OL The Journal of Research , g;J|ON THE Lepidoptera Volume 39 2000 (2006) The Journal of Research on the Lepidoptera ISSN 0022 4324 Published by: The Lepidoptera Research Foundation, Inc. 9620 Heather Road Beverly Hills, California 90210-1757 TEL (310) 399 6016 E-MAIL: Editorial: [email protected] Business: [email protected] Technical: [email protected] Founder: William Hovanitz (1915-1977) Edu'orial Staff: Andrew Warren, editor E-MAIL: [email protected] Rudolf H.T Mattoni, assistant editor E-MAIL: [email protected] Nancy R. Vannucci, managing editor E-MAIL: [email protected] Associate Editors: The Lepidoptera Research Eoundation is in process of reorganization. A board of associate editors will be established. Manuscripts and notices material may at present be sent to the assistant editor, Rudolf H.T. Mattoni, E-MAIL: [email protected]. The editor is in process of relocation, btu either the assistant or managing editors can be contacted at the addresses given. Please note the instructions to atuhors on the back inside cover of this Journal M.vn.aginc editor at the address: Nancy R. Vannucci, Maza 3340, Moron B1708GOP, Buenos Aires, Argentina The journal is sent to all members of the Eoundation. Classes of membership: Regular (Individual) $ 25.00 year (vol.) Contributing $ 30.00 or more year (vol.) Student/ Retired-Worldwide $ 20.00 year (vol.) Stibscription Rate/ Instittitions $ 35.00 year (vol.) Life % 300.00 STATEMENT OF OWNERSHIP AND MANAGEMENT THEJOURNAL OE RESEARCH ON THE LEPIDOPTER\ will be published two times a year by the LEPIDOPTERA RESEARCH FOUNDATION, INC. Publication and business offices are located at the Beverly Hills, California address given above. The Foundation is a non-profit organization incorporated in the State of California in 1965. The president is Rudolf H. T. Mattoni, the vice-president is Jeremiah Ceorge, the secretary-treasurer is Leona Mattoni. The board of directors (2005-) is comprised of Konrad Fiedler, Dan Rubinoff, Jeremiah Ceorge, and Rudolf H. T. Mattoni. Past issues are .available .at our temporary website : www.doylegroup.harvard.edti/~carlo/JRL/jrl.html jnumul of Research on the Lepidoptera 39; 1-7, 2()()() (2006) Total sperm ejaculation in monandrous (Papilio machaon) and polyandrous {P. xuthus) swallowtail butterflies (Lepidoptera: Papilionidae) restricted to larval stage-derived nutrients Mamoru Watanabe Institute oi Biological Sciences, University of Tsuknha, 'Isukuba, Ibaraki 305-8572, J K-mail: [email protected] Isukuba. ar.jp Taihei Kobayashi Institute of Biological Sciences, University of Tsukuba, 'Isukuba, Ibaraki 305-8572, Japan Abstract: Potential capacities of male swallowtail butterflies for ejaculation containing siDerin were examined in laboratory studies in monandrous {Papilio machaon) and |5(dyandrons {P. xuthus) species. Virgin males transferred a spermato|thore and accessor)' substances that corresponded to an average of 2.4% and 2.1% of their body weight at eclosion in P. xuthus And P. machaon, respectively. The spermatophore contained about 41 and 120 eupyrene sperm bundles and 247,000 and 202,000 apyrene spermatozoa for P. xuthus -And P. machaon, respectively. Individual eupyrene and apyrene spermatozoa of P. machaon were smaller than those of P. xuthus. I lowever, when the males re mated at two days after the first mating, they |troduced a spermatophore of 55% and 32% of full si/e in P. xuthus and P. machaon, respectively. The ntunber of eupyrene sperm Irundfes in the second spermatophore increased while the apyrene .s|)ermatozoa decreased in P. xuthus. I'he larger number of eupyrene sperm Itundles and the relatively smaller number of a|tyrene spermatozoa in the first and the second mating of P. machaon compared to P. xuthus are discussed from the viewpoint of female monandr)' and polyandry. Key words: apyrene sperm, eu]5yrene .S|)ei'm bntidle, Papilio machaon, Papilio xuthus, second mating, spermatophore. Introduction butterfly females can use such male-derived nutrients for somatic maintenance, to enhancement of their In many bntterfly species, the male’s second sper- fecundity, and to increase fitness of their offspring matophore is significantly smaller than his first (Svard (Friedel & Gillott 1977; Watanabe 1988; Boggs 1990). & Wikhmd 1986, 1989; Oberhanser 1988). Before The potential importance of male-derived nutrients production of a second spermatophore of full size, might correlate positively with the size of the male a resting period of several days is required, during ejaculate and the degree of female polyandry (Svard which the male’s activity consists of feeding, flying, & Wikhmd 1989). When the male re mates, he must avoiding predators, and so on (Watanabe & Hirota jjroduce another spermatojihore of sufficient size 1999). The copula duration of the second mating is with a sufficient number of sperm in order to fertilize often significantly longer than that of the first, [irob- as many eggs as possible. Therefore, the duration of ably due to the size of the ejaculate (Svard Sc Wikhmd the male’s recovery time and the rate of lecovery in 1986; Kaitala & Wiklimd 1995). terms of spermatophore size and sperm number are Among the yellow swallowtail butterflies in Japan, important for males under sperm competition (Wa- females of Papilio xuthus Linne (Papilionidae) mate tanabe & Hirota 1999; Wedell & Cook 1999a). The three times during their life span while females of former correlates with the duration of the female’s P. machaon hippocrates C. et R. Felder are apt to be non-receptive refractory period following mating monandrous (Watanabe & Nozato 1986). For poly- (Kiiitala & Wikhmd 1995), and high sperm numbers androus species like P xuthus, each mating provides seemed to be advantageous in sperm competition the female with nutrients and some alkaloids with (Parker 1992). Rather than utilizing the sperm of all both transferred to her via the male’s spermatophore her mates, the female uses predominantly those from (LaMimyon & Eisner 1994). It has been suggested that the last partner in P. dardanus (Simmons &: Siva-Jothy 1998). On the other hand, for monandrous species Recehied: 13 December 2003 like P. machaon, the female may not exercise such Accepted: 14 February 2005 postcopulatory sperm selection (Sims 1979). Intact . /. lifs.Lepid. spermatophores have been observed in the bursa ing the larval stage is butterfly mass. co])nlatrix of aged females of inonandrous species, Because the mating ability of males on the day of such as P. machaon (impnblisbed) and Lycaena phlaeas eclosion is low, we hand-paired one-day old males with (Watanabe & Nisbimnra 2001), suggesting that these virgin females (one to three-days old) in the morning. females did not use male ejaculate to obtain the nu- Hand-pairing is widely used in the laboratoiy rearing trients necessary for reprodtictive success. of butterflies (West 1983; Scriber & Lederhouse 1988; Most butterfly species produce two types of sperm, Watanabe & Hirota 1999). In this study, we brought enpyrene and apyrene, which can often represent up the tips of the male and female abdomens together, to 90% of the total sperm number (Cook & Wedell squeezed the male in order to open his claspers, and 1996; Watanabe et al. 1998a, b) . Males vary the size of then joined him with the female. After joining, the spermatophores produced as well as the mimber of pair was removed to a small cage and the copula dura- both types of sperm transferred de})ending on their tion was recorded. mating status (Cook & Wedell 1996; Wedell & Cook Males were returned to the flight cages immediate- 1999b). Previously mated males of /I xu//m.v produce ly after their hrst mating and held to attempt a second spermatophores that are about half the size of those copulation three-day.s-later with virgin females. A total they [jrodnced as virgins, when they used nutrients of 26 mated males for P. xiith us and 24 mated males derived only from their larval stages (Watanabe & for P. machaon were hand-paired. Spermatophore Hirota 1999). However, there have been no studies mass and the number of enpyrene sperm bundles on sperm competition in swallowtail butterflies. The and apyrene spermatozoa in the spermatophore were aim of this study was to compare male’s exploitation determined. Becau.se the accessory substances were of larval stage-derived nutrients for spermatophore ejaculated otUside the spermatophore into the bursa production between monandrous and polyandrous copulatrix of the female, we could easily separate them swallowtail butterfly species in terms of copula dura- during the di.ssection of the bursa copulatrix. tion, spermatophore mass, and the number of apyrene Immediately after copulation, the females were de- and enpyrene spermatozoa produced. capitated and di.ssected under a stereo microscope to measure the weight of ejaculates in the bursa copula- Materials and methods trix. The intact spermatophore and gel-like accessory substances were weighed (accuracy, 0.001 mg). Males General methods that had successfully mated twice were returned to flight cage to attempt a third copulation. One and three-day-old males were mated with vir- gin females of both P. xuthus And P. machaon. Sperm Sperm counting procedure transfer h'om the males to spermatophore was assessed immediately after copulation. All animals were ob- The procedure used for sperm counting has tained from a continuously breeding culture reared been described in detail iit Watanabe et al. (1998a, in the laboratoiy at room temperature in the summers b). We evaluated the number of both types of sperm of 2001 and 2003. Adults were collected in the held, derived in females by counting the number of sperm and females were allowed to oviposit on leaves of Citrus in the transferred spermatophore. Immediately after unshu (Rutaceae) for P. xuthus'Awd those of Heraclmm copulation, the enpyrene sperm are packed in the lanatum (Umbelliferae) for P. machaon. Larvae of both spermatophore in a bundle, whereas the apyrene species were reared in the laboratory under an 18-h bundle has already dissolved, and thus tlie apyrene light (>h dark regime at 25°(i to avoid diapause.