Here, I first considered the validity of the P2 value obtained in this study. If the gamma ray radiation successfully sterilized males without damaging other traits except for the post-fertilizing potential of the sperm, the P2 value must be constant regardless of the mating order (whether the last male is R or NR). However, the results in this study were not so, but instead showed that the gamma ray radiation damaged the viability of the sperm or male. Therefore, I averaged the two P2 values and analyzed the sperm precedence of B. dorsalis based on the averaged value, though it is rather qualitative. The mean P2 value (0.7) indicates that the last transferred sperm was used predominantly for fertilization. Considering the poor fertilizing ability of the R male, the averaged P2 value is probably underestimated, and sperm of the last mated male must be used at a higher probability. I could not determine whether the females chose larger males which may afford to invest more solely from the mating behavior exhibited in the female choice experiment. Namely, the classical and behavioral criterion on mate choice could not detect female choice in B. dorsalis. On the other hand, females that had received more investment (seminal fluid) refrained from mating for a longer period (Fig. 2). B. dorsalis females lay eggs one at a time throughout their life time (Takakura, unpublished data). Therefore, the longer mating interval, resulting from greater investment by males, will lead to predominant use of the sperm of greater investing males. Furthermore, as shown in Fig. 2, the refractory period gained per weight of seminal fluid became longer with heavier seminal fluid; because the intercept is negative, the increase is always positive but gradually decreases to zero. In addition, a smaller amount of seminal fluid (0.5 mg) did not prolong the refractory period at all. All these facts explain why B. dorsalis males produce exclusively large amounts of seminal fluid. On the contrary, if males invest less but copulate many times, the mated females should remate soon and fertilize disproportionally fewer eggs by sperm deriving from less investing males. In short, B. dorsalis females did not choose greater investing males as their mates on a behavioral basis, but they preferentially used sperm of greater investing males by prolonging the refractory period according to the amount of investment. In nature, it is probably difficult for the females of low density species like B. dorsalis to choose the greater investing male after sequentially comparing multiple males. Furthermore, females seem to hardly assess the male’s investing potential accurately only by the semblance. In such a situation, the mechanism of post-copulatory female choice as shown in this study is an adequate strategy, which enables the females to choose sperm of greater investing males on a simple basis. Adopting this mechanism, the females can decide how much and whose sperm is used even after they receive the investment. Moreover, a deceitful signal or cheating by males may be difficult to evolve. In fact, the different gradient between the 2 feeding treatments of males in Fig. 2 and the significant interaction effect in Table 1 revealed that poorly-fed males could prolong the refractory period of the mated females far less than well-fed males could even if the same amount (weight) of ejaculate was invested. This implies that females can assess not only the amount of seminal fluid but also the nutrients contained. The post-copulatory assessment of the investment may enable B. dorsalis females to respond accurately. It is not known how B. dorsalis females assess the amount and the quality of seminal fluid. It was reported in some insect species that nutrients of seminal fluid gradually assimilated into hemolymph (e.g. Huignard, 1983). If such is the case in B. dorsalis, females should remate when the size of the spermatheca decreases to at some threshold level, i.e. the mating interval is the time spent for assimilation of the seminal fluid. There is no evidence that justifies this hypothesis, but it can explain why females received low concentration seminal fluid remate sooner because such seminal fluid may be assimilated in a shorter time. The hypothesis, furthermore, explains the phenomenon whereby females that had received little seminal fluid (0.5 mg) remated soon, because the volume of the spermatheca was below the threshold.
For studies on female choice, however, this type of post-copulatory female choice is bothersome (Birkhead, 1998). The choice can not be detected from mating behavior alone. Observations must be done not only on the mating behavior but also on post-copulatory sperm use to elucidate the female choice, particularly, for species where females mate multiply.
In courtship-role-reversed insects, investing males are favored either by pre-copulatory female choice (in a katydid, Gwynne, 1982), by prolonging mating to transfer more sperm (in a scorpion fly, Thornhill, 1976), or by repeated mating (in a giant water bug, Smith, 1979). Pre- or post-copulatory guarding of females may be effective for ensuring paternity, and is easy to observe. On the other hand, observation of mating behavior did not reveal post-copulatory female choice as in B. dorsalis. In this sense, the process of female choice in B. dorsalis is “cryptic” (Thornhill, 1983, 1984).
Nevertheless, this process partly depends on female behavior and thus can not be called cryptic female choice in the sense of Birkhead (1998). Finally, sex-role-reversal in courtship is known for many species of insects, and the evolutionary and ecological background of the phenomenon has been intensively studied (e.g., Gwynne, 1981, 1982). In spite of the accumulated knowledge, the evolutionary origin of courtship-role-reversal is still vague. To tackle the issue, it is critical to distinguish the two types of male investment: mating effort to attain greater mating success and paternal effort to enhance offspring fitness, though these are not necessarily mutually exclusive. If the former is the case, courtship-role-reversal may have evolved through sexual selection. If the latter is the case, courtship-role-reversal may have derived from a trade-off between male mating success and paternal investment. Huignard (1983) reported that in a bean weevil (Acanthoscelides obtectus), nutrients in the seminal fluid were hardly assimilated into eggs within 24 h, but were assimilated maximally at 36 h after copulation. If this is true, B. dorsalis males can not nurture their own offspring by the seminal fluid, because the mating interval was approximately 20 h at longest. In other words, B. dorsalis males may sire eggs to which other previously mated males provided nutrients. Thus, male investment in B. dorsalis is probably a mating effort not a paternal effort, and has been favored through high reproductive success of males as shown in this study but not high fitness of offspring. This process in turn has promoted the evolution of cryptic female choice for greater investing males, though the contribution to offspring fitness by paternal investment remains to be fully examined. In other words, the current lines of evidence support the evolutionary scenario that courtship-role-reversal in B. dorsalis has derived from the interplay between malemale competition over greater mating success and cryptic female choice for greater male investment. Thus, the direct factor of courtship-role-reversal in B. dorsalis could be suggested from the view point of intrasexual competition. However, it is not known why the sex role in courtship is reversed only in B. dorsalis, whereas nutritional donation by males is known in some bean weevils (Huignard, 1983; Boucher and Huignard, 1987). A comparison of behaviors and selection processes in courtship is probably necessary to examine this problem.
ACKNOWLEDGEMENTS
I thank Dr. T. Nishida of Kyoto University for critical reading of this manuscript and valuable comments. I thank also Dr. E. Kuno for his suggestions and encouragement during this study. I relied on Dr. Y. Okumoto of Kyoto University for irradiation of insects, for which I thank him also.
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