Post-teneral nutrition as an influence on reproductive development, sexual performance and longevity of Queensland fruit flies

Authors


Phillip W. Taylor (corresponding author), Department of Biological Sciences, Macquarie University, NSW 2109, Australia. E-mail: Phil.Taylor@mq.edu.au

Abstract

Adult Queensland fruit flies, Bactrocera tryoni (Froggatt), require adequate post-teneral nutrition to complete reproductive development, to perform sexually and for maximum longevity. Recent research has focussed on nutritional requirements of adults released in sterile insect technique (SIT) programmes that are used to manage these pests. Several studies have suggested benefits of providing yeast hydrolysate (YH) in addition to sugar during the 24- to 48-h pre-release holding period. Current evidence suggests that provision of YH can induce faster development, increased mating probability, longer copulations, increased sperm storage by mates, higher levels of sexual inhibition in mated females and increased longevity. We here review research on adult B. tryoni nutritional requirements, assess the potential application of this information in the context of SIT programmes and highlight future research that will help to determine whether YH, or other supplements, should be included as pre-release treatment in B. tryoni SIT.

Introduction

At first glance, the sterile insect technique (SIT) used for integrated management of fruit fly pests is a disarmingly simple process. The basic principle entails rearing and releasing many millions of sterile male flies that copulate with, and induce reproductive failure in, fertile wild female flies, thereby reducing population levels in the next generation (Knipling 1959; Krafsur 1998). But closer inspection soon reveals a complex series of biological and operational processes. As has been presented most clearly for the Mediterranean fruit fly (Ceratitis capitata) (Yuval et al. 2002, 2007), fruit flies released in SIT programmes face a daunting series of challenges that they must overcome for the SIT to succeed. Having passed through the strong selection pressures encountered during domestication and mass rearing, after release into the field sterile males must (i) accrue resources needed to complete development, (ii) survive long enough to mature sexually, (iii) join wild flies in mating aggregations, (iv) attract wild females, (v) secure copulations and (vi) complete a normal copulation that entails transfer of both sperm and accessory gland fluids that induce sexual refraction in their mates. In this sequence, only those flies that succeed at each step progress on to the next one. Hence, poor performance of released sterile male flies at any one of these challenges will prevent them from achieving their ultimate goal of curtailing the reproduction of wild females.

Tephritid fruit flies typically emerge with immature reproductive organs and must forage for the nutrients required to complete reproductive development (Fletcher 1987; Drew and Yuval 2000). The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), Australia’s most widespread and economically destructive insect pest of horticultural crops (May 1961; Sutherst et al. 2000), follows this general pattern. Several research groups, each with its own distinctive approach and contribution, have explored the importance of diet in B. tryoni natural history, domestication, mass rearing, sexual biology and management. Adult diet plays a central role in B. tryoni biology, and there is growing evidence that nutrition contributes to performance at each of the challenges faced by B. tryoni in SIT programmes. Bactrocera tryoni are usually provided refined cane sugar (hereafter ‘sugar’) as a source of carbohydrate and water prior to release in SIT programmes (Anon 1996; Meats et al. 2006), but are not provided other nutrients required to sustain reproductive development and somatic maintenance such as amino acids, sterols, vitamins and minerals. Although there have been some studies of pupal release (e.g. Andrewartha et al. 1967; Dominiak et al. 2003; Reynolds et al. 2010), all ongoing B. tryoni SIT programmes use adult release methods. In B. tryoni SIT, flies emerge from the pupal stage in bins held in controlled environment rooms and are then held for approximately 24–48 h before release (Anon 1996; Meats et al. 2006; Worseley et al. 2008). This provides a window of time to provide pre-release dietary supplements that might enhance the ability of mass-reared insects to perform after release into the field.

In this review, we outline current evidence for the role of nutrition at each of the challenges faced by B. tryoni during SIT application and specifically assess the potential value of yeast hydrolysate (YH, often referred to in the literature as ‘protein’) as a pre-release dietary supplement that might increase the performance of B. tryoni released in the SIT. Yeast hydrolysate is a rich source of amino acids, carbohydrate, sterols, vitamins and minerals, as well as indigestible fibre and water (Barry et al. 2007; Lee et al. 2008; Chang 2009) and is used as a source of adult nutrition in mass-rearing facilities for most tephritid flies. Numerous studies have investigated the potential value of YH as a supplement for other tephritids, including Mediterranean fruit flies (Yuval et al. 2002, 2007; Shelly and McInnis 2003; Shelly and Edu 2008; Gavriel et al. 2010), Anastrepha ludens (Loew) (Mexican fruit fly), Anastrepha obliqua (Macquart) (West Indies fruit fly), Anastrepha suspensa (Loew) (Carribean fruit fly) (Aluja et al. 2001; Teal et al. 2007; Liedo et al. 2013), Bactrocera dorsalis (Hendel) (Oriental fruit fly) (Shelly et al. 2005) and Bactrocera cucurbitae Coquilett (melon fly) (Haq et al. 2010a,b). In a pupal release study, Dominiak et al. (2003) reported attraction of emerging sterile B. tryoni to a mixture of water, sucrose and YH, indicating that nutrition is high on the priorities of newly emerged adult B. tryoni. Finally, we highlight additional research that would help to determine what pre-release treatments should be provided to B. tryoni in SIT programmes.

Queensland Fruit Fly Nutrition in Nature

Adult fruit flies generally require carbohydrate, amino acids, minerals, sterols, vitamins and water (for reviews, see Bateman 1972; Fletcher 1987; Drew and Yuval 2000). Despite the importance of nutrition in the biology of tephritid fruit flies, the question of how fruit flies, including B. tryoni, obtain these nutrients in nature remains poorly resolved. In the context of the present review, it is important to understand the availability of nutrition in the field to ascertain the potential benefits of pre-release supplements in the SIT.

Drew and Yuval (2000) have suggested that the most likely food sources for Dacinae are extrafloral exudates, plant leachates and bacteria of the family Enterobacteriaceae. Bactrocera tryoni have been reported foraging on fruit and over plant surfaces (Drew et al. 1983; Drew and Lloyd 1987), and it is likely that they fit this general profile. Other fruit flies have been reported to also feed on honeydew, pollen, bird faeces and yeast (e.g. Hendrichs et al. 1993; Manrakhan and Lux 2006), and these cannot be discounted as at least occasional elements of the B. tryoni diet in the field. Weldon and Taylor (2011) have found that wild and domesticated B. tryoni will forage on bat guano, bird faeces and pollen in the laboratory but, when provided as a supplement to sugar, none of these natural foods provided significant improvements in reproductive development or longevity. Natural diet is an area in need of substantial further research.

Evidence from feeding behaviour, morphology, crop contents and faeces suggests that bacteria are especially important in the nutritional ecology of adult B. tryoni (Drew et al. 1983; Lloyd et al. 1986; Drew 1987; Drew and Lloyd 1987, 1991; Prokopy et al. 1991; Murphy et al. 1994; Vijaysegaran et al. 1997; Thaochan et al. 2010) as they also appear to be for larvae (Fitt and O’Brian 1985). Drew et al. (1983) maintained mixed sex groups of flies with sugar and water alone or supplemented the diet of separate groups with bacterial cultures from the crops of field-collected adult flies, bacterial cultures from leaf surfaces or YH and found that each of these supplements increased oviposition rate substantially (75–900 fold, depending on supplement). Suitable bacteria may sometimes be in short supply in the field, and low availability of bacteria as food has been suggested to sometimes constrain wild populations (Courtice and Drew 1984). To understand the nutritional challenges faced by mass-reared sterile flies released in SIT programmes, it would be particularly valuable to have greater understanding of the conditions under which key elements of diet might be limiting in the field and how food availability varies in time and space.

Domestication and Mass Rearing

The nutritional requirements of mass-reared B. tryoni may be quite different from those of their wild counterparts, especially once irradiated, and this must be kept in mind when comparing studies. Rather than the fruit juices, floral exudates, plant leachates and bacteria eaten by wild flies, in domesticated colonies, the adult flies are typically provided water, sugar as a source of carbohydrate and YH as a source of amino acids and other nutrients (Bateman 1972; Meats and Leighton 2004; Meats and Kelly 2008). Meats et al. (2004) investigated the changes that take place in nutritional responses of female B. tryoni from the initial establishment of new colonies until complete domestication. During the domestication process, the rate of YH consumption more than doubled and the efficiency with which this intake was converted to eggs increased more than fourfold. That is, they became much more efficient at converting YH into eggs. The transition from wild to mass-rearing adult diet may yield flies that are better equipped for life in mass-rearing colonies but adaptation for artificial diets could render them less equipped to survive, develop and perform on food available in the field.

One way to reduce the nutritional challenge faced by released flies might be to rear flies with diets containing some key elements of natural diet. Meats et al. (2009) considered whether it might be possible to decrease the intensity of diet-related selection on females in new colonies by providing bacteria as primary food, supplements, stimulants or attractants. Bactrocera tryoni colonies of different ages were provided two different bacteria of the gram-negative family Enterobacteriaciae, Klebsiella oxytoca and Klebsiella pneumoniae, both of which had been previously isolated from wild and laboratory B. tryoni populations. Although findings of Drew et al. (1983) suggest that these bacteria should be a good source of nutrition, and even as effective as YH for supporting development and fecundity, Meats et al. (2009) found no evidence that these bacteria enhanced development or oogenesis when offered with sucrose or provided any benefit when provided as a supplement to the standard mix of sucrose and YH.

Much remains to be learned about B. tryoni dietary requirements and how they are met in the field by wild flies and by released sterile flies. How capable are domesticated flies released in SIT operations at locating and utilizing natural sources of nutrition in the field after many generations of severe adaptation for YH? Might the brief pre-release holding period be sufficient for dietary supplementation with YH to significantly influence reproductive development and sexual performance of flies released during SIT application? Recent studies of reproductive development and sexual performance are promising.

Reproductive Development

Provision of YH in addition to sucrose continuously from adult emergence throughout adult life has a strong positive effect on the development of reproductive tissues in both male and female B. tryoni (Vijaysegaran et al. 2002; Weldon and Taylor 2011). Whereas YH-fed females show a rapid increase in growth of ovaries and accessory glands and accumulation of fat around spermathecae, females provided only sugar and water show negligible development of these reproductive tissues. Similarly, compared with YH-fed males, YH-deprived males had slower development and smaller accessory glands and erecting and pumping organs, and weaker odour release upon rupture of the pheromone reservoir.

While male and female B. tryoni both benefit from the inclusion of YH in their diet, females have greater need (Drew 1987; Pérez-Staples et al. 2007b, 2009). Drew (1987) set up laboratory cages containing groups of 3-week-old YH-fed males and YH-deprived males for 3 days to mate with 3-week-old YH-fed females or YH-deprived females, and fertility of eggs laid at the end of this period was then assessed. While treatments including YH-deprived females produced no eggs, treatments including YH-deprived males showed high levels of egg fertility. Drew (1987) concluded that females require protein from YH to produce eggs, whereas males are fertile regardless of diet. While the absence of statistical comparisons prevents firm conclusions, the values presented in tables suggest that groups of males denied access to YH do suffer a modest reduction in fertility in comparison with groups in which males had received even small amounts of YH. In such group studies, it is not possible to infer whether this trend stems from failure of certain nutrition-dependent males to mature (i.e. a smaller proportion of males were mature in these cages and so some eggs were laid by virgins) or from reduced performance of sexually mature males (i.e. proportion of males maturing was similar in the presence and absence of YH, but ejaculate of YH-deprived males was deficient) or a combination of these explanations. Although they may need less than females, dietary supplements of YH do enhance development of male reproductive organs (Vijaysegaran et al. 2002; Weldon and Taylor 2011) and sexual activity. Exploring the design of Drew’s (1987) experiment provides some additional insights into why effects of YH in the diet were found in female fecundity but were not apparent in male fertility. Although the flies were maintained in single sex groups on one diet in the period prior to mating, they were allowed to interact freely with YH-fed flies of the opposite sex for 3 days before eggs were collected. The YH-fed flies would have defecated during this time, making more complete meals available to the flies of the YH-deprived treatment (for discussion of this issue in studies of sexual competition between YH-fed and YH-deprived tephritids, see Blay and Yuval 1997; Shelly et al. 2006). If male B. tryoni have a substantially smaller need for nutrition from YH, this may have been sufficient time for males to obtain enough resources to attain sexual maturity and to be able to mate and transfer sperm to females, but insufficient time for most females to become fecund. The lower nutritional needs reported for males are very promising for the SIT as it suggests that males of bisex strains released in the field should be more likely than females to secure sufficient nutrition to complete reproductive development.

Male-only releases can be far more effective than bisex releases at inducing reproductive failure in wild populations (Vreysen et al. 2006). This is because the released males in male-only releases are all focussed on competing for copulations with comparatively few wild females, whereas the released males in bisex releases might mostly copulate with the millions of released females. However, if the released sterile females have comparatively poor development rates, then bisex releases may effectively operate more like male-only releases than the released 1 : 1 sex ratio might suggest (for an interesting parallel case of male-biased development enhancement from methoprene supplements in Anastrepha fraterculus, see Segura et al. 2009).

Given their lower dietary requirements, it seems that released males are more likely than females to mature in the field regardless of pre-release provisioning with YH. However, the lower dietary requirements of males suggest that it might be possible to use YH supplements during the 24-to 48-h pre-release holding period to achieve substantial enhancements in reproductive development of males without triggering nearly the same effect for females. As long as these effects are borne out in patterns of sexual activity, then pre-release supplementation with YH has the potential to not only increase the number of sexually active males but also skew the operational sex ratio in favour of males. Studies of sexual activity are key.

Sexual Activity

Bactrocera tryoni are sexually active only in the evening, initiating copulations during approximately 30 min around dusk (Barton-Browne 1957; Tychsen 1977). Males call and court females with a complex of acoustic, pheromonal and visual signals (Bellas and Fletcher 1979; Mankin et al. 2008).

Matching their data for reproductive development, Vijaysegaran et al. (2002) also investigated the effects of ad libitum access to YH on the emergence of sexual activity. In a series of experiments carried out when flies were two or three weeks of age, Vijaysegaran et al. (2002) found (i) in laboratory cages containing YH-fed and YH-deprived males and females, most matings were between YH-fed pairs, suggesting that YH is important for the sexual maturation of both sexes; (ii) cages of YH-fed males competing with YH-deprived males for matings with YH-fed females showed a similarly strong advantage for YH-fed males at both 2 weeks and 3 weeks of age; and (iii) when tested at 20 days of age, males fed YH for just the last 10 days were as sexually competitive as males fed YH throughout their lives, indicating that male B. tryoni can recover from early nutritional deprivation.

Pérez-Staples et al. (2007b) compared the mating tendency of YH-fed and YH-deprived males and females that were paired individually with sexually mature YH-fed partners. Importantly, this study included both fertile and sterile flies. From day 5, virgin YH-fed sterile and fertile flies of both sexes showed a marked increase in mating tendency until peaking at 10–12 days of age. On the other hand, YH-deprived flies of all groups showed little increase in sexual receptivity over this period. While males were sexually active slightly sooner than females, the difference was very small and the overall patterns were similar for the two sexes and for fertile and sterile flies. Even at 30 days of age, YH-deprived males and females showed quite low levels of mating; 20–40% vs. 70–100% for YH-fed flies.

Following on from Pérez-Staples et al. (2007b), Prabhu et al. (2008) considered the effects of various proportions of YH and sugar on sexual activity of male B. tryoni. Over the first 2 weeks, as was also found by Vijaysegaran et al. (2002) and Pérez-Staples et al. (2007b), males receiving no supplement rarely mated (<25%). In contrast, all levels of supplement from 9% to 83% of total diet weight showed a rapid increase in mating tendency from 6 days of age. Interestingly, flies fed diets containing 50% YH or more then suffered a sharp decline in mating tendency from 10 days of age and flies fed only YH never mated. Flies fed such high proportions of YH in their diet tend to die at a very young age, and so this reduction in mating tendency is most likely an indication of pathology. In addition to clear advantages for YH-fed males in mating probability, Pérez-Staples et al. (2007b) and Prabhu et al. (2008) both found that YH-fed males mated sooner after the onset of dusk (decreased ‘mating latency’) than YH-deprived males, suggesting either that they were more prompt or persistent in their efforts or that females resisted mounting attempts of YH-deprived males for longer. Detailed behavioural observations of calling, courtship (see Mankin et al. 2008) and mating of YH-fed and YH-deprived flies are required to address this question.

It is clear that if released flies fail to find sufficient nutrition in the field, then very few will engage in the sexual competition that is required for effective SIT application. If nutrition is at least sometimes a limiting factor (see Courtice and Drew 1984), this leads naturally to the question of whether the 1- or 2-day period during which flies are held before release can be sufficient for YH supplements to have a lasting effect on sexual performance of flies released in SIT. Recent work of Pérez-Staples et al. (2008, 2009) and Weldon et al. (2008) has addressed this question, with compelling results.

In a series of laboratory experiments, Pérez-Staples et al. (2008) assessed sexual activity of fertile male B. tryoni that had been fed only sugar and water or had been supplemented with 24 or 48 h access to YH and then restricted to sugar. In a close reflection of earlier studies in which flies had been supplemented continuously, males provided this simulation of pre-release access to YH supplements had a massive mating advantage over their YH-deprived counterparts, especially when young. While 48 h of supplementation provided some added benefits over 24 h of supplementation at the earliest ages tested (8 and 12 days), this gap closed as the flies aged and converged at around 20 days. Similar to previous laboratory studies of Pérez-Staples et al. (2007b) and Prabhu et al. (2008) in which males with continuous access to YH had short mating latency, Pérez-Staples et al. (2008) found shorter mating latency for males that had only 24 or 48 h access to YH.

Pérez-Staples et al. (2009) extended this line of research closer to operational SIT, investigating the ability of YH-fed and YH-deprived sterile mass-reared flies in mating competition with wild flies in field cages containing natural foliage. This study is important because it is the only one so far to consider the effects of dietary supplementation on sterile B. tryoni mating performance in outdoor conditions and in competition with wild type flies. Simulating the bisex B. tryoni releases, both males and females of each group were released. Sterile flies had either no access or 48 h access to YH, whereas wild flies had continuous access to YH. Sterile males that had received only sugar did very poorly, attaining only 12% of matings, but sterile males that received 48 h of access to YH immediately after emerging and only sugar thereafter secured as many matings (50%) as wild males that had received YH throughout their lives (38%). Because there is no unisex strain of B. tryoni, both sexes are released in SIT programmes and the massive abundance of sterile females has the potential to substantially dilute the effect of SIT (Vreysen et al. 2006). While there is currently no method available to bias the actual sex ratio of released flies in favour of males, the results of Pérez-Staples et al. (2009) do lend support to pre-release YH supplementation as a potential method for biasing the operational sex ratio of released flies in favour of males in the field.

As well as being an important prerequisite for released sterile males to participate in mating and control of wild populations, sexual maturity is also important in monitoring. Cue-lure is the main attractant used to monitor wild and released B. tryoni populations (Meats 1998a,b). Males are only attracted to cue-lure once sexually mature and perhaps for several days beforehand, and so recapture rates reflect not only prevalence but also sexual maturity of a monitored population. Recapture rates of sterile B. tryoni are highly variable and generally very low (Meats et al. 2003, 2006). One possibility is that rather than reflecting poor survival, these low capture rates instead reflect poor rates of sexual maturation, possibly as a consequence of released flies failing to find sufficient nutrition in the field. Given that a brief period of post-teneral YH feeding has a strong positive influence on sexual maturation, it seems likely that pre-release YH feeding would increase recapture rates in cue-lure traps. Weldon et al. (2008) investigated this in field cages and found that males provided just 24 or 48 h of access to YH, or continuous access, were caught in cue-lure traps much more often than were males denied access to YH. These results were consistent for fertile and sterile males.

Copula Duration

While mating between released sterile males and wild females is an essential step for the SIT, it is by no means the last. There are still challenges that males might fail to overcome after the onset of copulation, and these have received far less attention than the steps leading up to copulation. A copulation that makes no impact on lifetime fecundity or fertility of wild females is of no value to the SIT.

Copula duration has not been linked to any measures of post-copulatory performance, such as sperm transfer, fertility or female sexual inhibition, but it is nonetheless highly variable and the possibility of relationship to ultimate success cannot be ruled out. For example, long copulation may serve as mate guarding to prevent females from remating on the same evening or may increase paternity should females remate. Copula duration appears to be largely under female control in B. tryoni (Pérez-Staples et al. 2010), and so to the extent that copula duration is linked to post-copulatory success, wild females could discriminate against sterile males by constraining copula duration.

Three studies have considered the effects of post-teneral diet on copula duration, and these have been highly consistent in their findings. In the first of these, Pérez-Staples et al. (2007b) found that YH-fed flies had much longer copulations than YH-deprived flies. While the effects were stronger for males than for females over the first 2 weeks, at later ages the sexes showed similar effects. Of relevance to SIT application, all effects of diet on copula duration reported by Pérez-Staples et al. (2007b) were similar for fertile and sterile flies. Prabhu et al. (2008) only considered fertile males, but found results very similar to those of Pérez-Staples et al. (2007b). From ages 6–14 days, males that were allowed to choose freely between sugar and YH or were restricted to diets containing between 9% and 83% YH all had copulations lasting on average 90–100 min, whereas males provided no access to YH had copulations that lasted on average only approximately 30 min. Pérez-Staples et al. (2009) also considered only fertile males. Rather than manipulating proportions of YH in the diet, they assessed the performance of males that had been provided only sucrose or had been provided access to YH for 24 or 48 h after emerging and provided only sugar thereafter. Across all ages tested (6–28 days), males supplemented with YH for 48 h had longer copulations (mean 83 min) than males supplemented for only 24 h (mean 63 min), and both supplemented groups had longer copulations than males fed only sucrose (mean 40 min).

Sperm Storage

Bactrocera tryoni have two major sperm storage organs, a pair of spermathecae and a ventral receptacle (=bursa copulatrix). The spermathecae resemble those described in detail for the olive fly, Bactrocera oleae (Gmelin) (Dallai et al. 1993). They are used for long-term storage and contain the large majority of sperm (Pérez-Staples et al. 2007a). The ventral receptacle appears to serve as a short-term storage organ, containing comparatively few sperm that are used to fertilize eggs (Pérez-Staples et al. 2007a).

Several studies have highlighted that mates of sterile male B. tryoni store far fewer sperm than do the mates of fertile male B. tryoni (Harmer et al. 2006; Radhakrishnan et al. 2009b). It seems highly likely that low sperm numbers transferred by sterile males will mean higher fertility of wild females should they later remate with a wild male that transfers a normal complement of sperm. Accordingly, treatments that increase the success of sterile males at filling the female’s spermathecae with sperm might aid the SIT.

While not investigated directly, fertility studies of Vijaysegaran et al. (2002) implied effects of male diet on sperm storage of mates. Groups of 10 females were maintained with 40 males, various proportions of which had been fed only sucrose or supplemented with YH (male ratios 0 : 40, 10 : 30. 20 : 20, 30 : 10, 40 : 0). Higher fertility was reported for groups that contained relatively more YH-fed males, and this was interpreted as most likely reflecting superior sperm transfer perhaps as a consequence of the larger erecting and pumping organ of the more fully developed YH-fed males (Vijaysegaran et al. 2002). Pérez-Staples et al. (2008) is the only study to have directly investigated the effects of male B. tryoni diet (sugar only, supplemented with YH for either 24 or 48 h) on sperm storage by mates. Two aspects of sperm storage were investigated in the mates of fertile males; the probability of at least some sperm being stored and the number of sperm stored by mates of males that succeeded. Both measures of male success were strongly affected by diet. First, mates of males fed YH for 48 h were more likely to store sperm (88%) than were mates of males fed YH for just 24 h (79%) or provided no supplement (70%). Among females that stored at least some sperm, those mated by males provided either 24 or 48 h of supplementation stored far more sperm than those mated by males that had been denied supplements. These diet differences in probability of sperm storage and number of sperm stored persisted to 28 days, the oldest age of male flies tested.

While this is a very promising set of findings, especially given the persistence of effects induced by pre-release YH exposure that simulates the 24- to 48-h period available in current SIT practices, the interpretation of these findings from the perspective of SIT application is constrained because only fertile males have been considered in studies of dietary influences on sperm storage. Even when provided access to YH throughout their lives, sterile males transfer far fewer sperm than fertile males (Harmer et al. 2006), and because they are unable to replenish supplies, this difference becomes ever more exaggerated over sequential matings such that most sterile males transfer no sperm at all if they have mated one or more times previously (Radhakrishnan et al. 2009b). Because both sexes are released in B. tryoni SIT, males that mate with wild females may often have mated previously with a sterile female, and have severely diminished sperm supplies. Given the overwhelming effects of irradiation on sperm supplies, there may not be much scope for improving the SIT through the effects of diet on sperm transfer. Given the poor sperm capacity of sterile male B. tryoni, it is particularly important that females mated by sterile males exhibit low remating tendency. This is another challenge for released flies in which pre-release YH might play a role.

Remating Inhibition

While mating is more readily recognized as important for successful SIT programmes, the ability of males to prevent their mates from subsequently remating is almost as important; if wild females mated by sterile males later remate with wild males, then they will very likely succeed in producing viable offspring. Given the paucity of sperm stored by mates of sterile male B. tryoni (Harmer et al. 2006; Radhakrishnan et al. 2009b), this is a particular concern.

Pérez-Staples et al. (2008) investigated the effects of male diet on the remating tendency of their mates. Females that were mated by males that had been supplemented with YH for 24 or 48 h after emerging were less likely to later remate with another male than were females mated by males that had only received sugar and water. What mechanisms might link diet to remating inhibition? It could be that the low numbers of sperm stored by the mates of YH-deprived males are important. However, Harmer et al. (2006) and Radhakrishnan et al. (2009b) found no difference in remating tendency of females mated by fertile males (many sperm) and sterile males (few sperm), effectively dismissing a direct causal link between sperm storage and remating inhibition in B. tryoni.

On the other hand, Radhakrishnan and Taylor (2007) provide strong evidence supporting the involvement of accessory gland fluids transferred with the ejaculate as mediators of sexual inhibition (see also Radhakrishnan et al. 2008, 2009a). Materials from the male’s ejaculate pass through the female’s reproductive tract and disperse through her body while the pair mates (Radhakrishnan et al. 2008). Virgin females injected with extracts from male accessory glands exhibit sexual inhibition similar to that of mated females. After mating, male accessory glands are greatly diminished in size and recover their original size gradually over the following day (Radhakrishnan and Taylor 2008). These changes in accessory gland dimensions presumably reflect volume of contents, as fluids are transferred to females during copulation and then replenished in time for the next mating opportunity (Radhakrishnan et al. 2008). Virgin YH-deprived males have smaller accessory glands and smaller erecting and pumping organs (ejaculatory apodemes) than YH-fed males (Vijaysegaran et al. 2002; Weldon and Taylor 2011). If accessory gland size reflects the volume of contents (Radhakrishnan and Taylor 2008), it would seem that YH-deprived males suffer from low accessory gland fluid reserves. The poor performance of YH-deprived males at inhibiting female sexual receptivity is likely explained by a smaller supply (and perhaps lower quality) of accessory gland fluids and an incompletely developed pump with which to transfer them.

Longevity

It is important that flies released in SIT programmes survive long enough in the field to mature and to maintain pressure on wild populations between releases. Again, this is an area where YH may provide a means to enhance the SIT.

Drew et al. (1983) considered the effects of diet on longevity of mixed sex cages of B. tryoni. Compared with a diet containing only sugar and water, cages of flies supplemented continuously with YH or bacterial cultures showed similar, substantial, lifespan extension. Drew (1987) presented some similar experiments based on single sex cages of flies that received diets of sugar and water alone or were supplemented with continuous access to YH. These experiments showed a marked increase in the proportion of both male and female B. tryoni surviving to 28 days of age when diets were supplemented with YH.

Pérez-Staples et al. (2007b) began the task of exploring the potential of YH supplementation to enhance longevity of sterile B. tryoni used in SIT programmes. In addition to comparing the effects of YH on longevity of fertile males and females, as had been carried out by Drew et al. (1983) and Drew (1987), Pérez-Staples et al. (2007b) also considered the effects of irradiation in both sexes. Longevity of fertile males was not affected by continuous YH supplementation (unlike Drew 1987 and Prabhu et al. 2008 who both found male longevity was increased by YH), whereas longevity of fertile females was substantially increased. Of particular note for SIT application, the longevity response of sterile flies was somewhat different from that of their fertile counterparts. For both sexes, sterile flies had much shorter lives than fertile flies when fed sucrose only but showed a greater increase in longevity when provided YH to the extent that longevity of sterile flies provided YH was very similar to that of fertile flies provided YH. Alternatively stated, sterile flies suffered a much greater reduction in longevity when deprived of YH. Hence, irradiation greatly increased the sensitivity of both male and female B. tryoni to nutritional deprivation. Pérez-Staples et al. (2007b) remains the only study to have considered the effects of YH supplementation on longevity of sterile B. tryoni; this is an area that clearly warrants further investigation. In the meantime, there are further studies of fertile flies that provide some progress.

In a subsequent laboratory study that more closely approximates the pre-release context of SIT programmes, Pérez-Staples et al. (2008) considered the effects of 24 or 48 h of post-teneral access to YH on longevity of B. tryoni males. Both of these brief periods of supplementation yielded substantial increases in the proportion of males surviving to 28 days of age (approximately 90%) when compared with males that received only sugar and water throughout their lives (approximately 60%). Given the greater sensitivity of sterile flies to nutritional deprivation (Pérez-Staples et al. 2007b), it would be very interesting to see this experiment repeated for sterile males as it seems highly likely that they would show even greater differences. Further, given the absence of a single sex B. tryoni strain, it would also be interesting to compare results for males and females given 24 or 48 h access to YH. It is conceivable that a brief exposure to pre-release YH supplementation might enhance survivorship of sterile males significantly more than females and thereby bias the released population in favour of males through differential attrition.

While there are clear potential longevity benefits of YH supplementation under laboratory conditions, it is possible to provide too much, and this needs to be considered in operational settings. Prabhu et al. (2008) restricted small groups of male B. tryoni to dry diets containing various ratios of sugar/YH and found marked reductions in longevity if diets contained more than 50% YH. Using liquid diets containing fixed ratios of these same nutrients, Fanson et al. (2009) similarly found a marked reduction in longevity of female B. tryoni that were restricted to diets containing high proportions of YH. Given the ample capacity of B. tryoni to self-regulate intake to maintain nutritional targets when allowed to choose freely between separate sources of sugar and YH (Fanson et al. 2009), in operational conditions, the safest option may be to provide these dietary components separately.

Results of these laboratory studies of longevity are encouraging, but further research is still needed. In particular, there is a need for more data on how pre-release protein supplements influence survivorship of sterile B. tryoni in laboratory, field cage and field settings. Given that YH supplements can increase vulnerability of tephritids to starvation (Kaspi and Yuval 2000; Maor et al. 2004; Yuval et al. 2007; Gavriel et al. 2010), there is also a need for data on the consequences of pre-release diet supplements on the ability of B. tryoni to tolerate periods of nutritional deprivation and other stress in the field.

Meats et al. (2003) held sterile B. tryoni for a week in large cages with access to YH in addition to sugar and water, bringing them close to sexual maturity before release. Interestingly, recapture rates were very low in cue-lure traps. Given the link between sexual maturation and attraction to cue-lure (Weldon et al. 2008), high recapture rates might be expected with such a pre-release treatment. While stress during the holding period or unsuitable weather are possible explanations, another possibility is that pre-release YH treatment rendered the sterile males more vulnerable to nutritional deprivation after release. From studies of Pérez-Staples et al. (2008), we know that B. tryoni provided YH for 24 or 48 h and then only sugar thereafter show good longevity in the laboratory, but substantial further work is required to ascertain the effects of supplementation should flies be unable to find adequate sources of carbohydrate and/or water in the field. There is a need for further studies investigating the field nutritional ecology of released sterile B. tryoni (and other tephritids) both to ascertain the need for supplements and also to ascertain the risks of inducing a physiological state that leaves them vulnerable.

Summary and Future Directions

The emerging picture of benefits from pre-release YH supplementation in B. tryoni SIT is a promising one. However, much still remains to be done to more fully assess the benefits and to assess potential trade-offs, such as increased vulnerability to starvation should food prove limiting in the field. There is also a need for experiments that bridge the current gaps between our understanding of nutrition in basic B. tryoni biology and the application of this knowledge to the SIT. Many experiments have been carried out with just one sex or with fertile flies only. While these studies have brought substantial progress in the past decade, studies are still needed to place this work more firmly in the context of operational SIT. At this time, there is also a need to move further towards the field setting. Much of the work to date on development and sexual performance needed to be carried out under the controlled context of the laboratory, but there is now a need for further field cage studies similar to those of Pérez-Staples et al. (2009) to explore development, behaviour, mating performance and survival under semi-natural conditions. Finally, field studies will provide the ultimate test of whether pre-release dietary supplementation enhances sterility induction in B. tryoni SIT programmes.

We have focussed here on YH supplementation, but work on other tephritids has highlighted other supplements that also promise benefits and should be investigated in B. tryoni. First, while data for the effects of bacterial supplements in B. tryoni have been mixed (Meats et al. 2009), there is some suggestion that probiotic diets could enhance performance of sterile Mediterranean fruit flies (Niyazi et al. 2004; Ben Ami et al. 2010; Gavriel et al. 2011) and this possibility deserves further attention in B. tryoni.

Mature male B. tryoni (3 weeks old) that feed on raspberry ketone or cue-lure incorporate raspberry ketone in the pheromone glands and are sexually advantaged (Tan and Nishida 1995). Perhaps pre-release supplements containing raspberry ketone or cue-lure might enhance mate attraction ability of male B. tryoni. Bactrocera fruit flies can be broadly classified in their responses to raspberry ketone/cue-lure and methyl eugenol as lures. Oriental fruit fly responds to methyl eugenol, and pre-release dietary supplements of methyl eugenol and methyl eugenol-containing plant material have been found to enhance sexual performance in this species (Shelly et al. 2005, 2010; Shelly and Edu 2007). Perhaps, similar responses to raspberry ketone might be expected in B. tryoni. Access to fruits has been found to be beneficial in some tephritids (e.g. Anastrepha spp. Aluja et al. 2001; Liedo et al. 2013). Beneficial effects from fruit may be explained by nutrition from the fruit, bacteria or semiochemicals. Fruits, and other likely elements of natural diet, merit some consideration as potential dietary supplements.

Juvenile hormone, or its analogues methoprene and fenoxycarb, has been demonstrated to accelerate maturation in numerous Anastrepha species and melonflies (Teal et al. 2007; Pereira et al. 2009, 2010; Haq et al. 2010a,b; Segura et al. 2013). Apart from one promising pilot study (see Smallridge et al. 2008), the potential value of pre-release treatment with juvenile hormone products, in conjunction with nutritional supplementation, has not been investigated in B. tryoni and should be a high priority.

Finally, there is even a possibility that the olfactory environment during pre-release holding could be manipulated to enhance sterile male performance. In Mediterranean fruit flies, ‘aromatherapy’ with products containing α-copaene (e.g. ginger root oil, angelica seed oil) has been found to substantially enhance male sexual performance (Shelly 2001; Morelli et al. 2013) and positive effects on the SIT efficacy have been reported from the field (Shelly et al. 2007). There has been no investigation into the potential of aromatherapy in B. tryoni, but the promising results for Mediterranean fruit flies suggest that this is an area that could hold some value for B. tryoni as well.

Acknowledgements

Our research reviewed in this paper was facilitated by funding from Horticulture Australia Ltd (HAL) in partnership with the Australian Apple and Pear, Cherry, Vegetable, Melon, Table grape and Summerfruit industries (HG06040), and the Citrus industry (CT05002). The Australian Government provides matched funding for all HAL R&D activities. Diana Pérez-Staples was supported by a UNESCO-L’Oréal Co-sponsored Fellowship for Young Women in Life Sciences. For helpful comments on earlier drafts, we thank Bernie Dominiak, Katina Lindhout and Boaz Yuval.

This article was published online on 1 June 2011. The references for Liedo et al. (2013), Morelli et al. (2013) and Segura et al. (2013) have now been updated to show the correct citation details for J. Appl. Entomol. Vol. 137, Suppl. 1 and the reference for Gavriel et al. (2011) has been updated to show the correct citation details for J. Appl. Entomol. Vol. 135.

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