Ihsan ul Haq (corresponding author), Insect Pest Control Laboratory, FAO/IAEA Agriculture and Biotechnology Laboratories A-2444, Seibersdorf, Austria. E-mail: firstname.lastname@example.org
The application of methoprene and access to protein in adult diet has been shown to enhance mating success in male melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), supporting their incorporation into operational area-wide programmes integrating the Sterile Insect Technique (SIT). The objective of the present study was to investigate the effect of methoprene, diet including protein and feeding days on male starvation survival to determine the minimum number of feeding days required prior to male release in the field. The study was performed in the laboratory by treating males with (i) both protein and methoprene (M+P+), (ii) only protein (M−P+), (iii) only methoprene (M+P−) or (iv) untreated (M−P−). The males were starved after exposure for an increasing number of days (1–7) to their respective treatment. Mean longevity was highest after 3-day post-emergence feeding duration for M+P+, M+P− and M−P− males, but 4 days of feeding for M−P+ males. Additional feeding days after 4 days, did not increase male survival and feeding for 7 days decreased starvation survival of sugar-fed males. Application of methoprene and/or access to diet including protein had no adverse effect on starvation survival but feeding duration had a significant positive effect on starvation survival. To the contrary, the current study provides a strong evidence for the benefits of methoprene application and protein incorporation into the adult diet of sterile males. Treated males achieve higher sexual success, reach sexual development several days earlier, and are therefore much closer to sexual maturity when released in SIT action programmes after being held in the fly emergence and release facility for a post-emergence feeding duration of at least 3 days.
The melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), is a serious pest of economic importance causing severe direct losses in many fruits and vegetables (White and Elson-Harris 1992). Because of its quarantine status, its presence seriously interferes with the international marketing of fruits and vegetables and it has been the target of a very successful area-wide programme integrating the Sterile Insect Technique (SIT) to eradicate it from all of Japan (Koyama et al. 2004). In suppression programmes that incorporate the SIT against this species, mass reared sterile males are fed and held in fly emergence and release facilities before release. Ideally they are held until they reach sexual maturity, which can be up to 7–10 days (McInnis et al. 2004); however, such a long-holding period would be extremely expensive in terms of time, energy cost and space, adding considerably to the cost of the programme. In some tephritids, such as the Caribbean fruit fly Anastrepha suspensa (Loew) the application of methoprene can accelerate the rate of sexual maturation (Teal et al. 2000; Pereira 2005), but in the melon fly methoprene application is only effective in accelerating sexual maturity when sterile males are also provided with dietary protein (Haq et al. 2010). Apparently the methoprene treated males require this additional source of nutrition to be able to accelerate their reproductive development.
In the successful SIT programme in Japan, sterile flies were fed only sugar and water prior to release (Nakamori and Kuba 1990), but recent findings have shown significant improvement in mating behaviour when the males are treated with methoprene and fed protein (Haq et al. 2010), suggesting that this protocol could be incorporated into future SIT programmes. However, in these operational programmes, males can be fed on a sugar and protein diet for only 3–4 days in the emergence and release facility, and protein deprivation once in the field may have a negative impact on their survival. This study addresses the following questions: (i) what is the effect of methoprene treatment and access to protein in the adult diet on the post-feeding survival after release in B. cucurbitae? (ii) Does increasing the number of feeding days on protein diet enhance post-feeding male survival? (iii) What is the optimal duration for protein feeding in maturing males in terms of post-feeding survival? Although in the field, males have access to and actively forage for water and nutrients such as honeydews, plant exudates, bird faeces, extrafloral nectarines, and microbes on plant surfaces (Drew et al. 1983; Drew 1987; Hendrichs and Hendrichs 1990; Prokopy et al. 1993), in this experiment post-feeding survival was measured under the extreme situation of full absence of any food or water following removal of the flies from their feeding regime.
Materials and Methods
Strain and rearing
The genetic sexing strain of B. cucurbitae developed by USDA ARS, Hawaii (McInnis et al. 2004) was used for all experiments. The colony was maintained on wheat-based modefied standard Seibersdorf diet (Hooper 1987) at the FAO/IAEA Agriculture & Biotechnology Laboratories, Seibersdorf, Austria. The flies were maintained under low stress conditions (four larvae per gram of diet and ∼100 flies in 20 × 20 × 20 cm adult cages) and pupae sorted according to size for the experiments. The flies used in the experiments were therefore all obtained from pupae of average size (2.2–2.3 mm diameter). Following emergence, the flies were maintained in the laboratory with a photoperiod of 14L : 10D at 24 ± 1°C and 60 ± 5% RH.
There were four treatments for adult males:
1 topical application of the juvenile hormone analogue, methoprene (M), and sugar and hydrolysed yeast (MP Biomedicals Inc.; http://www.mpbio.com) (protein source) as adult food (M+P+);
2 no methoprene application but sugar and hydrolysed yeast (protein source) as adult food (M−P+);
3 topical application of methoprene and only sugar as adult food (M+P−);
4 no methoprene application and only sugar as adult food (M−P−).
The methoprene was applied topically 3–4 h after adult emergence at a rate of 5 μg in 1 μl acetone solution per male by immobilizing males in a net bag (as in standard marking techniques, FAO/IAEA/USDA 2003) and the solution applied via pipette through the net onto the dorsal surface of the thorax. No anaesthesia was used to immobilize the flies. One hundred males from each treatment were maintained in separate 12 × 10 × 20 cm Plexiglass screened cages (20 males per cage) with the type of food assigned for each treatment. In treatments without protein feeding (P−) only water and sugar ad libitum were supplied to the flies. In the treatments with protein (P+), hydrolysed yeast was added to the sugar in a proportion of 3 : 1, sugar:hydrolysed yeast, and the flies were supplied with this diet and water ad libitum; this diet is considered a high-quality diet for anautogenous tephritid fruit flies (Hendrichs and Prokopy 1994; Drew and Yuval 2000).
Measuring effect of days of feeding on post-feeding survival
Five cages of 20 males from each of the four treatments were exposed for an increasing number of days (1–7) to their respective treatment (for a total of 35 cages/treatment). At the end of each period of feeding (from days 1 to 7), the flies from the five cages from each treatment were transferred to clean 12 × 10 × 20 cm Plexiglass screened cages with no food or water and post-feeding survival was assessed. Dead males were removed from all cages and counted every 12 h until all the flies had died.
The data were analysed using survival analysis and the Cox proportional hazard model (Everitt and Pickles 2004) by considering the treatments methoprene, diet including protein and feeding days as factors with all possible interactions. Cox proportional hazard model was fitted for comparative survival of each treated males (M+P+, M+P− and M-P+) with control males (M−P−), and survival of all type of males at each feeding duration was compared with 1-day feeding duration. Mean longevity was calculated by constructing the life tables (Carey 1993). Post-feeding survival for each combination of male treatment and number of days of feeding was compared with 95% confidence intervals. Statistical analyses were performed using r software (http://www.r-project.org/).
General effects of male treatment on post-feeding survival after feeding for a different number of days
Male’s starvation survival curves for each feeding duration are shown in fig. 1. Cox proportional hazard model showed that application of methoprene, diet including protein, and their interactions had no significant effect on starvation survival. However, feeding duration had significant effect on males starvation survival (χ2 = 27.21, P < 0.001). The starvation survival of treated males (M+P+, M+P− and M−P+) in comparison with control males (M−P−) at the same feeding duration (table 1), showed that M+P+ males had similar starvation survival to that of M−P− males at 1–5 days of feeding but significantly higher starvation survival after 6 and 7 days of feeding. The males M+P− showed similar starvation survival to that of M−P− males after 1–7 days of feeding. The males M−P+ males showed a variable response; significantly reduced starvation survival after 2 and 3 days of feeding, similar starvation survival after 1, 4 up to 6 days of feeding but significantly increased starvation survival after 7 days of feeding as compared with that of M−P− males at same feeding duration.
Table 1. Starvation survival analysis of M+P+, M−P+ and M+P−Bactrocera cucurbitae males1 compared with M−P− males at each feeding duration (1–7). Within each column, comparisons with P < 0.05 are significantly different (Cox proportional hazard model). Negative Z-value indicates higher survival
Starvation survival after each feeding duration
1Males were treated with either methoprene and protein (M+P+), no methoprene and protein (M−P+), methoprene and only sugar (M+P−), or no methoprene and only sugar (M−P−). All males had access to sugar and water.
Specific effects of number of feeding days on survival of males for the different treatments
Feeding for different number of days has a significant effect on starvation survival of M−P− males (χ2 = 71.21, P < 0.001). Feeding these males for 2, 3 and 4 days significantly increased their post-feeding survival in comparison with the males fed for 1-day only, but feeding for 5 and 6 days did not increase survival and feeding for 7 days significantly decreased post-feeding survival (table 2). The mean post-feeding longevity was highest after 3 days of feeding (fig. 2).
Table 2. Starvation survival analysis of Bactrocera cucurbitae males1 treated with methoprene and/or protein and feeding during 2–7 days compared with 1-day feeding. Within each column, comparisons with P < 0.05 are significantly different (Cox proportional hazard model). Negative Z-value indicates higher survival
1 day feeding
1Males were treated with either methoprene and protein (M+P+), no methoprene and protein (M−P+), methoprene and only sugar (M+P−), or no methoprene and only sugar (M−P−). All males had access to sugar and water.
2 days feeding
3 days feeding
4 days feeding
5 days feeding
6 days feeding
7 days feeding
Differential feeding days also had significant effects on the survival of M+P− males (χ2 = 56.98, P < 0.001). Feeding M+P− males for 2, 3 and 4 days significantly increased post-feeding survival compared to males fed for 1 day. Feeding for 5 and 6 days had no effect and removal of food after 7 days significantly decreased the survival of males (table 2). The mean post-feeding longevity was highest in males fed three days (fig. 2).
Different feeding days had significant effects on starvation survival of M−P+ males (χ2 = 14.47, P= 0.02). Feeding for two or three additional days did not increase survival, but feeding for 4 days did improve post-feeding survival. Feeding for 5, 6 or 7 days also did not increase survival (table 2). The mean post-feeding longevity was highest following 4 days of feeding (fig. 2).
Similarly, starvation survival of M+P+ was significantly affected by the feeding duration (χ2 = 68.63, P < 0.001). The post-feeding survival of M+P+ males was higher following feeding for 2, 3 and 4 days, but feeding for 5 or 7 days did not improve survival when compared with 1-day feeding. Rather surprisingly males fed for 6 days had a higher starvation survival as compared with males fed for 1 day (table 2). The mean post-feeding longevity was highest in those males fed for 3 days (fig. 2).
Our results showed that the interaction between nutrition, longevity and stage-specific survival is complex. The response of survival did not increasing linearly with days of feeding as would have been expected. The optimal range of post-emergence feeding days in males lies between a duration of 3–4 days. A gradual increase in survival was observed from days 1 to 3, which can be explained as the flies’ need to build up reserves for optimal surviving and performance. Afterwards, the decline in post-feeding survival in spite of the possibility of accumulating additional reserves can possibly be explained because of the diversion and use of reserves towards physiological needs associated with sexual development (male cost of reproduction). The minor exception to this general trend was M−P+ males. These males attained maximum survival following a feeding duration of 4 days, rather than 3 days.
Although the general trend in starvation survival was similar for all four treatments, the possible explanation about the effects of access to protein in the diet or methoprene treatment could be different. The males (M+P−, M−P−) had similar post-feeding survival to that of M+P+ males until 5 days of feeding duration, but afterwards there was a declining trend and the lowest survival for these (sugar-fed) males was observed at 7 days of feeding. Sugar-fed males initiate sexual calling at the age of 9 days (Haq et al. 2010). Thus, increased mortality in food deprived males at 7 days of feeding (males were 9 days old) could be associated with physiological needs related to the start of sexual development.
No adverse effect of methoprene treatments on starvation survival was observed and this response was similar to that of C. capitata (Faria et al. 2008), A. suspensa (Pereira 2005) and Tenebrio molitor L. (Rantala et al. 2003). It could be expected that the application of this hormone would have an adverse effect on survival (Herman and Tatar 2001) because of the cost associated with earlier initiation of sexual development. However, the males M+P− showed similar starvation survival to that of M−P− during 1–7 days of feeding. The similar starvation survival by both type of males is understandable because M+P− males, were not significantly different from M−P− males for their sexual maturity age and mating success (Haq et al. 2010). But the males M+P+ exhibiting early sexual maturity and higher mating success (Haq et al. 2010), also showed a similar starvation survival as M−P− males until fed for days 1–5. A possible explanation for this similarity was that extra cost of sexual development in M+P+ males was compensated by the addition of protein in the diet.
However, M+P+ males had also a significantly higher survival after 2 and 3 days of feeding than M−P+ males following the same feeding duration and reached peak longevity after 3 days feeding, 1 day earlier than that of M−P+ males. The survival response of M−P+ males was more variable and they showed lower post-feeding survival than any other type of male when deprived of food after 2 and 3 days of feeding. These findings agree with those of Kaspi and Yuval (2000) and Maor et al. (2004), who reported increased mortality in protein-fed C. capitata males, deprived of protein after 4 days of feeding. The higher mortality of M−P+ males following deprivation after 2 or 3 days of feeding may be due to the protein diet accelerating reproductive development and diverting resources for sexual signalling and development of accessory glands (Yuval et al. 2007). However, these males may not have attained at the time of food removal a certain developmental threshold, only reached until after 4 days of access to a diet containing protein, which then caused decreased post-feeding male survival. There are studies suggesting the negative effect of male sexual calling on starvation survival (Johansson et al. 2005). The males with access to protein in the diet initiate sexual calling at 5 days of age (Haq et al. 2010). Thus, decreased survival in food deprived males at 3 days of feeding (males were 5 days old) may also be due to the onset of their sexual calling. Our results further suggest that starvation survival corresponds more to deprivation before reaching a critical developmental metabolism threshold than to the specific type of food provided to males.
The balance in physiological activity between reproductive development and somatic maintenance has consequences for survival (Herman and Tatar 2001). Dingle (1997) suggested that JH may play a key regulatory endocrine role for moderating the plasticity of life history. M+P+ males may therefore have more balance in physiological activity between their reproductive and somatic metabolism than M−P+ males up to 2–3 days of feeding. But the similarity in post-feeding survival of M+P− and M−P− males with M+P+ at 3–4 days of feeding might be the consequence that these protein deprived males have only somatic maintenance cost up to 4 days of feeding. The least survival of M+P− and M−P− males after a feeding duration of 7 days may be the result of an imbalance between costs of reproductive and somatic maintenance caused by lack of sufficient and appropriate food reserves.
This study provides insight into the response of imaginal males to methoprene application and protein feeding and represents a step towards the resolution of the nutrition – survival paradox of male tephritids (Carey et al. 1999; Kaspi and Yuval 2000). We confirmed that access to protein in the adult diet is a major advantage for male development and performance, although the timing of deprivation of protein feeding can be critical in B. cucurbitae as it is in C. capitata (Maor et al. 2004; Yuval et al. 2007), and this sensitivity can be overcome by calibrating the post-emergence feeding duration to bridge the decisive pre-copulatory maturation period of the males.
Our findings have implications for area-wide B. cucurbitae control programmes integrating the SIT. Ongoing programmes release M−P− sterile males, whereas based on our results it can be proposed that they be exposed to a M+P+ regime after emergence. In addition, they should be fed for at least 3 days prior to release to optimize their survival ability in the extreme assumption that they will not find nutrients in nature. This is of course often not realistic because sterile flies have been shown to have a capacity to forage for resources, and in any case regularly need to obtain in the field new resources to be able to participate in energetically costly lekking and courtship activities (Maor et al. 2004). On the other hand, immature sterile males, while maturing sexually under natural conditions, suffer significant losses, mainly caused by predation. Thus, the age of sterile male release needs also to take into account such other competing issues, suggesting the postponement of the sterile male release to when they are close to achieving sexual development to maximize numbers surviving to sexual maturity. This further supports the use of methoprene, because males without methoprene treatment would have to be maintained at the emergence and release facility for a much longer time, increasing costs and reducing their relative survival ability. Alternatively they will continue to be released much before sexual development as M−P− males, therefore increasing their mortality losses in the field prior to attaining their full mating potential.
Once sterile males attain sexual maturity, increased longevity is only of relative importance since they have limited sperm availability and therefore can be effective in only few matings (Itô and Yamagishi 1989). Fortunately, there is evidence now that dietary protein and the application of methoprene can also increase the mating success of mass reared B. cucurbitae males (Haq et al. 2010). This additional benefit also supports the incorporation of methoprene and protein into SIT operations. It would also result in less cost of holding males in emergence and release facilities as compared to non-treated males that would have to be held for at least 7 days until sexual maturity, or as standard practice, releasing them after 3–4 days as M−P− males when they are several days away from sexual maturity.
Further research is needed to address the following questions: (i) Does sterilization of males affects the findings of this study? (ii) Does a M+P+ regimen affect the ability of sterile males to search for food and leks, and to disperse and survive as well as non-treated sterile males? (iii) Will M+P+ treated sterile males compete successfully with wild males for mating with wild females?
We thank V. Wornoayporn, Amirul Islam and Sohel Ahmed for providing technical assistance in the rearing of flies and logistical support.