diet have shorter copulations than protein-fed conspecifics. By contrast to these trends for Q-flies, in C. capitata and in A. striata, males that are fed protein tend to have shorter copulations than flies fed sucrose only (Blay & Yuval, 1997; Pérez-Staples & Aluja, 2004) and, for)C. capitata it seems that males fed on protein may be able to transfer large ejaculates faster (Taylor & Yuval, 1999). In addition to these species with opposite effects of dietary protein on the duration of copulation, in A. serpentina, A. ludens and A. obliqua this appears to be unaffected by adult diet (Aluja et al., 2001b).

Although the consequences of copula duration were not investigated in the present study, some inferences from other recent work using flies from the same stock can be drawn. Using methods very similar to the present study, Harmer et al. (2006) found no relationship between the duration of copulation and the probability of sperm storage, number of sperm stored by females, or probability of females remating. In a subsequent study, Perez-Staples et al. (2007) similarly found no evidence of relationship between the duration of copula and the number of sperm stored among those copulations that did entail some sperm storage. However, unlike Harmer et al. (2006), Perez-Staples et al. (2007) did find some evidence that very short copulations are less likely to entail transfer of sperm (although there is considerable overlap in duration of successful and unsuccessful copulations).

Access to protein not only promotes high levels of sexual activity in fertile and sterile flies of both sexes, it also promotes longevity (especially in females). Likewise, access to protein increases longevity in Rhagoletis indifferens (Yee, 2003) and A. serpentina (Jacome et al., 1995). In C. capitata, access to protein has an opposite effect on female and male life-span. Females fed with protein have higher life expectancies than males fed on protein, whereas when both sexes are deprived of protein, males live longer than females (Müller et al., 1997). Although there is no evidence that sterilization of the Q-flies for use in SIT has any bearing on their protein requirements for sexual activity, there are marked differences between sterile and fertile flies in the effects of diet on longevity. Specifically, although both fertile and sterile flies have increased longevity if they had access to a source of protein, these effects are greater for sterile flies.

Male copulatory success is a key factor in the sterile insect technique (SIT) and these findings have important implications for SIT used to manage wild Q-fly populations in Australia. Currently, flies are released at 1–3 days of adult age and typically are provided with water and sucrose, but not a source of protein during the prerelease holding period. Access to protein in nature is probably very limited in ephemeral sources such as bacteria in bird faeces and leaf leachates (Drew et al., 1983; Hendrichs et al., 1993). If flies have difficulty finding protein, in nature, then a very large proportion of released sterile flies are likely to never enter sexual activity, eventually dying as virgins and making no contribution to the control of wild populations. If provision of a protein source in the prerelease holding period enables more of the released flies to reach sexual maturity, this may dramatically increase the effective sterile/wild over-flooding ratio without increasing the number of flies released. There is little point in including sexually inactive flies in calculations of over-flooding ratios. Further research is required to determine whether a brief window of 1–2 days of access to dietary protein (the current prerelease holding period) is sufficient to yield a substantial increase in sexual performance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported by funds from Horticulture Australia Ltd to P.W.T. and an Endeavour Research Fellowship from the Australian Department of Education Science and Training and a UNESCO-L’OREAL Fellowship to D.P.S. We are especially grateful to Laura Jiang and Selliah Sundaralingam of New South Wales Department of Primary Industries for supplying flies from the stocks used in SIT. We also thank Maria Castillo-Pando for help with sexing and maintaining the flies. Andrew Jessup, Cathy Smallridge and Bernie Dominiak as well as two anonymous referees provided valuable comments on the manuscript.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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