In many animals, males may maximize their reproductive success by fertilizing as many females as possible (e.g. Bateman 1948). The number of females a male can mate is often limited by male competition for access to females, sperm competition and the cost of sperm production (Pitnick and Markow 1994a; Rittschof et al. 2012). Especially, recent studies have shown that sperm production is more costly than previously expected, and also that males readily experience sperm depletion by multiple mating in a short period even in synspermatogenic species, in which males produce sperm throughout their adult life (Partridge and Harvey 1992; Van Voorhies 1992; Pitnick and Markow 1994b; Pitnick et al. 1995; Olsson et al. 1997; Preston et al. 2001; Rubolini et al. 2007; Radhakrishnan et al. 2009; Dowling and Simmons 2012; Macartney et al. 2021). Therefore, how long it takes for sperm replenishment after the depletion would be important in determining the optimal number of mating and reproductive strategy in males of synspermatogenic species.
Tetranychus urticae Koch is a spider mite infesting various crops, and famous as a cosmopolitan pest (Helle and Sabelis 1985). In this species, females often mate with several males, but they use the sperm only from the first mating for egg fertilization if there are no abnormalities in the first mating (Boudreaux 1963; Helle 1967; Potter and Wrensch 1978; Satoh et al. 2001). Therefore, males compete for virgin females, and it makes competition among males very intense. As a result, males often mount females in the teleiochrysalis stage, which is the stage just before molting to adult female (premating guard) (Potter et al. 1976a, b). Males also show alternative reproductive tactics (Sato et al. 2013, 2014, 2016; Schausberger and Sato 2019, 2020; Schausberger et al. 2021). Therefore, in the mite, the number of females a male can fertilize is limited mainly by male competition for access to females. However, in the absence of rivals, males mate with a large number of females and experience sperm depletion. For example, a previous study reported that a 0-day-old male mated with 5–25 females (12–13 females on average) but did not inseminate females after around the 11th mating (Kobayashi et al. 2022). Mating without sperm transfer does not produce any offspring, although it takes time and energy. Therefore, the question arises why males of this mite species continue to mate even after sperm depletion.
Continuing to mate after sperm depletion in males is observed in several species of parasitoid wasps (Simmonds 1953; Laing and Caltagirone 1969; Gordh and DeBach 1976; Nadel and Luck 1985; Ramadan et al. 1991). In Trichogramma evanescens Westwood, it is found that mating without sperm transfer reduces ability of the mated females to store sperm from other males (Damiens and Boivin 2006). In Anisopteromalus calandrae (Howard), it is found that mating reduces females’ subsequent mating receptivity regardless of the amount of sperm transferred (Abe 2019). Therefore, in parasitoid wasps, sperm-depleted males may continue to mate in order to interfere with the reproduction of rival males, so as to increase their own relative fitness. In the spider mite, mated females readily remate with males, and only when the first mating is abnormal (e.g. shorter copulation than in normal mating), their eggs can be fertilized by sperm from remating (Helle 1967; Potter and Wrensch 1978; Satoh et al. 2001; Morita et al. 2020). Therefore, mating without sperm transfer may not be expected to function such as to reduce subsequent mating receptivity in females. However, remated spider mite females produce fewer fertilized eggs (Rodrigues et al. 2020). So, mating without sperm transfer likely has a function to interfere with reproduction of females fertilized by other males. However, in the first place, if sperm can be replenished very quickly and given that the premating guarding phase takes several hours, males may continue to mate even after sperm depletion in order not to lose mating opportunity. In a previous study, a T. urticae male that mated 25 times in 3 hours could inseminate the 23rd female slightly although the male failed to inseminate the 13th to the 22nd female consecutively (Kobayashi et al. 2022). Therefore, it is highly expected that sperm can be replenished in a short period in T. urticae males.
In this study, we investigated how long it takes for sperm replenishment in T. urticae males. We allowed males to have a rest for 3 h, 6 h, 12 h and 24 h after sperm depletion, then provided virgin females to them, and determined if sperm were replenished by checking insemination status of mated females. Spider mites are haplodiploid: females develop from fertilized eggs and males develop from unfertilized eggs. Hence, we used the production of female offspring in the determination of insemination status. Since it is known that copulation duration without sperm transfer is much shorter than that with sperm transfer (Kobayashi et al. 2022), we also checked and compared the copulation duration.