Sperm present in the female's sperm storage organ (spermatheca, see Fig. 3.3) appear to have a long-term effect on female receptivity. In the butterfly Pieris rapae, the presence of sperm in the spermatheca appears to cause neural triggering of female unreceptivity (Obara et al., 1975). Similarly, in several other species of Lepidoptera, the presence of sperm in the spermatheca is required to switch off female receptivity and stimulate oviposition (e.g. Giebultowicz et al., 1991a,b; Karube and Kobayashi, 1999). For example, an inverse relationship between the quantity of sperm in the spermatheca and female remating is reported (e.g. Proshold, 1995; Shantharam et al., 1998). Both the spermatheca and the bursa copulatrix are sensory innervated, suggesting that sperm and seminal fluid may play a role in switching off female receptivity and stimulating oviposition (Sugawara, 1979; Kingan et al., 1995). For example, in Plodia interpunctella, females receiving few sperm from a male on his second or third mating are more likely to remate (Cook and Gage, 1995), and in the gypsy moth Lymantria dispar, remating is more likely to occur if there are few sperm in the spermatheca (Proshold, 1995).
Male butterflies and moths produce two types of sperm: normal, fertilizing 'eupyrene' sperm, and a large number of non-fertile, anucleate 'apyrene' sperm (Meves, 1902; Friedländer, 1997). Apyrene sperm typically represent 90% or more of the total sperm number (Cook and Wedell, 1996; Solensky, 2003). Fertilizing eupyrene sperm are transferred in the spermatophore to the female in bundles of 256 sperm per bundle (Virkki, 1969; Phillips, 1970; Richard et al., 1975; Witalis and Godula, 1993). Non-fertile sperm represent a significant investment by males, since they are transferred to females in large numbers. Apyrene sperm have different behaviour from eupyrene sperm, being highly active at ejaculation, whilst the eupyrene sperm usually remain in bundles. Apyrene sperm also appear to reach the female's spermatheca before the fertile sperm in both butterflies and moths (Silberglied et al., 1984; Tschudi-Rein and Benz, 1990; Watanabe et al., 2000; Marcotte et al., 2003). Non-fertile sperm seem to be critical to male reproductive success, because males do not decrease investment in apyrene sperm relative to eupyrene sperm when reared on a restricted diet (Gage and Cook, 1994; Cook and Wedell, 1996).
Various hypotheses have been proposed to explain apyrene sperm function (reviewed in Silberglied et al., 1984). Many of these suggest that apyrene sperm have a physiological role, for example in aiding eupyrene sperm transport or activating the eupyrene sperm (e.g. Osanai et al., 1986, 1987). In Bombyx mori, they appear to be important for successful fertilization, possibly by aiding transfer of the fertile sperm to the spermatheca and dissociation of the eupyrene sperm bundles (Sahara and Takemura, 2003). Alternatively, they may represent a nutrient source either for the fertile sperm in the spermatheca, or for the female and the developing zygotes. However, Silberglied and co-workers (1984) have argued that these hypotheses do not account for the fact that apyrene sperm reach and persist within the spermatheca and do not appear to be digested. In some species non-fertile sperm may be stored separately in the different parts of the spermatheca (the lagena and utriculus, see Fig. 3.3) (Holt and North, 1970; Miskimen et al., 1983). If apyrene sperm were only involved in eupyrene sperm transport or activation in the spermatophore, it seems unlikely that they would then be stored, and even less likely that they would be sorted and stored in particular areas in the spermatheca. Furthermore, it has been suggested that if apyrene sperm have a physiological role, a given number of non-fertile sperm should be needed for the activation or transport of a single fertile sperm, and therefore the proportion of the two sperm types should be constant within a species (Cook and Gage, 1995). This does not appear to be the case. For example, in at least two species there is a significant increase in the proportion of fertile sperm over the first two matings: in Plodia interpunctella, the proportion of eupyrene sperm increases from 7.5% to 10% (Gage and Cook, 1994), and in Pieris rapae the increase is from 11% to 15% (Cook and Wedell, 1996).
In their landmark paper, Silberglied et al. (1984) suggested that apyrene sperm play a role in sperm competition. They specifically suggested that apyrene sperm may displace or inactivate rival sperm, or, by remaining in the females' spermatheca, they may delay female remating. Both these sperm competition hypotheses predict that apyrene sperm numbers should increase with increased risk of sperm competition. If apyrene sperm displace or inactivate rival males' sperm, they may increase in response to the presence of rival male sperm. In P. interpunctella, males provide non-virgin females with more eupyrene, but not apyrene, sperm (Cook and Gage, 1995), whereas in the green-veined white, Pieris napi, males provide both a higher number of eupyrene and apyrene sperm to mated females (N. Wedell and PA. Cook, unpublished data). On the other hand, if apyrene sperm influence female sexual receptivity, we expect their numbers to be related to female remating behaviour. It is, of course, possible that non-fertile sperm may play both these roles.
A recent study on the green-veined white, P. napi, suggests that the number of non-fertile sperm in the spermatheca is responsible for reducing female receptivity (Cook and Wedell, 1999). Once-mated females were given the opportunity to remate up to 10 days after their first mating (females rarely remate after this time), dissected upon remating (before the sperm from the second mating reached the spermatheca), and the number of eupyrene and apyrene sperm present in the spermatheca were counted. Females that did not remate within 10 days were also dissected. There was no difference in the number of fertile sperm in the spermatheca of the females that did remate compared with those that did not. However, females that did not remate had significantly more non-fertile sperm in their spermatheca (Fig. 3.4). Moreover, within the females that did remate, there was a positive relationship between the length of time until remating and the number of apyrene sperm in the
Fig. 3.4. The relationship between number of non-fertile (filled circles) and fertile (open circles) sperm stored by female Pieris napi butterflies in relation to probability of remating. Means ± se (redrawn after Cook and Wedell, 1999).
spermatheca. There was no such relationship with eupyrene sperm number. These results suggest that non-fertile sperm are involved in influencing female sexual receptivity by filling their sperm storage in this species. There is genetic variation between females in their tendency to store non-fertile sperm, which correlates with the duration of their refractory period (Wedell, 2001). Females may have a mechanism to detect the presence of sperm in their spermatheca (e.g. by the presence of mechano-receptors; Lum and Arbogast, 1980) in order to ensure high fertility. Males may have evolved to take advantage of this system: rather than transferring large numbers of nucleate eupyrene sperm, the males transfer large numbers of non-fertile sperm that fill the female's sperm storage organ, thereby delaying female remating and hence reducing sperm competition. It is possible that production of non-nucleated sperm is more efficient than a similar investment in eupyrene sperm to switch off female receptivity, although this is yet to be confirmed.
In P. napi, there is a conflict over female remating rate, as males transfer nutrients, increasing female fecundity (Wiklund et al., 1993). Females may have responded to the males' manipulation of their reproductive system by evolving a better detection system to monitor sperm in storage and regain control over their mating rate. This imposes further selection on males to increase the number of non-fertile sperm transferred to females. A coevolutionary arms race between males and females over female receptivity may have resulted in the elaboration of male P. napi ejaculates to the point where they now consist of mainly non-fertile sperm, reducing female remating and thus reducing sperm competition for the first male to mate with the female. Similarly, in the armyworm moth, the regaining of female receptivity is associated with a pronounced decline in the number of apyrene, but not eupyrene, sperm, in storage (He et al., 1995), and the presence of motile apyrene sperm in the spermatheca temporarily switches off female receptivity in Heliothis zea (Snow et al., 1972).
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