Why do females prefer elaborate male ornaments and displays in species where females receive only ejaculates from males? Under the pervasive assumption that "fertility is seldom likely to be limited by sperm production" , indirect genetic benefits are typically evoked. This has resulted in the relative neglect of an obvious alternative, that females choose ornamented males to ensure they receive sufficient sperm or other components of the ejaculate to fertilise their eggs.
In T. dalmanni, eyespan and all other external morphological traits are permanently fixed in size shortly after eclosion  and cannot change in response to the adult environment. In contrast, the accessory glands and testes only start to develop after eclosion. In this paper, we established a range of adult nutritional stresses which affected the size and growth rate of the male accessory glands and testes. We then investigated variation in male eyespan (after controlling for body size), the principal trait subject to female mate preference [18–20]. Despite this mismatch in developmental periods, male eyespan was a strong predictor of internal reproductive organ size. How can this association be explained?
Previous work on T. dalmanni has demonstrated that male eyespan is highly sensitive to stress during the larval stage [21, 23], and certain genotypes are capable of producing large eyespan across a range of larval environments . In more stressful environments, variation in eyespan is greater  and causes an amplification of genotypic differences between males . These results are consistent with the handicap principle, which assumes that high quality males pay lower marginal costs for the production of larger eyespan than do low quality males .
Could male internal reproductive organ size be subject to similar condition-dependent costs? The handicap principle predicts that under harsh (0% corn) adult conditions, high quality males should still be able to develop large accessory glands and testes, whereas low quality males will be unable to do so (because of the higher marginal cost of reproductive organ size development felt by low quality males). But under benign adult conditions, all males will get access to sufficient resources independent of their quality. So differences between low and high quality males in the size of accessory glands and testes should be reduced under benign (75% corn) adult conditions (because the marginal cost of reproductive organ size development is much more similar in low and high quality males). Assuming that male eyespan is an indicator of quality, the correlation between eyespan and internal reproductive ornament size should decline as adult conditions become less stressful. Our results do not support this prediction. Large eyespan males consistently developed larger accessory glands and testes than small eyespan males under high (0% corn), medium (25% corn) and low (75% corn) adult food stress (Fig 1). We also can be sure that the 75% corn treatment was benign as our preliminary experiment showed the same growth profile for accessory glands and testes for both less (50%) and more (100%) corn (Additional file 1).
An alternative possibility is that the level of larval stress induces long lasting physiological changes that affect the allocation of resources to reproduction and survival in adults (e.g. ). In our study, high quality males that invested in larger eyespan, may thereby have adopted a life history which requires the production of larger accessory glands and testes. In T. dalmanni, males with larger eyespan are successful in agonistic interactions with other males  and are attractive to females [18–20]. They mate more frequently than males with smaller eyespan during both the dusk and dawn mating periods, and so benefit more from greater investment in their reproductive organs. Larger accessory gland size is strongly associated with higher male mating frequency [13–15] and larger testes presumably allow high mating males to maintain sperm numbers per ejaculate. This may explain why, even in benign conditions of ad libitum adult food supply, we still found that males with larger eyespan had larger accessory glands and testes.
In line with this explanation, we found that female fertility was positively correlated with the eyespan of their mating partners. In our assay a single large or small eyespan male was placed with 8 females for 24 hours and we recorded the number of fertile eggs laid by the group of females over the next 30 days, by which time most females would have exhausted their sperm stores  (note, we again controlled for body size variation of females in this experiment). This assay gives an indication of the number of sperm successfully transferred when a male has the opportunity for multiple mating. The number of fertile eggs laid increased approximately 8% per millimetre increase in male absolute eyespan. These findings suggest that female preference for large eyespan males potentially could result in higher fertility under natural conditions. In T. dalmanni, several females settle on nocturnal aggregations controlled by a single male [12, 20, 43, 44]. The lek holding male mates with the females in his aggregation at dusk and at dawn prior to female dispersal. This corresponds to the conditions in our assay in which fertility was assessed for a single male kept for a limited time with multiple females.
However, we should be cautious about this interpretation. It is unclear what mechanism contributed to the fertility advantage gained by females in our assay. The greater fertility of large eyespan males probably reflected their larger accessory glands which are associated with higher mating frequency, and larger testes which are positively correlated with the number of sperm stored by females following a single copulation . So the group of females housed with large eyespan males probably collectively mated more and received larger amounts of sperm, and as a consequence had higher fertility. However, our experiments do not allow us to distinguish the importance of extra matings, the transfer of more sperm (or other substances) per mating, female effects or other factors contributing to the elevated fertility of large eyespan males. In addition, meiotic drive in T. dalmanni is likely to exaggerate differences in fertility in nature and may play an additional selective role with respect to female preference if its occurrence is linked to male eyespan  (flies in our experiment lacked any meiotic drive). Further experimentation is required in order to establish the exact mechanism of fertility enhancement by large eyespan males and its relevance to the mating system of T. dalmanni in the wild. Our results contrast with a previous study which found no association between male eyespan and the number of fertile eggs laid following a single mating . However, in that study, females were allowed only a single copulation with a large or small eyespan male, whereas multiple mating is typical in this species .
Of wider importance, attractive, large eyespan males are likely to suffer from greater ejaculate and sperm depletion due to having more mating opportunities in the dawn and dusk mating periods. Such a loss of male fertility occurs in several other species in which males mate multiply over short periods of time (e.g. [47, 48]). Although the reduced fertility of male T. dalmanni following mating is known to recover across days , this must present an evolutionary dilemma for females as fertility assurance may be higher with an unattractive male rather than with an already mated attractive male. Further understanding of the trade-off between fertility and number of matings is needed to identify the fertility benefits arising from mate choice.