Table Of ContentReference: Biol. Bull. 198: 367-378. (June 2000)
A Morphological Study of Nonrandom Senescence
in a Colonial Urochordate
ROBERT J. LAUZON1 *, BARUCH RINKEVICH2, C. W. PATTON3,
AND IRVING L. WEISSMAN4
1Department ofBiological Sciences, Union College, Schenectcidy. New York 12308: 'Israel
Oceanographv and Limnological Research, National Institute ofOceanography. Tel Shikmona,
Haifa, Israel 31080: 3Hopkins Marine Station ofStanford University, Pacific Grove,
California 93950: and ^Department ofPathology, Stanford University School
ofMedicine, Bcckman Research Center, Stanford, California 94305
Abstract. Botryllus schlosseri is a clonally modular as- indicate that senescence is accompanied by unique morpho-
cidian, in which individuals (zooids) have a finite life span logical changes that occur systemically, and which are dis-
that is intimately associated with a weekly budding process tinct from those occurring during takeover. We discuss our
called blastogenesis. Every blastogenic cycle concludes findings in relation to current experimental models ofaging
with a synchronized phase of regression called takeover, and the possible role of a humoral factor in bringing about
during which all zooids in a colony die, primarily by apo- the onset of senescence.
ptosis, and are replaced by a new generation of asexually
derived zooids. We have previously documented that, in Introduction
additiontothiscyclicaldeathphase, entire coloniesundergo
senescence during which all asexually derived individuals The colonial urochordate Botryllus schlosseri (Tuni-
in a colony, buds and zooids, die in concert. In addition, cata, Ascidiacea) is a ubiquitous, filter-feeding inhabitant
when a specific parent colony (genet) is experimentally of shallow waters and harbors worldwide, where it is
separatedintoanumberofclonal replicates (ramets), ramets found on a wide variety of hard substrates (Sabbadin,
1969). Each colony arises from a sexually produced,
frequently undergo senescence simultaneously, indicating chordate larva that attaches itself to a substratum and
tHiinhfeaorlrtena,ibmaoo,wrreatttahldoaitrotycye-uxmmhacieiabnnnitttamianannomeindofrrecpasohntlodolnoioitmgesieslmcfofarrlitonapmloinrtMtoyorn.anrittNaeonrondfeorysmaeBnnadfeyaso,scmheCinaosclnee-.- udgtionradottewehrtleghyospeabfrseergnomitmen.tsiaWtmstiootlrahptpiehrnrooasldaiubwscoa.eultlTbhaouennddesreawrsteeuhleagttkeinnoegbrteiiotgcowiazneloaeltoyneidi1dae6sinmtomiaucenta-d-l
nescence proceeded according to a series of characteristic 20C, the oozooid regresses and the first asexual gener-
changes within the colony over a period ofabout one week. ation of buds simultaneously reaches functional maturity
These changes included systemic constriction and conges- (Sabbadin. 1969). This first generation of blastozooids
tion ofthe vasculature accompanied by massive accumula- produces its own buds, which in turn mature in about a
tion ofpigment cells in the zooid body wall (mantle), blood week and subsequently die after giving rise to an addi-
vessels, and ampullae; gradual shrinkage of individual zo- tional set of zooids. Each such cycle, called blastogene-
oids; loss ofcolonial architecture, and ultimately death. At sis, is entirely temperature-dependent and of shorter du-
the ultrastructural level, individual cells exhibited changes ration at higher temperatures (Milkman. 1967; Sabbadin.
typical of ischemic cell death, culminating in necrotic cell 1969; Rinkevich et al., 1998). During the last stage of a
lysis rather than apoptosis. Collectively, these observations typical blastogenic cycle (the takeover stage), all zooids
in a colony regress over a 24- to 30-h period through the
*To whom correspondence should be addressed. E-mail: lauzonr@ death of all visceral tissues, primarily by apoptosis
union.edu (Lauzon et al., 1992, 1993). Thus, the life history ofa B.
367
368 R J I U/ON // \l
schlossericolony ischaracterized by a long scries of such indicating that senescence is reproducibly associ-
blastogenic cycles, which succeed one anothct pcnoih ated v\ ith characteristic morphological changes that are con-
cally. In time, a colony can grow up to hundreds ol ,] IIOMI colons to colony and appear identical to those
genetically identical blaslozooids. with all /muds .nul in iicld colonies. Furthermore, since field studies
buds in the colony being developmental!) synchronized only described macroscopic changes occurring at the
(Milkman. 1967). Zooids are typically arranged in star level iBrunetti, 1974; Chadwick-Furman and Weiss-
shaped groups called systems, which are embedded in a man. 1995a). it has become imperative todocument cellular
gelatinous tunic. Blood continuously Hows ilnm uul subcellular features occurring during nonrandom senes-
the systems, zooids. and buds, and into the peripheral cence, as a means of understanding its underlying mecha-
endings of blood vessels called ampullae. nism. Here, we provide evidence that the histological and
In addition to this week.lv death process, individual col- ultrastructural features of cell death during nonrandom se-
onies have finite life spans, Colonies grovwi under tield nescence are distinct from those occurring during takeover
conditions are characterized hs short, sub-annual lite spans and ultimately reflect systemic changes within the colonial
(Berrill, 1950; Millar. I^2. Hrunetti. 1974: Brunetti and vasculature.
Copello. 1978: Grosherg. 1988; Chadwick-Furman and
Weissman, 1995a, b). For instance, in K. \dilosscn'colonies Materials and Methods
from Monterey Bay, maximum life span ranges from about
3 months for animals born in the sprint; to just over 8 Liihtiruton- manculturc ofBotryllus schlosseri
months for those born in the fall (Chadwick-Furman and Animals were maintained between 16 and 17C under
Weissman, 1995a). Senescence appears to be the primary laboratory conditions, as previously described (Boyd ct
cause of mortality in field-raised colonies, and involves a ill.. 1986; Rinkevich ct til.. 1992). In our initial study on
series ofcharacteristic degenerative changes that culminate senescence and mortality in this species, we had observed
in the death ofthe entire colony (Brunetti. 1974; Chadwick- that senescence in laboratory-maintained colonies could
Furman and Weissman. 1995a). In contrast, laboratory- occur in either a random or a nonrandom fashion
maintained colonies from Monterey Bay have been docu- (Rinkevich et al.. 1992). To document the morphological
mented toexhibit life spans ofmore than 7 years (Rinkevich changes that are consistently associated with senescence.
and Shapiru. 199X1. and counterparts from the Venetian we have focused solely on the latter type. For the animals
Lagoon (Italy) were reported to have been kept alive for up reported in this study, individual colonies (genets) were
to 3 years (Sabbadin. 1969). It is well documented that this separated with the use ofa razor blade into several clonal
species exhibits tremendous plasticitv. and that life-history replicates (ramets). Each ramet was attached to a slide,
traits are subject to variation by changes in environmental after which they were maintained separately or within the
conditions (Chadwick-Furman and Weissman, 1995a, b; confines of the same tank. Our previous findings had
Rinkevich el ai, 1998). For instance, colonies grown under indicated that, within the group of colonies exhibiting
field conditions attain large sizes, form compact structures, nonrandom senescence, the placement of ramets within
and reproduce rapidly, whereas laboratory-maintained col- the same or different tank did not affect the onset of
onies grow slowly, often fragment into subcolonies, and senescence. That is. ramets derived from the same genet
may not undergo sexual reproduction (Chadwick-Furman underwent simultaneous degenerative changes that led to
and Weissman. 1995b). Vet. it is precisely in the laboratory their death, regardless of whether they were placed in the
environment that this animal can best be manipulated and same tank or kept in separate tanks (Rinkevich cl til..
studied as a model svstem of invertebrate immunity (re- 1992). Dates of subcloning. colons number, and numbers
viewed in Magor ct til.. I999i. development (Milkman. of ramets were recorded as previously described (Rinke-
1967; Lauzonm//.. 1992; Kinkevicli ct ,il.. 1992; I.au/on ct vich t'l nl.. 1992). Life span was recorded as number ol
ai. 1993), cell and molecular biology (DeTomaso </ <//.. hiastogenic cycles until death. Observations of colonies
1998), and evolutionary studies (Stoner ct til.. |999i. We and determination of zooid length were carried out will)
I ih.n lahoralorv colonies (genets) and the (he use of a stereomicroscope (Stemi SV 6, Carl Zeiss,
lets) derived horn these genets uiulci Germany) equipped with an ocular micrometer.
at the end of then life span that
Fforl (Kmkcvich i'I ill.,
to ilu death ol prev louslv
'iHili.mcouslv within Colonies undergoing nonrandom senescence were sepa-
indom nature ol this laled into individual systems (star-shaped groups of zooids)
proce , , mild be incdi with a razor blade on the third day alter the onset of
.;/. 1992). I he systemic vasculature constriction and increased pigmenta-
findings i .,, pii-v,,,us obser- tion ol /oniils. Day 3 was selected as representative of an
SENESCENCE IN A COLONIAL ASCIDIAN 369
early stage of senescence. The systems were processed for Y8 63.0
electron microscopy as previously described (Lauzon etal.,
1993). In brief, the systems were fixed in a solution of 1%
glutaruldehyde, 1% acrolein (freshly distilled), and 80% Y7 51.0 2.0
artificial seawater (ASW) for 2 h at room temperature.
Individual systems were washed several times in ASW for
51.3 10.5
a total of 1 h and were then post-fixed in 1% osmium 404 b
tetroxide for 1 h in ASW at room temperature. The speci-
mens were subsequently dehydrated in a graded acetone
series and embedded in Spurr's resin (Polysciences, War- 275 35.0
jjr
rington, PA) according to the manufacturer's specifications.
Ultrathin sections were cutwith adiamond knife (EM Corp,
Chestnut Hill, MA), placed onto Formvar-coated 300-mesh
37.0
nickel grids, stained with 2% aqueous uranyl acetate and 1% 403d
EM
lead citrate, and examined with a Phillips 201 trans-
mission electron microscope. 386 58.0
i
Histology
68.4 1.3
Individual systems from senescent colonies (days 3 and 248 hsi
5) were isolated with the use of a razor blade and fixed in
1% paraformaldehyde-filtered seawaterfor2 h at4C. Days 39.0
433 z
3 and 5 were selected as representative ofearly and middle
senescence respectively. Specimens were subsequently de-
hydrated in a graded ethanol series, and embedded in JB-4 435 h 35.0
plastic (Polysciences, Warrington, PA) according to the
manufacturer's specifications. Sections ranging between 2 56.0
360 c
and 3 /u.m in thickness were generated with a Histoknife
(Diatome Inc., Switzerland) along the anteroposterior axis
of zooids, as previously described (Lauzon et al., 1992).
Individual sections were stained with 0.01% toluidine blue- 433 af 49.3 5.5
sodium bicarbonate, mounted with Permount (Fisher Scien-
tific, Springfield, NJ), and observed under bright-field mi-
croscopy (Olympus BH-2 model. New York/New Jersey
Scientific, Inc., Middlebush, NJ).
6 8 10 12 14 16
AGE
(months)
Results
Senescence is a heritable process in some Botryllus rivFeidgculroena1l.repLliifceastpesanthoaft"epxaprreensts"nBoontrryalnlduosmscmholrotsasleitryi.cTohloenigeesneatndnudme--
schlosseri colonies bersareindicatedontheleftmargin.Thelifespanoftheoriginalparentpart
is indicated by a thick horizontal line, whereas lifespan from subcloned
In our previous study of senescence in B. schlosseri ramets derived from the original parent part is indicated by a thin line.
colonies maintained under laboratory mariculture, 17 of41 When multiple ramets were subcloned on a given day, their litespans are
genets were documented to exhibit nonrandom mortality indicated by parallel horizontal lines originating from a common perpen-
(Rinkevich et al.. 1992). The life spans of some additional dicular line. The numbers above each thick line represents the mean
flaoblolroawtionrgy-abnoirmnalsanudntirlaitsheedenctoilroeniceoslowneyrmeandiefteesrtmeidnseednebsy- neraautcmohbryegrecnoeontfd.ibtEliaoanscsth.ogbelnaisctocgyecnliecscuynctliel ldaesattsh7 daystsanatdar1d6-de1vi7aCtiounnd(eSrD)labfoo-r
cent features indicative of impending death. Eleven genets
were selected on the basis of their ability to undergo non-
random mortality. As illustrated in Figure 1, death was
synchronous between all ramets in 7 of 1 1 colonies (Y8, which diedeither4(Y7) or6 (248hsi and433af)blastogenic
275jjr, 403d. 386i, 433z, 435h, 360c). occurring within the cycles earlier. In colony 404b, two ramets died before the
same blastogenic cycle. In 3 of the remaining 4 colonies others one 2 blastogenic cycles earlier and the other 24
(Y7, 248hsi and 433af). all ramets died simultaneously, cycles earlier(in this ramet, death wasprobably unrelatedto
with the exception of one ramet from each of the genets. senescence).
370 R J l.AUZON I I \1
Stereomicroscopic observations ofBotryllus schlosscn ical stimulus. By day 7. all zooids in the colons were dead.
colonies undergoing nonrondom senescence essed by lack of heartbeat and shutdown of the si-
phons (Fig. 2d). At day 9, the colony was in an advanced
The findings in this report are based upon state of decay and zooids were no longer recognizable.
senations, over several years, ofcharacteristic Protozoa were observed swimming on the surface of the
preceded death of individual colonies. H softened tunic, presumably contributing to the dccav pro-
senescence could not be accurately pred L-IVCII cess (data not shown).
genet or raniet. colonies were ol these In some instances, the onset of senescence was also
characteristicchanges. When observed Micros- observed during the takeoverphase ofthe blastogenic cycle.
copy, ramets derived from tlu- d a senes Takeover is characterized by the simultaneous regression of
of regressive changes over It jieriod. These the previous generation of zooids and the increase in size
changes resulted in the near simultaneous death of a genet and maturation of the new generation of pre-fiinctional
following a given numlvi oi !i|:;siogcnic cycles. zooids. In nonsenescent colonies, takeover begins with the
The earliest recogni/ablc features ot nunrandom senes- shutdown of the oral and excurrent siphons, followed by
cence involved the constriction of blood vessels throughout polarized zooidcontraction: it is generallv completed within
a colony, resulting in the accumulation of pigment cells 24 to 30 h between I6-18C (Lauzon a <//.. 1992). At the
ssithin the zooid mantle, buds, blood vessels, and ampullae conclusion oftakeover, the zooid remnants appear as small
(Fig. 2). In nonsenescent colonies, ampullae serve as reser- vesicles, and a new blastogenic cycle begins with the open-
voirs forpigmentcellsduring takeover, and thus accumulate ing of oral and excurrent siphons in the next generation of
within them only during this phase ofthe blastogenic cycle zooids (Lauzon et <//.. 1992). In contrast to the process seen
(Lau/on el nl.. 1992). In senescent colonies, however, this in nonsenescent colonies, zooids were never complete!),
morphological feature was consistently observed to occur resorbed in colonies undergoing nonrandom senescence. In
independently of takeover. The ampullae also began to arepresentative colony atday 5 ofthe senescent process, the
retract from the leading edge of the colony, perhaps as an previous zooid generation was still visible 60 h after the
indication of declining health (Fig. 2b). At this stage of onset of takeover, although considerable resorption had
senescence, zooids were still actively feeding, as assessed already taken place (Fig. 2e). At this stage, the next asexual
by the functional nature ol both oral and excurrent siphons. generation of zooids had open oral and excurrent siphons
but had begun to shrink in size relative to their nonsenescent but exhibited signs of fragmentation, in that the) failed to
counterparts. The average /ooid length for randomly se- form complete star-shaped systems. Instead, zooids were
lected zooids from nonsenescent coloniesduring the asexual found individually or assembled into groups oftwo or three
growth phase ofthe blastogenic cvele was 2.34 mm 0.29 with a common excurrent siphon. In addition, the systemic
(n = 85). compared to 1.69 mm 0.15 forearly senescent vasculature had become sluggish, and pigment cells
zooids (n == 105: P < 0.05 bv paired Student's i test). which normally leave the ampullae at the conclusion of
Blood flow slowed considerably between days 1 and 3. and takeover remained within them. At 110 h after takeover,
as a result of heavy congestion of the vessel walls with the entire colony was dead, with resorption products still
pigment cells, blood circulation became progressively more visible (Fig. 20.
sluggish between days 3 and 6 of nonrandom senescence.
The first vessels to be affected were the marginal \csscN Histological observations ofnonrandom senescence
near the periphery of the colony. Buds were also affected
during this process: they gradually separated from the par- Histological studies have previously been carried out on
ent zooids (data not showni. eventually becoming develop- nonsenescent zooids during blastogenic growth (Lauzon et
mentally arrested. By day 6 of nonrandom senescence the i//., ll>l)2). In contrast to those nonsenescent zooids. indi-
ystems had become highly disorganized, with concomitant vidual zooids at day 3 of nonrandom senescence promi-
loss of the common excurrent siphon, and zooids had sep- nently displayed congested visceral tissues and vascular
arated from one another (Fig. 2c). Zooids at this degenera- spaces (Fig. 3a). A large proportion of cells from visceral
mm =
te furthershrunken ( 1.01 0.17.;; 102; tissues also appeared swollen with a hollow appearance.
Miuleni's I test), displayed a more These cells invariabh took up verv little stain (weakly
i UK! were scattered individu- basophilic) and were at times harboring pyknotic nuclei. In
small groups of two to three some cases, cells with large, open spaces suggestive ofcell
I individual zooidsexhibited lysis were also found. At this stage of senescence, visceral
' impending death. tissues (Fig. 3a. b. for stomach, intestine, endostyle: pha-
in senescence the oral ryngeal shis and neural complex not shown) were still
sively more dilated confined In an intact perivisceral epithelium, and thus lew
-i-nsitivity to mechan- cells were free in the perihraiichial cavity (Fig. 3b). In
SENESCENCE IN A COLONIAL ASCIDIAN 371
Figure 2. Bnnyllus scli/osseri colonies at different stages of nonrandom senescence, (a) Appearance of
star-shaped groupsofzooids in a nonsenescentcolony, (b)Arepresentative colony 3 days following the onset
ofsenescence. Note the dark pigmentation pattern within zooids and accumulation ofpigment cells in blood
vessels and ampullae, (c) Atday 6ofnonrandom senescence, there is lossofcolonial architecture. The zooids
withinindividual systemshavedisconnected fromoneanotherfollowing lossofthecommonexcurrentsiphon.
Note that the siphons from individual zooids have greatly dilated. Systemic blood flow has almost shut down
except forthe central partofthecolony, (d)The end stage ofsenescence (day 7) ischaracterized by complete
stasis of the colonial vasculature and dead zooids with closed siphons, (el A different colony at day 5 of
nonrandomsenescence,observedat60hoursfollowingtheonsetoftakeover(bottom leftcorner). Notethatthe
zooids ofthe previous asexual generation are still visible (black arrows).(f) The same colony observed at 110
hours following onset oftakeover, depicting dead zooids with resorption products still visible (black arrows).
Panelsa-c anddrepresentdorsal and ventral views respectively ofzooids from different portionsofthe same
colony (orange colormorph) undergoing nonrandom senescence. Panelse-faredorsal views ofthe same part
ofadifferentcolony (blue/browncolormorph).The numbersatthe bottom leftportion ofpanelsa-d represent
thedaysfollowingonsetofnonrandomsenescence.Abbreviations: am,ampulla;bv,bloodvessels;es,excurrent
siphon;os,oralsiphon;t,tunic. Asterisksrepresenttheregionoccupiedby thecommonexcurrentsiphon. Scale
bars equal 1 mm for panels a-d and e-frespectively.
372 R J I.AU7.ON T AL
te
Figure 3. Histological observations of Botryllus schlosseri zooids and buds at day 3 of nonrandom
senescence,(a)Ventralsectionofasenescentzooid.Atthisstage,zooidsarecongestedandvisceralorgansha\c
a spongy appearance, characterized by large empty spaces or swollen cells with weakly basophilic cytoplasm,
(b) Higher magnification of the endostyle near the ventral sinus depicting the swollen cells that are weakly
basophilic (large black arrows). Cells with multiple nuclei are widespread along the anteroposterioraxis ofthe
zooid. indicativeofextensivephagocytosisofdyingcells, (c) Primary and secondary budsofaday 3 senescent
colony appearmorphologically normal ascompared with buds from a nonsenescentcolony (d). Abbreviations:
bb. branchial basket; bl. blood; en, endostyle; epi. epidermis; h. heart; int, intestine; pbc. peribranchial cavity;
pbe. peribranchial epithelium; ps. pharyngeal slits; pv. perivisceral epithelium; sec. hud. secondary bud; sto,
stomach; t. tunic; te. testes. Scale bars = 240 /j.m (a. c. d) and 120 /xm (b).
contrast to the features associated with zooids at day 3 of ferring a congested appearance (Fig. 4c) not seen in nonse-
senescence, primary and secondary buds were morpholog- nescent colonies during blastogenic growth (Fig. 4d). In
ically indistinguishable from buds of nonsenescent animals addition, phagocytic cells containing many ingested cells
(Fig. 3c, d). with pyknotic nuclei became more prevalent overthe course
By day 5 of senescence, the squamous perivisceral and of senescence (Fig. 4a. c). By day 5, the buds began to
perihranchial epithclia and the epidermis of x.ooids was exhibit morphological degeneration similar to that seen in
visibly swollen, thus exhibiting degenerative changes sim- /.ooids two days earlier, and the colonies usually died at day
il.n tu those observed lor other cells at day 3 (Fig. 4a). 7, often loaded \\ilh trapped eggs anil sperm (data not
Ill . hanges presumably led to the release of individual shown|.
cells in toups of cells from the viscera or blood into the
perihranchial cavity (Fig. 4a). In contrast. Miter-feeding
/ooids Inmi MMIsenescent animals retained intact squamous
perivisceral ami peribranchial epithelia (Fig. 4b). Through- The ultrustructure of senescent /ooids revealed a number
out the pidL'K n "I senescence, as blood vessels con- of features diagnostic of ischaemic cell ileaih \\ith cellular
stricted and blood lln\>. became sluggish, pigment cells and necrosis as an end-point; these supported the histological
other blood cells ai ilated \Milun ampullae, thus con- profile seen by light microscopy. At day 3. small groups of
SENESCENCE IN A COLONIAL ASCIDIAN 373
.vv
*."
-
\
1
Figure4. Histologicalobservationsofzooidsatday5ofnonrandomsenescence, (a)Dorsalsectionofaday
5 senescent zooid near the neural complex, outlining the progressive disorganization of the pharyngeal slits
within the branchial basket,and the sheddingofindividual cellsorgroupsofcells intotheperibranchialcavity,
(b) Pharyngeal slits in a comparable dorsal section from a nonsenescent colony. Epithelia (peribranchial
epithelium andepidermis) fromnonsenescentzooids are squamous, whereasduring senescence, the epithelium
swells,(c)Cross-section through aperipheral ampullafromasenescentcolony atday5 ofsenescence. Notethe
congestedappearanceofbloodcellswithin theampullaascomparedtoanampullafrom a nonsenescentcolony
(d). Abbreviations: apc, ampullarpadcell; bb, branchial basket; bl. blood; c.cilia; epi,epidermis; fc, freecells
inperibranchialcavity;ng,neuralgland;pbc,peribranchialcavity;pbe,peribranchialepithelium;pc,phagocytic
cell; ps, pharyngeal slits. Scale = 60 fim.
cells interspersed within visceral tissues began a sharp de- Golgi apparatus, seen primarily in those cells in which
crease in cytoplasmic density accompanied by pronounced cytoplasmic density was markedly reduced. In contrast, the
distension of the endoplasmic reticulum (Fig. 5a, b: plasma membrane remained intact at this time, and contacts
branchial basket; Fig. 5c, d: intestine). Within these cells, with neighboring cells were maintained (Fig. 5a-d).
the inner and outer leaflets of the nuclear envelope sepa- During the takeover phase of blastogenesis, visceral tis-
rated, but the chromatin configuration remained nearly nor- sues from the zooid undergo apoptotic cell death, and mi-
mal as compared with unaffected neighboring cells (Fig. 5a. tochondrial architecture is generally preserved throughout
d). The nuclear hallmarks of apoptosis, such as chromatin this process (Lauzon elai, 1993). However, examination of
condensation and margination against the nuclearenvelope, the mitochondria during nonrandom senescence revealed
were not observed in the senescent tissues examined by that they too underwent pronounced degenerative changes,
electron microscopy (Fig. 5: branchial basket and intestine; appearing swollen, with their cristae both distended and
Fig. 6: endostyle and stomach). Another change associated disorganized (Fig. 5b-d). More importantly, these changes
with the nonrandom senescent phenotype was a prominent frequently preceded the loss ofcytoplasmic density (Fig. 5a,
374 R J. LAl'/ON /:/ ,!/..
Figure 5. I'ltrasiructural analysis of visceral cell Jcalh al day 3 ofnonrandoni senescence. Cross-sections
throughthebranchialbasket(a.b)andintestine(c,cl)atday3ofnonrandonisenescence. UHrastructural features
are diagnostic of ischennc cell death, and include the separation ofthe nuclear envelope (black arrowheads in
a.c.andd),apronounceddecrease incvtoplasmicdensity(a-d).distensionoftheendoplasmic reticulum(h-d).
swelling ol mitochondria with concomitant distension and disorganization ol cristae (h-d). and appearance ot
a prominent Golgi apparatus (a. b). Note that mitochondnal disruption is also observed in cells with normal
cuoplasmic density (large black arrows in a.cl. Incontrast, thechromatin appears normal at this stage, and the
plasma membiaiie remains intact (a-d). Abbreviations: er. endoplasmic reticulum; g. Golgi apparatus; m.
mitochondria; n. nucleus; mi, nucleolus; pm. plasma membrane. Scale bars = 1 /iin.
o. Interestingly, this degeneration did not occur simulta- Discussion
neous!;, in all cells ul a given visceral tissue (Fig. 5a) or We have previously documented the longevity ol 41
other tissues (Fig. 6h, d). Other ultrastruetural changes thai independent, laboratory genets and observed that senes-
vscie observed at day 3 of nonrandoni senescence included cence in lioirvlln.\ .s<7;/c.v\<T/ could manifest itself in either
accumulation ol large intra-cytoplasmic lipid inclusions random or nonrandoni fashion (Rinkevicli ft <il.. IW2).
ilnoiiL'lioul cells ofthe \ iscera and hlood (Fig. 6a), fusion of Dining nonraiidom senescence, specilic parental colonies
microvilli at the brush border of the digestive epithelium and iheir subcloned ramets died within a defined period of
(Fig. Mi), and extensive phagocytosis of dying cells time, often within the same blastogenic cycle. Here, we
through .1 the anleroposterior axis ofthe /ooid (Fig. 6c). In have described a morphological profile of nonraiidom se-
addition ' .iimal activity was inferred to be an important nescence al the level of the genetic individual, the genet.
component in I! death in some instances, as autophago- Under laboratory conditions, specific colonies had finite life
somes were h -nily observed within cells ol the en- spans, and most of ihe ramets from a given genet exhibited
dostyle near the ntral sinus, independent!) of the necrotic in-concert, degenerative changes that signaled imminent
changes described .ibove (Fig. Ml death within about 7 days. The nonraiidom senescent phe-
SENESCENCE IN A COLONIAL ASCIDIAN 375
Figure6. Furtherultrastructural alterationsin zooidsatday 3 ofnonrandom senescence, (a) Individualcell
withprominent lipidinclusion found inbloodspaceof/.ooid. Suchcellsare found randomlydispersedinblood
and viscera throughout the dorsoventral plane ofindividual zooids. (b) Cross-section through the brush border
ofthe gastric epithelium reveals fused microvilli. These cells do not yet display any disruption ofthe nuclear
membrane,butsomelossofcytoplasinicdensitycanbeobserved(largeblackarrowheads),(c)Phagocytefound
inthebranchialbasketwithmultiplephagolysosomes.(d)Longitudinalsectionoftheendostyle.neartheventral
sinus,revealsprominentmembranewhorlsindicativeofautophagy. However,thetightjunctionshaveremained
intact. Abbreviations: ap.autophagic vacuoles; mv. microvilli:ec,engulfedcell; n, nucleus; pi.phagolysosome;
=
pv, perivisceral epithelium; tj. tightjunctions. Scale bars 1 (xm.
notype in this species was defined by characteristic mor- dilation of siphons: and separation of buds from the parent
phological changes that were identical from colony to col- zooids. The concordance between our macroscopic obser-
ony, and which began with the systemic constriction ofthe vations oflaboratory colonies and those resulting from field
colonial vasculature. followed by cellular necrosis as an work are important because this species exhibits tremen-
end-point. Macroscopic, senescence-associated changes dous plasticity of life-history traits in response to environ-
similar to the ones reported here have been documented in mental variation (Brunetti. 1974; Chadwick-Furman and
field-grown B. schlosseri colonies (Brunetti. 1974; Chad- Weissman, 1995b; Rmkevich et ai, 1998). Furthermore,
wick-Furman, 1995a). For instance, Brunetti (1974) de- our studies are the first to document in detail the morphol-
scribed a mortality process with rapid onset, inB. schlosseri ogy of senescence at the cellular and subcellular levels in
colonies from the Venetian Lagoon (Italy). The features of this species.
this process included slowing of blood flow: storage of In the clonally modular ascidian B. schlosseri, death
pigment cells within blood vessels, ampullae, and zooid processes can be investigated at three levels; the individual
body wall; inability of zooids to form complete systems; zooids, the ramets, and the senet. Studies on individual
376 R. J. LAL'ZON tT AL
zooids (Milkman, 1967: Sabbadin, 1969; Lau/on et al.. ated by reactive oxygen species (oxidative stress), genomic
1992) have revealed the existence of a weekly, svnchro- instability, telomere shortening, cell death, genetically pro-
nized cycle of asexual development and regression, known grammed life extension, and systemic control of aging (re-
as the blastogenic cycle, which concludes with massive viewed in Jazwinski, 1996; Sohal and Weindruch. 1996:
visceral apoptosis (Lauzon el al., 1993). On the other hand, Johnson etal.. 1999). The relevanceofsuch processes in the
senescence atthe level of the ramet has not been extensively regulation of life span in Botryllus is unknown. However,
investigated. In contrast to nonrandom senescence, senes- the universality ofthe nonrandom senescent phenotype in B.
cence at the ramet level can manifest itself at random, and schlosseri genets argues for the existence of an underlying
thus may occur at any blastogenic cycle, independently of genetic program controlling the aging process. In some
thegenet (Rinkevich etal.. 1992). This processalsoexhibits aspects, nonrandom senescence of Botryllus genets resem-
a characteristic morphology; this includes synthesis of bles other senescence and life-span phenomena seen in
fewer than one bud or/ooid per blastogenic cycle, constric- some aclonal invertebrates. It is well documented that the
tion of blood vessels, accumulation of pigment cells within timing of expression of life-history stages is an important
zooids and ampullae, dispersal of zooids within systems. factor regulating aging and senescence in animals with
degeneration of the tunic and loss of zooid resorption po- complex life cycles (Miller and Hadtield. 1490). For in-
tential during takeover (Brunetti and Copello. 1978: stance, the expression ofa genetic program associated with
Rinkevichetal.. 1998). These features resemble nonrandom aging early in the larval life ofa nudibranch mollusc (Miller
senescence documented in B. schlasxeri genets (Rinkevich anil Hadtield. |49()i or prior to the subcloning ofa Botryllus
el al.. 1942: this study). There are, however, two major colony (Rinkevich et al.. 1442; this study) may already set
differences between nonrandom and random senescence. the limit for the maximal longevity of a genet. The obser-
First, nonrandom senescence is fast, being completed in 7 vation that ramets often die simultaneously within the same
days from onset of morphological changes, as compared to blastogeniccycle, months following theirseparation, further
the weeks and months of gradual deterioration that is typi- validates this hypothesis. Recent studies have further impli-
cally associated with random senescence oframets. Second. cated genetic elements, and their often complex interac-
random senescence may be followed by rejuvenation. De- tions, in the manifestation ofsenescence in mammals (Haan
generating ramets undergoing random senescence otten do et iii. 1998) and invertebrates (Orr and Sohal. 1994: Gems,
not die (Rinkevich a al.. 1998). Several weeks (sometimes 1999: Taub et al.. 1999). One such studv documented the
more than 2 months) after the manifestation of senescent life spansofindividual mice originating from a recombinant
teatures. colonies may return to a last growth rate charac- inbred strain. Their findings indicated that, despite a homo-
teristicofyoungcolonies (Rinkevich etal.. 1998). Although geneous genetic background, life spans fell into two tem-
senescence is regarded as an unavoidable part of the life poral groups, revealing a locus that causes variability in the
histories of many organisms, it seems that some clonal rate at which members of a population die (Haan et al..
organisms that produce genetically identical modules may 1998). In the nematode Caenorhabditis elegant, at least 39
escape aging at the level ofthe genet (Gardner and Mangel. genes have been demonstrated to significantly affect worm
1997). According to this concept, ramets may senesce. but life span (Murakami and Johnson. 1998: Gems. 1999; re-
the further production of new ramets max enable the genet viewed in Johnson et al.. 1444; Taub et al.. 1999).
to escape senescence. Many colonial marine invertebrates
have been documented to undergo periodic phases of de- 'I'lie role of tilt' vascular s\stei in programmed
generation and regeneration. These include bryo/.oans mortalities
(Palumbi and Jackson. 1483) and clonal species of sponges,
corals, hydro/oans. the related hotryllid ascidian liotryl- An intriguing observation reported here concerns the
lniile\ li'iithi and main other ascidians (Jackson. 1985; systemic nature of vasculature constriction and thai this
Rinkevich et al., 1996). The mechanisms regulating such change appears to underlie all subsequent events within
processes are unknown, and clearly deserve further atten- senescent colonies. These findings raise the possibility that
tion. in li. .sclilouM'ri. nonrandom senescence could be triggered
by a humoral factorthat initiates the vascular anomalies and
that may be transferred throughout the colony via blood.
l\ ntmiiiinliiiH \ene\cence geneticallyprogrammed?
Our previous findings in B. schlosseri chimeras (Rinkevich
The life span of an organism can best be conceptualized el <//.. 1442) indicated that the onset of senescence occurred
as the sum <>1 h.nmlnl changes and compensatory mecha- synchronously in both partners at the time of the earliest
nisms that either delay or limit such changes, thus promot- senescent partner. These observations were completely re-
ing longevity (Johnson et al.. 1444). A number of mecha- produced with vascularchimeras grown in the field, hut not
nisms have been described as potentially affecting aging in the majority of laboratory cohorts (Chadwick-Furman
and senescence in eukaryotes. These include damage medi- and Weissman, I995h). The reason for the discrepancy
