and the retention of eggs and embryos (Strathmann 1990; Ryland and Bishop. 1993). In contrast, the majority of unitary (non-budding) ascidians release both.
Laboratory Studies of Mating in the Aplousobranch Diplosoma listerianum
John 0.0. Bishop1.2, Andrew J. Pemberton'·3, A. Oorothea Sommerfeldt1.2 and Christine A. Wood' 'Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hili, Plymouth PLI 2PB, UK 2 Department of Biological Sciences, University of Plyrnouth, Drake Circus, Plymouth PL4 8AA, UK 3 Department ofZoology, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB242TZ, UK
Summary. The didemnid DiplosoTrUl listerianum exemplifies colonial ascidians in releasing sperm into the water to be captured by neighbours to fertilize brooded eggs. This mating process has been the subject of aseries of studies using colonies in laboratory culture, and the findings are summarized here alongside those of published electron microscopy investigations of events in the female tract. Sperm travel through the oviduct of a zooid to reach the ovary, allowing true internal fertilization around the time of ovulation of a single oocyte into the colonial tunic, where the embryo is brooded. Cross-fertilization is the norm, but is selective: along with self sperm, non-self sperm from incompatible sources are blocked within the oviduct and prevented from reaching the ovary. The receipt of compatible sperm triggers vitellogenic egg growth, and sperm may be stored in the ovary for weeks for the fertilization of successive ovulations. Sequential mating by two males at an interval of eight days yields a succession of paternity within progeny arrays indicative of first-in-first-out utilization of stored sperm. With a shorter mating interval (24 h), this pattern is lost. Key words: DiplosoTrUl, Sperm, Oocyte, Compatibility, Fertilization
Introduction Various sessile, suspension-feeding marine invertebrates release sperm which are filtered from seawater by conspecific individuals to fertilize their brooded eggs. This pattern of mating offers little scope for control by an acting fe male over the origin of sperm that are received; rather, a mixture of sperm from a variety of 305
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sources will typically be collected (Bishop and Pemberton 1997). Such "obligatory polyandry" might promote female selectivity in the utilization of sperm if progeny vary in fitness between different potential fathers. Among ascidians as a whole, a remarkable correlation exists between the occurrence of budding to produce a colony of inter-linked individuals or zooids and the retention of eggs and embryos (Strathmann 1990; Ryland and Bishop 1993). In contrast, the majority of unitary (non-budding) ascidians release both sperm and eggs for external fertilization. Reproduction in the brooding colonial stolidobranch Botryllus has been the subject of important studies over' a long period, while mating in aplousobranchs has received much less attention. Here we summarize the findings of a decade of laboratory-based studies that have started to reveal the pattern of sperm utilization in the widespread didemnid Diplosoma listerianum (Milne Edwards 1842). The ability to grow the species in the laboratory and to maintain colonies in true reproductive isolation has been vital to these investigations, and we begin by detailing the culture techniques involved.
Laboratory Culture Minor modifications have been made to the culture protocols outlined by Ryland and Bishop (1990). Colonies are grown on the inward- and slightly downwardfacing sides of 76 x 38 mm glass microscope slides c1ipped near-vertically in tanks arranged in a single row along a shelf. Each tank holds 800 ml of filtered, UV-sterilized seawater. The tanks on a shelf are stirred by vertical reciprocating paddles (35 mm wide, stroke of c. 30 mm, c. 30 oscillations per minute) driven via horizontal rods cranked by a central electric motor. The rods are simply suspended by cords from the shelf above. Each tank is given a daily ration of 3 ml of a mixture of the cultured microalgae lsochrysis galbana Parke and Rhinamonas reticulata (Lucas) Novarino, generally in morning and evening instalments, supplemented by Liquifry Marine (Interpet Ltd., Dorking, England) if rapid growth is required. Weekly water changes, brushing the tank to remove faecal material, suffice for routine maintenance of stocks, but more frequent changes improve the general vigour of experimental material. Stirring rather than aerating the tanks abolishes the risk of sperm transfer with aerosol particles, while vertical partitions on the shelves prevent splashes between tanks. Given the efficient capture and storage of very dilute sperm by the species (Bishop 1998), additional precautions to maintain reproductive isolation are necessary during feeding, water changes and experimental manipulation. Separate containers and instruments (pipettes, brushes, forceps, razor blades, etc.) are used for each genotype, and are rinsed in tap water and dried completely between uses. Any splashes, drops and smears in the working area are completely removed, and hands thoroughly dried, before the next genotype is processed.
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The culture rooms are at about 1TC with a 15:9 light: dark regime. The lifecycle can be completed in c. 8 weeks in laboratory culture, with matings at any time of year. The oldest laboratory clones are now more than nine years old. Chimerism is prevalent in wild colonies (Sommerfeldt and Bishop 1999), so laboratory clones are founded from isolated new metamorphs. Grown colonies may be divided, and thus clonally propagated, by vertical cutting with razor blades, and the pieces removed from the slide by running ablade under the colony, and cleaned by shaking in seawater. Stirring the pieces in a cylindrical dish for 12-24 h to keep them in suspension may be worthwhile, promoting the healing and rounding-off of the cut tunic. This aids reattachment, which involves either allowing the pieces to settIe onto horizontal slides in still water for 24 h, or trapping each sub-colony against a slide in normal (stirred) culture using a loop of nylon monofilament. Reduction of colonies and transfer onto clean glass in a fresh tank every six to eight weeks is necessary to maintain healthy growth. DiplosoTlUl listerianum is capable of slow movement and may undergo natural colony fission (Della Valle 1908), and re-culturing onto new slides generally triggers this process. Colony division mayaIso occur in culture at other times, but an unpublished undergraduate project (AI Pemberton 1995) showed no detectable effect of reduction in salinity, oxygen tension or food ration on the rate of fission.
Sperm Release and Uptake, Ovulation, and Fertilization Mature sperm are stored in the seminal vesicle adjacent to the two-Iobed testis, and reIeased as discrete but rapidly dispersing wisps of a few thousand gametes which pass out through an exhalant opening (Bishop and Ryland 1991; Bishop 1998). There appears to be no clear daily peak of sperm release by a colony (unpublished observations). The route of entry of sperm into a recipient colony is unknown, but is presumed to involve the inhalant current. Sperm then enter a duct running from the ovary of a zooid to open into the colonial cloacal space adjacent to the anus. This is anatomically the oviduct, although ova are not discharged into it (see below). The complex structure of DiplosoTlUl listerianum sperm is described by Burighel et al. (1985). Sperm within the oviduct have undergone a reaction or metamorphosis involving the shortening of a spiral den se groove to occupy only the front half of the head, which acquires a corkscrew configuration, restricting the mitochondrion to the region behind (Burighel and Martinucci 1994a). Phagocytosis of sperm is apparent throughout the female tract (Burighel and Martinucci 1994a). Self sperrn, originating from the recipient genotype, enter the oviduct but are generally haI ted a small distance along it, far from the ovary (Bishop 1996); it is presumed that there is relatively intense phagocytosis of sperm at this point. Non-self sperm from particular sources, depending on the recipient clone, are also blocked in the same region, resulting in sexual incompatibility, as confirmed by molecular paternity analysis (Bishop 1996; Bishop et al. 1996). In contrast, compatible sperm proceed right along the oviduct to the ovary, where
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they occur in the main lumen and in hollow blind-ended diverticula leading to individual oocytes (Burighel and Martinucci 1994a; Bishop and Sommerfeldt 1996; Bishop 1996). Acceptance and rejection of sperm from different sources can occur concurrently (Bishop et al. 1996). The ovarian diverticula pass through the follicle cell layers of oocytes and terminate next to the vitelline coat (Burighel and Martinucci 1994b). Penetration of the ovum by a sperm occurs shortly before ovulation (Bishop and Ryland 1991; Burighel and Martinucci 1994b). The metamorphosed sperm passes between the cells of the ovarian epithelium, despite the presence of intercellular tight junctions, to reach the vitelline coat (Burighel and Martinucci 1994b). The sperm head, less cytoplasmic organelles, is engulfed by the ovum in a process resembling phagocytosis, with delayed fusion of the respective plasma membranes (Burighel and Martinucci 1994b). The ovum/zygote is ovulated directly into the tunic when the follicle cells separate opposite the ovarian diverticulum and retract onto it, forming a corpus luteum (Burighel and Martinucci 1994b). The zygote passes down the strand of tunic from the zooidal abdomen into the basal tunic of the colony, where brooding occurs. Embryonic development lasts c. 13 days in our culture conditions (Ryland and Bishop 1990), although slightly longer times have been reported (Berrill 1935; Brunetti et al. 1988). Ova are generally ovulated one at a time by a particular zooid, at intervals of four to 20 days (Bishop and Ryland 1991). Sperm may remain viable for over seven weeks in the ovary (Bishop and Ryland 1991; Bishop 1998), enabling the fertilization of successive ovulations and the production of progeny over several weeks following abrief period of sperm uptake (Bishop et al. 20ooa). Vitellogenic egg growth does not generally occur in reproductive isolation (Ryland and Bishop 1990). The receipt of compatible sperm triggers egg growth, which is maintained while sperm remain in storage (Bishop et al. 2oo0b).
Patterns of Mating Diplosoma listerianum can maintain maximal production of outcrossed zygotes at ambient sperm concentrations down to 102 compatible spermIrni, which would cause fertilization failure through sperm limitation in almost all externally fertilizing marine animals (Bishop 1998; Al Pemberton et al., in prep.). This may reflect efticient capture of sperm (probably during feeding), the longevity of sperm following release (half-life c. 8 h) coupled with storage after capture, and the low rate of ovulation of the individual zooids of a colony (Bishop 1998). Nowadays. larvae are only very occasionally produced by our colonies cultured in reproductive isolation. We intend to use microsatellite markers currently under development to assess wh ether these progeny result from selting or from inadvertent transfer of sperm between tanks. The moderate production of larvae from supposedly isolated specimens in the first experiments reported (Ryland and Bishop 1990) probably resulted from partial failure of early isolation protocols.
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Fig_ 1. Paternity within progeny arrays resulting from sequential exposure to two acting males. First-male paternity above horizontal line, second-male paternity below. Left: eight-day interval between the initiation of matings; right: 24 hinterval
There is a high rate of sexual incompatibility between unrelated clones in the laboratory cultures studied to date, which are derived from Queen's Dock, Swansea, UK (Bishop et al. 1996 and unpublished observations). Of the 132 trials in a 12 x 12 matrix of reciprocal matings, 52 (39%) showed complete incompatibility, with restricted fecundity in an additional 27 (20%). When two acting male clones are mated to an acting female with a delay of eight days, the earliest progeny are sired almost exclusively by the first male, while progeny in later weeks are largely attributable to the second male (Bishop et al. 2000a) (Fig. 1). This indicates that the population of stored sperm within the ovary remains structured with respect to its time of arrival, and is utilized on a queueing basis. However, this succession of patemity within progeny arrays is lost if the interval between matings is reduced to 24 h (Fig. 1).
Conclusions and Prospectus Diplosoma listerianum shows remarkable sophistication in its handling of sperm, which it can extract from very low ambient concentrations, subject to selective scrutiny in the oviduct, and store for long periods in an age-structured ovarian sperm population which triggers female investment in the form of vitellogenic egg growth. Remaining questions abound: the level of sperm supply and the pattern of patemity in natural populations; the effect of relative sperm abundance upon patemity of competing males; the exact nature of interactions between sperm and the female tract; the origin and present significance of the observed sexual incompatibility; the extent of incompatibility in other populations and in interpopulation matings; and so on. We hope to be able to investigate so me of these.
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References Berrill J (1935) Studies in tunieate development. Part I1It>Oifferential retardation and acceleration. Phil Trans R Soc Lond B 225:255-326 Bishop JOO (1996) Female control of patemity in the intemally fertilizing compound ascidian Diplosoma listerianum. I. Autoradiographic investigation of sperm movements in the female reproductive tract. Proc R Soc Lond B 263:369-376 Bishop JOO (1998) Fertilization in the sea: are the hazards of broadcast spawning avoided when free-spawned sperm fertilize retained eggs? Proc R Soc Lond B 265:725-731 Bishop JOO, Pemberton AJ (1997) Sessile animals: attached, but prorniscuous? Trends Ecol Evol 12:403 Bishop JOO, Ryland JS (1991) Storage of exogenous sperm by the compound ascidian Diplosoma listerianum. Mar Bio1 108: 111-118 Bishop JOO, Sommerfeldt AO (1996) Autoradiographic investigation of uptake and storage of exogenous sperm by the ovary of the compound ascidian Diplosoma listerianum. Mar Biol 125:663-670 Bishop JOO, Jones CS, Noble LR (1996) Female control of patemity in the intemally fertilizing compound ascidian Diplosoma Listerianum. H. Investigation of male mating success using RAPO markers. Proc R Soc Lond B 263:401-407 Bishop JOO, Pemberton AJ, Noble LR (2ooOa) Sperm precedence in a novel context: mating in a sessile marine invertebrate with dispersing sperm. Proc R Soc Lond B 267:1107-1113 Bishop JOO, Manrfquez PH, Hughes RN (2oo0b) Water-borne sperm triggers vitellogenic egg growth in two sessi1e marine invertebrates. Proc R Soc Lond B 267: 1165-1169 Brunetti R, Bressan M, Marin M, Libralato M (1988) On the ecology and biology of Diplosoma Listerianum (Milne Edwards, 1841) (Aseidiacea, Oidemnidae). Vie Milieu 38: 123-131 Burighel P, Martinucci GB (1994a) Sexual reproduction in the compound ascidian Diplosoma listerianum (Tunicata). I. Metamorphosis, storage and phagocytosis of sperm in female duct. Mar Biol 118:489-498 Burighel P, Martinucci GB (1994b) Sexual reproduction in the compound ascidian Diplosoma Listerianum (Tunicata). H. Sperm penetration through ovary wall and evidence of internal fertilization. Mar Biol 118:499-510 Burighel P, Martinucci GB, Magri F (1985) Unusual structures in the spermatozoa of the ascidians Lissoclinum perforatum and Diplosoma Listerianum (Oidemnidae). Cell Tissue Res 241 :513-521 Oella Valle A (1908) Osservazioni su a1cune ascidie dei Golfo di Napoli. Atti Accad Sei Napoli 2a 13:1-89 Ryland JS, Bishop JOO (1990) Prevalence of cross-fertilization in the hermaphroditic compound ascidian Diplosoma listerianum. Mar Ecol Prog Ser 61: 125-132 Ryland JS, Bishop JOO (1993) Internal fertilisation in hermaphoditic colonial invertebrates. Oceanogr Mar Biol A Rev 31 :445-477 Sommerfeldt AO, Bishop JOO (1999) Random amplified polymorphie ONA (RAPO) analysis revea1s extensive natural chimerism in a marine protochordate. Mol Ecol 8:885890 Strathmann RR (1990) Why life histories evolve differently in the sea. Am Zool 30: 197207
