sexual and asexual reproduction in the colonial ascidian botryllus schlosseri

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REVIEW Sexual and Asexual Reproduction in the Colonial Ascidian Botryllus schlosseri Fabio Gasparini, Lucia Manni,* Francesca Cima, Giovanna Zaniolo, Paolo Burighel, Federico Caicci, Nicola Franchi, Filippo Schiavon, Francesca Rigon, Davide Campagna, and Loriano Ballarin Department of Biology, University of Padova, Padova, Italy Received 23 April 2014; Revised 1 July 2014; Accepted 7 July 2014 Summary: The colonial tunicate Botryllus schlosseri is a widespread filter-feeding ascidian that lives in shal- low waters and is easily reared in aquaria. Its peculiar blastogenetic cycle, characterized by the presence of three blastogenetic generations (filtering adults, buds, and budlets) and by recurrent generation changes, has resulted in over 60 years of studies aimed at under- standing how sexual and asexual reproduction are coordinated and regulated in the colony. The possibility of using different methodological approaches, from classical genetics to cell transplantation, contributed to the development of this species as a valuable model organism for the study of a variety of biological proc- esses. Here, we review the main studies detailing rear- ing, staging methods, reproduction and colony growth of this species, emphasizing the asymmetry in sexual and asexual reproduction potential, sexual reproduction in the field and the laboratory, and self- and cross- fertilization. These data, opportunely matched with recent tanscriptomic and genomic outcomes, can give a valuable help to the elucidation of some important steps in chordate evolution. genesis 53:105–120, 2015. V C 2014 Wiley Periodicals, Inc. Key words: left-right asymmetry; blastogenetic cycle; budding; coloniality; cross-fertilization; self-fertilization INTRODUCTION Tunicates are filter-feeding marine invertebrates, both sessile and pelagic, and they represent the sister group of vertebrates (Delsuc et al., 2006, 2008). The name refers to the presence of the tunic, a peculiar tissue embedding the whole organism. Ascidians, the largest and most studied class of tunicates, contain both solitary and colonial species and are characterized by the presence of a free-swimming, tadpole-like larva. The latter shares the main chordate features, i.e., a support- ing dorsal rod or notochord in the tail (hence the alter- native name of Urochordata assigned to the taxon), a dorsal central nervous system in the form of a hollow tube that is anteriorly enlarged to form a brain, and a muscular tail (Burighel and Cloney, 1997). The ascidian larva metamorphoses into a sessile, filter-feeding oozooid lacking most of the chordate fea- tures. Solitary ascidians grow in size during their adult life; conversely, colonial forms propagate asexually, forming colonies of many small individuals (Brown and Swalla, 2012). Mechanisms of asexual development vary among colonial ascidian species in terms of bud- ding tissue and/or budding mode (reviewed in Brown and Swalla, 2012; Kurn et al., 2011; Nakauchi, 1982). In addition, various degrees of integration among zooids can be observed: they can share the basal stolon, the colonial tunic or the tunic and the colonial vasculature. This diversity, associated to phylogenetic evidences, suggest coloniality in ascidians occurred several times independently during evolution (Brown and Swalla, 2012). In the last few decades, solitary ascidians of the genus Ciona have been intensively used as model organisms *Correspondence to: Lucia Manni, Department of Biology, University of Padova Via Ugo Bassi 58/B, 35121, Padova, Italy. E-mail: [email protected] Contract grant sponsor: MIUR PRIN Projects, Contract grant numbers: 2009XF7TYT, 20109XZEPR; University of Padova Senior post-doc 2012 Project; GRIC120LSZ Published online 12 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/dvg.22802 V C 2014 Wiley Periodicals, Inc. genesis 53:105–120 (2015)

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REVIEW

Sexual and Asexual Reproduction in the ColonialAscidian Botryllus schlosseri

Fabio Gasparini, Lucia Manni,* Francesca Cima, Giovanna Zaniolo, Paolo Burighel,Federico Caicci, Nicola Franchi, Filippo Schiavon, Francesca Rigon,Davide Campagna, and Loriano Ballarin

Department of Biology, University of Padova, Padova, Italy

Received 23 April 2014; Revised 1 July 2014; Accepted 7 July 2014

Summary: The colonial tunicate Botryllus schlosseri isa widespread filter-feeding ascidian that lives in shal-low waters and is easily reared in aquaria. Its peculiarblastogenetic cycle, characterized by the presence ofthree blastogenetic generations (filtering adults, buds,and budlets) and by recurrent generation changes, hasresulted in over 60 years of studies aimed at under-standing how sexual and asexual reproduction arecoordinated and regulated in the colony. The possibilityof using different methodological approaches, fromclassical genetics to cell transplantation, contributedto the development of this species as a valuable modelorganism for the study of a variety of biological proc-esses. Here, we review the main studies detailing rear-ing, staging methods, reproduction and colony growthof this species, emphasizing the asymmetry in sexualand asexual reproduction potential, sexual reproductionin the field and the laboratory, and self- and cross-fertilization. These data, opportunely matched withrecent tanscriptomic and genomic outcomes, can give avaluable help to the elucidation of some important stepsin chordate evolution. genesis 53:105–120, 2015. VC 2014

Wiley Periodicals, Inc.

Key words: left-right asymmetry; blastogenetic cycle;budding; coloniality; cross-fertilization; self-fertilization

INTRODUCTION

Tunicates are filter-feeding marine invertebrates, bothsessile and pelagic, and they represent the sister groupof vertebrates (Delsuc et al., 2006, 2008). The namerefers to the presence of the tunic, a peculiar tissueembedding the whole organism. Ascidians, the largestand most studied class of tunicates, contain both

solitary and colonial species and are characterized bythe presence of a free-swimming, tadpole-like larva. Thelatter shares the main chordate features, i.e., a support-ing dorsal rod or notochord in the tail (hence the alter-native name of Urochordata assigned to the taxon), adorsal central nervous system in the form of a hollowtube that is anteriorly enlarged to form a brain, and amuscular tail (Burighel and Cloney, 1997).

The ascidian larva metamorphoses into a sessile,filter-feeding oozooid lacking most of the chordate fea-tures. Solitary ascidians grow in size during their adultlife; conversely, colonial forms propagate asexually,forming colonies of many small individuals (Brown andSwalla, 2012). Mechanisms of asexual developmentvary among colonial ascidian species in terms of bud-ding tissue and/or budding mode (reviewed in Brownand Swalla, 2012; Kurn et al., 2011; Nakauchi, 1982). Inaddition, various degrees of integration among zooidscan be observed: they can share the basal stolon, thecolonial tunic or the tunic and the colonial vasculature.This diversity, associated to phylogenetic evidences,suggest coloniality in ascidians occurred several timesindependently during evolution (Brown and Swalla,2012).

In the last few decades, solitary ascidians of the genusCiona have been intensively used as model organisms

* Correspondence to: Lucia Manni, Department of Biology, University

of Padova Via Ugo Bassi 58/B, 35121, Padova, Italy. E-mail:[email protected]

Contract grant sponsor: MIUR PRIN Projects, Contract grant numbers:

2009XF7TYT, 20109XZEPR; University of Padova Senior post-doc 2012

Project; GRIC120LSZPublished online 12 July 2014 in

Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/dvg.22802

VC 2014 Wiley Periodicals, Inc. genesis 53:105–120 (2015)

for the study of the molecular control of embryogenesisand development, and their sequenced genomes areavailable today (Dehal et al., 2002; Small et al., 2007).Unfortunately, the lack of genetic information on colo-nial ascidians has limited molecular studies on their sex-ual and asexual developmental pathways.

Botryllus schlosseri is a cosmopolitan colonial ascid-ian that is frequently found in shallow, temperatewaters, and can easily grow in aquaria. Colonies areembedded in a gelatinous, transparent tunic that allowsfor the direct and detailed observation of zooids underthe microscope (Fig. 1A–C).

FIG. 1. (A–C) Colonies of B. schlosseri and related sketches (A0–C0). A: Dorsal view. B: Ventral view. C: Details of a bud and its budlet in a col-ony at stage 9/8/4. In A0–C0: black: adult zooids; brown: stigmata in bud; dark blue: endostyle; gray: gut; green: peripharyngeal band; yellow:intersiphonal double band of pigment; lilac: budlet; orange: ventral cell islands; pale blue: tunic (in A0) and tunic vessels (in C0); red: bud; violet:oral siphon. (D–I) Metamorphosis of B. schlosseri larva. D: swimming larva; E: adhesion to substrate; F: early regressing tail; G: late regressingtail; H: bell stage, arrow: tail residual; I: oozooid, arrow: bud. (J) Life cycle of B. schlosseri (modified from Manni and Burighel, 2006).

106 GASPARINI ET AL.

ASEXUAL REPRODUCTION IN Botryllusschlosseri

The Colony of B. schlosseri

Each colony derives from the metamorphosis of atadpole-like larva into a sessile, filtering oozooid after afew hours of free swimming. Metamorphosis includesa set of characteristic steps that are summarized inFigure 1D–I.

The larva already bears the primordium of the bud,which is clearly evident on the right side of the oozooidand will become the first blastozooid (Fig. 1I and J) thatwill replace the oozooid at the generation change ortake-over (Sabbadin, 1955b). The latter is a colonialdevelopmental phase that lasts 24–36 h at 20�C, duringwhich old zooids are progressively resorbed andreplaced by buds that ultimately grow to an adult sizeand, finally, open their siphons to acquire functionalmaturity. The first blastozooid originates two palleal (pri-mary) buds, one on each side of the body, which can,then, form secondary buds or budlets on each side.Therefore, a colony usually contains three clonal blasto-genetic generations: adult zooids, grouped in star-shapedsystems and bearing primary buds that, in turn, have sec-ondary buds or budlets (Fig. 1C). This budding modality,called “palleal”, allows colony growth such that, underhealthy conditions, zooids of a colony bear two or more(up to four) buds. The colony is periodically renewed bycyclical (weekly at 20�C) generation changes. A blastoge-netic cycle is defined as the period of time from one gen-eration change to the next; a zooid, from its appearanceas a bud primordium to its resorption at the take-over,spans three blastogenetic cycles.

In a system, zooids have their oral siphon outwardsand their atrial siphons converging in a common cloacalchamber that opens to the exterior through a commoncloacal siphon. Zooids and buds are interconnected bythe colonial vasculature, deriving from the epidermisand crossing the tunic (Brunetti and Burighel, 1969;Gasparini et al., 2007). The colonial vessels form mar-ginal sausage-like blind termini, called ampullae, alongthe contour of the colony, which function as a reservoirof hemocytes. Ampullae of the colony growing edges(i.e., sides of colonial growth) are particularly elongatedand allow the adhesion of the colony to the substratum.

Since the original establishment of laboratory cultureconditions by Sabbadin (1955b), this species was used asa model organism for studies across several fields of bio-logical research. In particular, studies related to develop-mental biology were of particular interest due to thecontinuous, cyclical budding of this species that offersthe possibility to compare two alternative developmentalpathways related to sexual and asexual reproduction inthe same chordate species (Gasparini et al., 2011, 2013Manni and Burighel, 2006). The growing interest

towards the biology of B. schlosseri led to the release, in2013, of a first draft of the genome (Voskoboynik et al.,2013) and to the recent definition of the ontology ofanatomy and development (Manni et al., 2014).

Colony Rearing

According to Sabbadin’s method (Sabbadin, 1955b),for common and routine use, colonies can be gentlydetached from the substratum (bivalve shells, seagrassleaves) with a razor blade and allowed to adhere onclean glass slides. The latter are inserted in a numeratedsupport for the easy recognition of colonies and main-tained in a humid chamber for a couple of hours. Colo-nies are then gently transferred to aerated aquaria inthermostatic rooms and left undisturbed for an addi-tional day to allow stronger adhesion. Once stuck to theslides, colonies are kept upside down to keep themclean from their feces. They are fed with Liquifrymarine (Liquifry Co., Dorking, England) and unicellulargreen algae (e.g., Dunaliella sp.). The tunic of coloniesfrom the wild is usually embedded with sediment par-ticles and inhabited by various epibionts (polychaetes,amphipoda, nematodes, and ciliates). Therefore, twicea week, the colonies are cleaned with a soft brush, andthe old tunic is cut away with a razor blade, which isalso used to remove debris and the bacterial film fromthe supporting slides. As such, a new clean tunic isformed at the growing edges of the colonies, thus allow-ing for the direct observation of the zooids and buds.

When mature gonads appear in zooids, controlledcrosses can be obtained by transferring a couple of col-onies in small aquaria. Fertilization is internal andembryos develop into zooids. Mature larvae can be col-lected from the parental colony before their release(which normally occurs before the take-over) by cuttingthe body wall of the zooid with a thin tungsten needleto prevent the risk of losing the larvae. Alternatively,swimming larvae released from wild colonies in aquariacan be patiently collected with a pipette. The collectingeffort is facilitated by the observation that most of thelarvae are usually released at dawn and direct them-selves towards a light source.

Larvae are kept for a few minutes in low salinity(�15%) water to induce metamorphosis; they are thenrapidly reimmersed in normal seawater. After a fewminutes, when the tail resorption and anterior ampullaeelongation start, larvae are transferred on a glass slide ina drop of seawater and gently pressed to force the adhe-sion of the ampullae to the substratum. Slides are keptin a humid chamber for 1 h to allow the completion ofmetamorphosis and are finally immersed in aquaria.

Bud Development

Various authors have studied and described thedevelopment of B. schlosseri buds (reviewed by

REPRODUCTION IN A COLONIAL ASCIDIAN 107

Manni et al., 2007). A bud initially appears as a disc-likethickening of the parental mantle (i.e., the peribran-chial epithelium overlaid by epidermis and the connec-tive tissue between them). It then arches progressivelyto form a double vesicle; the outer component givesrise to the zooid epidermis, whereas the inner vesiclefolds to form the branchial and the peribranchial cham-bers, the gut, and the nervous system. The anatomy ofthe adult blastozooid is schematized in Figure 2.

Zooid development was divided in 11 stages by Ber-rill (1941a) and successively modified by Sabbadin(1955b) to render the staging method easier and hand-ier. Staging was then modified by Izzard (1973) in hisstudy on bud polarity and zooid bilateral asymmetry inblastogenetic potential. According to Sabbadin (1955b),stages 1–6 refer to budlets, stages 7–8 refer to buds, andstages 9–11 refer to adults. Stage 10 refers to sexuallymature zooids and is equivalent, with regards to zooidblastogenetic development, to stage 9, with the only dif-ference being the presence of mature gonads (Manniet al., 2007, 2014).

Bud development is influenced by seawater tempera-ture: the zooid life-span, which lasts approximately

three weeks at 18�C, reduces to 13 days at 26�C, andincreases to 65 days at 10�C (Table 1). Experiments ofcolony rearing at different temperatures showed thatthe duration of all of the developmental stages increasesas the temperature decreases (Rinkevich et al., 1998;Sabbadin, 1955b); the longer period is that of stage 9which lasts more than 5 days at 18�C (Fig. 3) (Sabbadin,1955b). The results show that, even at different temper-atures, buds progress in their development in a stereo-typed way and confirm the validity of the stagingmethod and its suitability for the study of asexualreproduction.

As stated before, the development of budlets, budsand adult zooids in a colony is synchronized, and variousdevelopmental phases can be identified during the colo-nial blastogenetic cycle. Phases are univocally defined bythe developmental stages of the three blastogenetic gen-erations and can be indicated by a series of three num-bers separated by slashes (e.g., 9/8/3), each referring tothe developmental stage of each colonial generation: thefirst to adult zooids, the middle to primary buds and thelast to secondary buds, respectively (Sabbadin, 1955b).The following developmental phases can be recognizedin a blastogenetic cycle: 9/7/1, 9/8/2–5, and 11/8/6 (thelatter corresponding to the take-over).

SEXUAL REPRODUCTION INBotryllus schlosseri

Sexual Reproduction and Germ Cells

Although B. schlosseri is attractive primarily for itscyclical and highly coordinated asexual reproduction,some aspects of its sexual reproduction have been stud-ied for many years. In particular, the correlation betweensexual reproduction (in term of germ cell recognition,gonad origin and differentiation, efficiency of embryo-genesis, and sexual maturation) and blastogenesis (interms of bud development and competition betweenblastogenesis and embryogenesis), under both naturaland laboratory conditions, were deeply analyzed.

In B. schlosseri, both the testis and ovaries form inthe mesenchymal space between the epidermis and the

FIG. 2. Sketch of an adult blastozooid of B. schlosseri (modifiedfrom Burighel et al., 1998).

Table 1Duration of Developmental Stages at Various Temperatures (According to Sabbadin, 1955b)

Developmental stage

Temperature

10�C 18�C 21�C 26.5�C

1–2 7.75 days 59 h 44 h 38 h2–3 4.75 days 35 h 26 h 23 h3–4 4.50 days 33 h 23 h 20 h4–7 7.00 days 64 h 44 h 31 h7–8 5.00 days 46 h 30 h 27 h8–9 13.00 days 97 h 76 h 60 h9–11 20.00 days 153 h 114 h 99 h11 3.50 days 30 h 20 h 17 hTotal duration 65.50 days 21.50 days 15.70 days 13.10 days

108 GASPARINI ET AL.

peribranchial epithelium of the right and left sides ofthe zooids. The testis consists of lobules filled with clus-ters of male germ cells at different stage of differentia-tion (Burighel and Martinucci, 2000). The ovary lacks adistinctive germinal epithelium and is composed ofonly a few functional ova with their envelopes (Izzard,1968; Manni et al., 1993, 1994; Sabbadin and Zaniolo,1979). In botryllids, the gonadal rudiment arises de

novo in each asexual generation in the form of a loosemass of undifferentiated cells (Kawamura et al., 2011;Manni et al., 1994; Mukai and Watanabe, 1976; Suna-naga et al., 2008). These are morphologically similar tothe hemoblasts migrating from the blood stream andfinding a niche in the mantle, where they differentiateand form the gonads; they are often accompanied byprevitellogenic oocytes migrating from an older asexualgeneration of zooids (Brown et al., 2009; Mukai andWatanabe, 1976; Sabbadin and Zaniolo, 1979). Undiffer-entiated cells located near the inner vesicle wallbecome a solid mass that is furnished with its own wall:the testis rudiment. Cells located in the periphery ofthe mass differentiate into the ovaries.

Sexual Reproduction in the Field

The sexual reproduction of B. schlosseri wasstudied in different localities in natural conditions(Brunetti, 1974; Chadwick-Furman and Weissman,

1995; Grosberg, 1988; Lo Bianco, 1909; Millar, 1952;Sabbadin, 1955a). In the Lagoon of Venice, sexual matu-rity was first analyzed by Sabbadin (1955a), who consid-ered the presence of gonads, embryos, and larvae inblastozooids from a large number of colonies collectedmonthly. Colonies were grouped on the basis of theirsize expressed as the number of systems in each colony.New colonies derived from sexual reproduction wereidentified as oozooids or colonies composed of a singleadult blastozooid.

The breeding season was assessed using both a directand an indirect method (Sabbadin, 1955a). The formerwas based on the identification of colonies with/with-out eggs/embryos/larvae. The indirect method eval-uated the variation in the frequency of colonies withdifferent sizes (from one to ten systems, for a total of1786 colonies) throughout a year. These observationsestablished that the breeding season in the Lagoon ofVenice ranges from April to November. Only during thisperiod, new colonies (i.e., colonies formed of a singleadult zooid) appeared; conversely, during the periodfrom January to March, the frequencies of all groups ofcolonies (each composed of a various number of sys-tems) were comparable. It is important to note that sex-ually mature colonies were recovered throughout theyear, even if the eggs were never fertilized.

Comparative studies on the annual growth and repro-duction of the same species in Scotland (Millar, 1952)

FIG. 3. Growth and relationships between the developmental stages of blastozooids belonging to successive generations of a colonyreared at 26.5�C. The generations represented are the fourth, fifth, sixth, and seventh (modified from Sabbadin, 1955b). Each cone repre-sents the average length of zooids (y-axis) during their asexual growth, from stage 1–11. Note that three generations coexist in the colony;therefore, the three cones partially overlap. During the take-over, regressing zooids can persist for few time after the appearance of newbudlets so that four generations of zooids can coexist. Zooid stages are color coded.

REPRODUCTION IN A COLONIAL ASCIDIAN 109

and the Gulf of Naples (Lo Bianco, 1909) allowed theidentification of the lower temperature limit (10–11�C)for the breeding of B. schlosseri, as defined by Orton(1920).

The colony life-span of B. schlosseri in the South–West coast of Scotland is one year (Millar, 1952),whereas in the Lagoon of Venice, sexual generations oftwo subsequent years normally coexist. Here, the col-ony life-span is 12–20 months, and most of the coloniesusually reproduce during two breeding seasons (Sabba-din, 1955a).

Sexual Reproduction in Laboratory

Accurate analyses of sexual reproduction in B. schlos-

seri were performed in laboratory conditions, where itis possible to observe gonad occurrence in new colo-nies starting from oozooids. In young colonies, gonadsare absent; only after some blastogenetic cycles germcells become recognizable in the early bud (Berrill,1941a,b). In particular, colonies follow this sequence ofsexual phases (Rinkevich et al., 1998; Sabbadin, 1960;Sabbadin and Zaniolo, 1979):

� asexuality, with no trace of gonads (up to ten blasto-genetic generations);� hypo-sexuality, with gonads that do not reach matu-

rity (few blastogenetic generations), with the follow-ing sub-phases: incipient sexuality (recognizableprimordial germ cells), hypo-femaleness (incompletedifferentiation of ovaries in the absence of testes),hypo-femaleness and hypo-maleness (incomplete dif-ferentiation of both ovaries and testes);� hypo-femaleness and maleness, with male sexual

maturity in one or more (up to nine) generations;� femaleness and maleness, with zooids maturing both

testes and ovaries for more than 20 generations.

The timing of ovulation, sperm release and fertilizationin a blastogenetic cycle of sexually mature colonies canbe summarized in the following points (Manni et al.,1993; Sabbadin, 1955b, 1960; Zaniolo et al., 1987):

� oocytes ovulate when the primary buds approachthe adult stage;� adults discharge most of the sperm one to two days

after ovulation (with sperm release continuing up tothe end of zooid life-span);� fertilization occurs just after the ovulation;� larvae are released just before the colonial take-over.

Because all maturing buds are at the same sexualstage, self-fertilization is prevented. If oocytes ovulatelater than usual and self-fertilization occurs, embryos donot reach the larval stage before the colony take-overand are resorbed by the parental zooids.

Gonadogenesis does not correlate with budletsize, and colonies of same age do not mature gonads

simultaneously (Sabbadin, 1955b). Unlike colonies fromthe fields, only a few blastozooids usually reach sexualmaturity in colonies reared in the laboratory; only in afew cases do they release mature swimming larvae. Inmost cases, eggs do not reach maturity, or adults withembryos are prematurely resorbed, thus preventing thecompletion of embryogenesis and larval hatching. It hasbeen shown that, differently from what has beenreported in nature, no colony mature eggs in laboratoryconditions at 13�C (Brunetti et al., 1984).

Investigations on the combined effects of tempera-ture and salinity on sexual and asexual reproductionhave demonstrated that the appearance of gonad pri-mordia in budlets is only a function of colony age. Con-versely, environmental parameters strongly influencecolonial growth and, in sexually mature colonies, gonadmaturation (Brunetti et al., 1984). Stress conditions cancause the absence of gonads for several generations(Sabbadin, 1955b).

Germ Cell Recycling

One of the most interesting and topical issue relatedto sexual reproduction in B. schlosseri is germ cellcycling within a colony. Indeed, germ line precursorsare mobile and can migrate to a niche in the secondarybud, where they expand and differentiate into gametes.Previtellogenic oocytes can also migrate through suc-cessive asexual generations until mature generationsappear; they then fix in a gonad and contribute to zooidsexual reproduction (Izzard, 1968; Mukai, 1977; Sabba-din and Zaniolo, 1979).

Exploiting the fact that contacting colonies sharing atleast one allele at a highly polymorphic fusibility/histo-compatibility (fuhc) locus can fuse their tunic and thecolonial vasculature and form a chimeric colony (Okaand Watanabe, 1957; Sabbadin, 1962), Sabbadin andZaniolo (1979) induced the fusion of the tunics and thecolonial vasculatures (parabiosis) of colonies homozy-gous at two loci for pigmentation genes and with oppo-site genotypes (AAbb and aaBB, respectively). Previousanalyses found that these two genes control two simpleMendelian characters: the orange pigmentation and thepresence of the intersiphonal double band (Sabbadinand Graziani, 1967). The orange pigmentation can betransferred from one colony to the other as pigmentcells circulate in the hemolymph; however, this is notthe case for cells responsible of band formation. Thepresence/absence of the intersiphonal double bandthus served as a marker to identify the two original col-onies in the chimera (Fig. 4A). Colonies were then sepa-rated after a few days and crossed with coloniescarrying the double recessive genotype (aabb), and theoffspring pigmentation was evaluated.

In addition to the expected phenotypes, offspringphenotypically Ab from aaBB 3 aabb crosses and

110 GASPARINI ET AL.

FIG. 4. A two-step experiment to study germ cell recycling (Sabbadin and Zaniolo, 1979). (A) The parabiosis step: 22 pairs of two differ-ently pigmented, fusible genotypes were fused and then separated to form ex-parabionts. Note that the transmissible character (orangepigment) is present in both ex-parabionts. (B) The cross step: 53 ex-parabionts were used as male and/or female and crossed with a doublerecessive genotype; the number of crosses, total, and heterochthonous offspring for each type of cross are indicated; the two percentagessummarize the heterochthonous offspring resulting from crosses, providing evidence for the transfer of both male (upper) and female (bot-tom) germ cells.

REPRODUCTION IN A COLONIAL ASCIDIAN 111

phenotypically aB from AAbb 3 aabb crosses wereobtained (heterochthonous offspring). These offspringwere derived from the germ cells (both male andfemale) exchanged during the transient parabiosis (Fig.4B). Heterochthonous offspring were collected up tothe 15th blastogenetic generation after the interruptionof the parabiosis. In a few cases, the entire offspringfrom certain generations were heterochthonous (germcell parasitism) (reviewed by Kawamura et al., 2011).This unequivocally demonstrated that germ cells, whichare capable of differentiating into gametes of either sex,circulate in the blood of B. schlosseri.

In recent years, the germ line development of colo-nial ascidians, such as Polyandrocarpa misakiensis

(Tatzuke et al., 2012) and Botrylloides violaceus

(Brown and Swalla, 2007), was investigated. However,most of the results focused on germ cell recycling andthe differentiation of B. schlosseri and the congenericspecies B. primigenus (Carpenter et al., 2011; Kawa-mura and Sunanaga, 2011; Kawamura et al., 2011; Rin-kevich et al., 2013; Rosner et al., 2013). In the latterorganism, expression studies of genes known to beinvolved in the germ line development of metazoans(primarily piwi, nanos, myc, and vasa orthologs) haveshown that (i) although nanos is expressed widely ingerm cells and somatic stem cells, it is mostly involvedin spermatogenesis; (ii) the transcription products ofvasa and piwi are distributed extensively in germ cells,but piwi is more widely dispersed in the hemocoel thanvasa; (iii) female germ cells and accessory cells can gen-erate from hemocoelic vasa-positive cells gathering inthe gonadal space; and (iv) both the germline stem cellsreservoir and the precursors of male gonads are amongthe hemocoelic piwi1/vasa-/myc1 cells (Kawamuraand Sunanaga, 2011; Kawamura et al., 2011).

In B. schlosseri, functional and expression studiesconsidering several genes involved in germ line differ-entiation (including DDX1, vasa, c-H2AX, cadherin,phospho-Smad1/5/8, piwi) indicated the presence ofvarious germ cell subpopulations (Rosner et al., 2013).Vasa-positive cells were also observed in the gonads ofB. schlosseri and in some circulating cells in both fertileand nonfertile colonies. Vasa was expressed in cell pop-ulations with high levels of ALDH activity that havebeen shown to contain functional germline precursors(Laird et al., 2005). Similar results were obtained in thesame work using an elegant and sophisticated approachbased on the transplantation of single undifferentiatedcells from a donor into a recipient colony: they argueagainst the presence of totipotent (both somatic andgermline) stem cells in colonies. Indeed, these singleundifferentiated cells contributed to either somatic orgermline chimeras, but not to both (Laird et al., 2005).Nevertheless, the presence of a population of mobilecells that can retain germline competence in recipientcolony after parabiosis (Sabbadin and Zaniolo, 1979;

Stoner et al., 1999; Stoner and Weissman, 1996) hasbeen recently and elegantly confirmed through the useof newly metamorphosed individuals far before sexualmaturity (Brown et al., 2009; Carpenter et al., 2011).Following their fusion with (or transplantation of theirhemocytes into) fertile colonies, the authors demon-strated the individuals/cells ability to reconstitute thegermline independently of the somatic progenitors. Toidentify and localize the source of the germline, vasa

expression and function was followed during embryo-genesis and at metamorphosis in oozooids and severalsubsequent blastogenetic generations. Collectively, thedata support the hypothesis that the germline progeni-tors specified during embryogenesis acquire an earlyfunctionality, independent of somatic progenitors, inmetamorphosed juveniles (Brown et al., 2009; Carpen-ter et al., 2011). The results also suggest the existenceof developmental cues that induce the germ cell pro-genitors to exit the circulation and enter a niche in thebudlet where they initiate germline formation. Morerecently, the formation of transient niches has beendescribed in adult zooids in which germ and somaticstem cells crowd together; the degradation of theniches at take-over enables the repeated trafficking andthe colonization of new sites of the following blastoge-netic generation and allow germ and somatic stem cellsto survive the individual zooids (Rinkevich et al., 2013;Rosner et al., 2013). The niches, called ventral cellislands (Manni et al., 2014), appear in the mantle flank-ing the endostyle of the adult zooids at phase 9/7/1 or9/8/2 and are no longer visible in adult zooid at phase9/8/5.

Self- and Cross-Fertilization in B. schlosseri

The analyses of self-fertilization in B. schlosseri wereinitially stimulated by Oka and Watanabe (Oka andWatanabe, 1957). They indicated that, in the relatedJapanese species B. primigenus, self-fertilization cannotoccur, and sexual incompatibility exists both betweenparental and descendant colonies and among coloniesderived from the same parents (“sister colonies”). Thisis an important issue because in colonial organisms,self-fertilization means fertilization both between game-tes of the same individual and between gametes fromdifferent zooids of the same colony, as zooids belongingto a colony are derived from the same metamorphosedlarva and are therefore genetically identical.

B. schlosseri is a protogynous hermaphrodite, and ina colony, eggs are ovulated approximately two daysbefore the peak of sperm emission. To verify the pres-ence and the role of self-fertilization in B. schlosseri,controlled crosses were performed, keeping subclonesof the same colony out of phase (through their previousrearing at different temperatures, Fig. 5A) in the sameaquarium in such a way that the maturation of sperm in

112 GASPARINI ET AL.

a subclone occurred contemporarily with the matura-tion of eggs in the other subclone due to protogynoushermaphroditism. Aquaria were filled with seawaterconserved in the laboratory for a few days to preventthe risk of the presence of contaminating sperms.Under normal laboratory conditions, the differencein phase was maintained through the successive blasto-genetic generations, and either subclone could

alternatively act as male or female. As controls, thesame colony was crossed with other colonies; the colo-nies were then separated and followed individuallylooking for larvae. The results obtained with thisapproach showed that, unlike B. primigenus, self-fertilization is possible in B. schlosseri.

The use of colonies that were homozygous at the twopigmentation loci and with opposite genotype (AAbb

FIG. 5. (A) Efficiency of self-fertilization in B. schlosseri (Sabbadin, 1969). Subclones reared at a lower temperature showed slower blasto-genic development. This strategy allowed for the collection of subclones out of phase, which could then be used alternatively as male andfemale. (B, C) Experiments of cross-(B) and self-(C) fertilization using homozygotic colonies for pigmentation characters. Descendantscould be easily attributed to self- or cross-fertilization due to their different pigmentation.

REPRODUCTION IN A COLONIAL ASCIDIAN 113

and aaBB, respectively) allowed for a better insight intothe problem (Sabbadin, 1969; Sabbadin et al., 1968). Inthis way, the offspring derived from cross- and self-fertilization was easily recognized as descendants fromcross-fertilization showed the phenotype AB, whereascolonies derived from self-fertilization had the pheno-types aB or Ab (Fig. 5B,C).

In addition, to verify the success of self-fertilization compared with cross-fertilization, threecolonies were put into the same aquarium

(Fig. 6A,B): two subclones out of phase from thesame parental colony (a1 and a2), and a third colony(b) at the same developmental stage as a1 (it could,therefore, cross only with colony a2). The followingoutcomes were expected (Fig. 6C): (1) eggs from a1

fertilized by sperms from a2 (self-fertilization), (2)eggs from a2 fertilized in equal number by spermfrom a1 (self-fertilization) and b (cross-fertilization),and (3) eggs from b fertilized by a2 (cross-fertiliza-tion). Collectively, the obtained data indicate that

FIG. 6. Comparison of the success of self-fertilization vs. cross-fertilization (Sabbadin, 1969). (A) a1 and a2 are subclones of the same col-ony at different blastogenetic phases; b is a different colony at the same phase than a1. (B) Possible crosses among selected colonies. (C)Possible crosses and descendants. The table on the right shows the results obtained from each cross.

114 GASPARINI ET AL.

a. Usually, self-fertilization either did not occur or wasexceptional.

b. Self-fertilization occurred with the same frequencyas cross-fertilization, when protogyny was avoidedusing colonies that were out of phase. This finding

confirms that in a colony, cross-fertilization aloneusually occurs because the sperm in the colony arereleased after the ripening of the eggs.

c. Self- and cross-fertilized eggs developed with thesame frequency, but the former showed a higher

FIG. 7. (A) Different vitality of dextral and sinistral buds in colonies reared at different temperatures. Bottom: position of blastozooids withrespect to the parental body side (dextral bud in light blue, sinistral bud in yellow) [data from (Sabbadin, 1955b)]. (B) Asymmetrical distribu-tion of gonadogenetic power in B. schlosseri (Sabbadin, 1955b). The figure is organized in two columns, each one for a different rearingtemperature. For each column: (i) upper row: temperature, number of analyzed colonies, range of studied blastogenetic generations; (ii)middle row: position of blastozooids with respect to the parent body side (dextral bud in light blue, sinistral bud in yellow), and position ofgonads (left side in red, right side in dark blue); (iii) lower row: results from zooids and gonads/eggs counts; the colors used to highlight thetable’s columns refer to the above sketch and indicate zooid position. In the table, red/blue bars refer to the presence of the gonad on theleft side (red) and on the right side (blue). Note that, at 26.5�C, most of the zooids form gonads.

REPRODUCTION IN A COLONIAL ASCIDIAN 115

frequency of anomalies during cleavage (23.11% vs.1.64%).

d. Larvae from self-fertilization metamorphosed at asignificantly lower percentage (51.55% vs. 87.82%);moreover, the growth of the colonies derived fromtheir metamorphosis was significantly slower (anaverage of 2.74 zooids per colony at the fifth blasto-genetic generation vs. 7.54 in colonies from cross-fertilization).

In addition, in the case of self-fertilization, the percent-age of abnormal larvae and of newly founded coloniesthat died between the first and fifth blastogenetic genera-tion was higher compared with cross-fertilization. Addi-tionally, the growth of surviving colonies, as measuredby the number of individuals at the fifth blastogeneticgeneration, was much slower than colonies derived fromcross-fertilization (Sabbadin, 1971).

Experiments were also performed with sexuallymature colonies collected in the Lagoon of Venice(reviewed in Sabbadin, 1972, 1973; Sabbadin et al.,1991, 1992) and the results were compared with thosepreviously yielded with the laboratory colonies (previ-ous experience has demonstrated that in laboratory-bred colonies, a certain depression of sexual reproduc-tion occurs in terms of gonad development, percentageof developed eggs, and progeny viability and growth).Colonies were crossed with subclones or alien coloniesout of phase, respectively, and the offspring of field col-ony was compared with that of laboratory colony.These results showed that:

1. The frequencies of fertilized and developed eggswere much higher for both self- and cross-fertilization in the series of field colonies comparedwith the series of laboratory colonies. Within eachseries, self- and cross-fertilization gave, overall,rather similar results.

2. In field colonies, the yield of normal larvae fromcross-fertilization was higher than that from self-fertilization. A less clear difference was foundbetween larvae obtained by cross-fertilization andthose obtained by self-fertilization as compared tothe laboratory series. Data referring to laboratorycolonies showed high variability between the sets(normal larvae from cross-fertilization vs. normallarvae from self-fertilization), consistent with thehypothesis of an inbreeding depression, which wasexpected to be different for different colonies.

3. Cross-fertilization gave a higher percentage of sur-viving colonies and a higher mean number ofzooids per colony with respect to self-fertilization,irrespective of the origin of parental colonies (fieldor laboratory).

Studies on self- and cross-fertilization declined in the1970s but still offered cues regarding the successiveinvestigations on the reproduction of B. schlosseri, pri-marily addressing the study of the morphology of thegonads and gametes (Burighel and Martinucci, 2000;Burighel et al., 1982; Manni et al., 1993, 1994; Sabbadinand Zaniolo, 1979), the success of fertilization in rela-tion to population density and sperm release (Johnsonand Yund, 2004; Phillippi et al., 2004; Stewart-Savageet al., 2001; Stewart-Savage and Yund, 1997), placenta-tion (Zaniolo et al., 1987), germ cell recognition andmigration (Ballarin et al., 2011; Brown et al., 2009; Rin-kevich et al., 2013; Rosner et al., 2013; Sabbadin andZaniolo, 1979).

ASYMMETRY IN REPRODUCTION INB. schlosseri

Asymmetry in Asexual Reproduction Potential

Budding is bilateral in B. schlosseri, but the two sidesare not equivalent in term of blastogenetic potential.The development of left buds is slightly slower than

Table 2Degenerated Buds at Various Developmental Stages [According to (Sabbadin, 1955b)]

TemperatureTotal number of

degenerated buds

Developmental stage at which buds degenerated

2 3 4–6 7 8 9

18�C 29 – 8 (27.6%) 11 (37.9%) 5 (17.2%) 5 (17.2%) –21�C 77 4 (5.2%) 33 (42.9%) 21 (27.3%) 7 (9.1%) 12 (15.6%) –26.5�C 87 10 (11.5%) 42 (48.3%) 20 (23.0%) 4 (4.6%) 10 (11.5%) 1 (1.2%)Total 193 14 (7.3%) 83 (43.0%) 52 (27.0%) 16 (8.3%) 27 (14.0%) 1 (0.5%)

Table 3Mean Number of Buds in Zooids of Various Blastogenetic

Generations

Blastogeneticgeneration

Number of zooids withMean numberof buds/zooid2 buds 3 buds 4 buds

1 14 2.002 14 2 2.123 13 3 4 2.554 22 11 4 2.515 58 9 6 2.296 68 25 2.277 55 17 2.24

Data refers to the analysis of blastogenesis in 14 colonies (Sab-badin, 1958).

116 GASPARINI ET AL.

that of the right buds, and a sort of competition occursbetween right and left budlets frequently leading to theatrophy of the smaller left buds (Fig. 7A).

Budlets of both sides can easily reach the develop-mental stage 3 (double vesicle stage), but from thisstage onwards, the majority of the left secondary budstend to stop their growth and undergo resorption. Mostof the resorbed buds do not overcome the developmen-tal stage 3 (Table 2). In this way, the ratio of zooids fromright and left buds is severely unbalanced towards theformer (Fig. 7A). This limits the growth of the colonybecause the number of its zooids is not doubled aftereach take-over. However, early budlets can split in two,especially those of the right side, so that a single budcan bear up to four budlets. The mean number of budsper zooid tends to stabilize to the value of 2.3–2.5, start-ing from the third blastogenetic generation (Table 3);zooids with 4 buds appear in the 3rd, 4th, and 5th blas-togenetic generation but disappear with the aging ofthe colonies (Sabbadin, 1955b).

Even the size of the buds increases from the 1st tothe following blastogenetic generations (Sabbadin,1958) (Table 4). According to Sabbadin (1955b), therearing temperature of 21�C seems to be the most favor-able for budding, as the ratio of right to left budsreaches the lowest value; in addition, the blastogeneticpotential of a bud is related to its original position withrespect to the parental zooids (Fig. 7A). Further analy-ses, carried out by Rinkevich and collaborators in theIsraeli population reared at 15, 20, and 27�C (Rinkevichet al., 1998), agree with the above results with regardsto a favorable temperature for budding.

Asymmetry in Sexual Reproduction Potential

Berrill (1941a, b) was first to analyze, in B. schlosseri,the position of gonads and to observe that the gonado-genetic potential is asymmetrical in the blastozooid.This finding was then verified by Sabbadin through con-tinuous observations of colonies for many blastogeneticgenerations and considering (i) the number of adultsand their origin from left or right buds, (ii) the appear-ance of mature eggs (on both sides or only on one sideof the zooid), and (iii) the number of eggs and theirlocation (left or right side) (Sabbadin, 1955b). Figure 7Bsummarizes the results of these observations in colo-nies reared at 21 and 26.5�C. The figure shows that thegonadogenetic potential varies in zooids at both

temperatures, being significantly higher on the left sideof zooids (gonads were never found on the right side ofzooids only).

Zooids developing on the right side of the parent pro-duce many more gonads than do those on the left sideat 21�C (gonads in 92% of zooids on the parental rightside versus 58% on the parental left side at 21�C; Fig.7B). A subsequent quantitative and comparative studyof gonad development in a huge quantity of coloniesreared at 18�C confirmed the asymmetry of gonadoge-netic power at this temperature (Sabbadin and Zaniolo,1979). This asymmetry is lost at 26.5�C. At this temper-ature, the frequency of zooids derived from the rightand left sides of the parent that show differentiatedgonads was similar: 95% versus 92%, respectively (Fig.7B) (Sabbadin, 1955b).

Collectively, these studies lead to the conclusionsthat

I. There is competition between the two sides of thezooids both in the blastogenetic and gonadoge-netic potential; the former is higher on the rightside, and the latter is higher on the left side;

II. Unlike what was hypothesized for Botryllus primi-

genus (Mukai and Watanabe, 1976), the appear-ance of gonads is not influenced by buddingbecause gonads form in zooids at stage 4, longbefore they produce their buds (at stage 7);

III. Temperature influences the bud gonadogeneticpotential.

CONCLUSIONS AND PERSPECTIVES

Colonial tunicates are the only chordates that are ableto reproduce both sexually and asexually. For this rea-son, these organisms offer the unique possibility toinvestigate how animals that exhibit recognized symple-siomorphies with vertebrates utilize different develop-mental strategies to build up their body. Amongcolonial tunicates, B. schlosseri remains the primarymodel species used to analyze and compare these strat-egies. Its introduction in the laboratory can be attrib-uted to Prof. Armando Sabbadin in Padova in the 1950s.Most of his initial work focused on the acquisition ofinformation on the B. schlosseri life cycle, which wasobtained through daily observations of a great numberof colonies, both in field and in laboratory. Sabbadin’s

Table 4Size (in mm) of Stage 8 Buds at Phases 9/8/2 and 9/8/5 (Sabbadin, 1958)

Colonial developmentalphase

Blastogenetic generation

2nd 3rd 4th 5th 6th

9/8/2 344.2 6 35.5 376.3 6 7.9 398.1 6 15.7 453.2 6 11.0 417.0 6 8.69/8/5 775.9 6 56.7 710.8 6 49.3 875.9 6 34.8 853.7 6 28.2 800.2 6 28.4

REPRODUCTION IN A COLONIAL ASCIDIAN 117

enduring and reproducible results provide a solid baseon which modern investigations are anchored.

For the past 60 years, the scientific community hasacquired a solid background in term of rearing, stagingand methodological approaches in B. schlosseri (Manniet al., 2007). The recent release of the B. schlosseri

genome (Voskoboynik et al., 2013) and the definition ofits ontology of anatomy and development (Manni et al.,2014) opens the possibility to study in greater detail itssexual and asexual reproductive strategies and their bal-ance in the colony. However, many questions remainunsolved. For example, how do somatic stem cells dif-ferentiate in the bud? How do germ cells recognize theniche where they localize and organize the gonad?Which mechanisms regulate the balance between sex-ual and asexual reproduction? How are the blastoge-netic and gonadogenetic asymmetric potentialsmaintained in the zooids? Which molecular mecha-nisms allow the evolution of the wide tunicate homeo-static power? The comprehension of these and otherbiological questions in the context of B. schlosseri willgreatly help the comprehension of chordate evolution.

ACKNOWLEDGMENTS

The authors thank Prof. A. Sabbadin for having transmit-ted them his great passion for ascidian biology and for hisscientific rigor. The authors have no conflict of interest todeclare.

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