J. Phycoi. 15, 154- 162 (1979)

DEVELOPMENT OF jANCZEWSKlA lV/ORlMOTOl (CERAl\HALES) ON ITS HOST LAURENCIA NIPPONICA (CERAMIALES, RHODOPHYCEAE) !

Arthur lVI . Nonomura Lab.:>ratnrlo de Flcologl Department of Bot an y, Univers ity of Ca lifo rn ia, Berkeley, California 94720 , D~pert ~H~1 e rrto.de Biolog ' F acu t.t a-d de C len c 11::1 ABSTRACT o AM Janczewskia morimotoi Tokida was successfully cul­ (Co urt 1977) show no evid en ce for uptake of host tured[rom spore to reproductive maturity on its host Lau­ phot osynthate by mature J. gardne ri Setchell. Court rencia nipponica Yamada. Th e spore penetrates the host (1977), the refore, suggests that J. ga rdne ri is an ob­ without requirementJar wound or abrasion sites, gTowing ligat e ep iphy te rather th an a h oloparasite. This between host cortical cell , and deueloping a superficial and viewpoint doe s not con sider th e possible tr ace re­ an endophytic system simultaneously. During the juvenile quirements for host substances othe r than ph oto­ period, when the parasite is nonpigmented, it difJerentiates syntha te o r host metabolit es in early parasite devel­ a cortex and the proliferating endophyticfilaments enlarge op me nt. causing a displacement of layers of host cells into the para­ Although investigations have been made on the sitic tissue. H ost cells contacted by cells oj the parasite ex­ pen et rati on of J. uerrucaejormis Solms-Laubach into hibit increased wall thickness, cytoplasmic density and ves­ th e h ost Laurencia obtu sa (H udson) Lamouroux icle[ormation. Pit connections between host and parasite (Solrn s-Laubach 1877, Falk enberg] 90 ] , Feldmann cells were rarely observed whereas penetration oj host cell and Feldmann] 958), int erpretati ons of its devel­ walls was seen commonly. As the parasite increases in size, op me nt co nflict, and no one has observed the entire its cells become pigmented evenly throughout the cortex and d evelopment. In the onl y previou s study of Jan­ host cell; show less obvious reactions to the parasite. At this czeushia infectivity, Feldmann a nd Feld rnann (1958) same time, the parasite deoelops branches and reproductive were un abl e to follow a nd cor rec tly int erpret devel­ structures. H ost plant segments less than 3 cm long Jailed o pme nt afte r rhizoid penet rati on. to gruw when injected with spores o] the parasite whereas The major objectives of th is inv estigation are to longer segments were not significantly affected by the par­ describ e th e entire development of janczewskia mo­ asite. In the absence oj the host, the parasite cannot com­ rimotoi T okida from spore infection to reproductive plete its development. Although J. morimotoi is well pig­ matu rity a nd to observe the effec ts of infection on mented at maturity, the absence oj pigmentation in the the gro wth of the host, Laurencia nipponica Yamada, juvenile stage, penetration of host cells, and eJJect on host in cu ltu re . J. morimotoi is d ist ributed from Kaiba-to, growth in culture strongly suggest that it is parasitic dur­ Saghalien southward th rou gh the T ohoku region of ing at least its early devel opment . H onshu, J apan . Its general habit a nd reproductive a natomy were fir st described by T okida (1947). This Key index words: Ceramiales; endophyte, J an czewskia; parasitic red alga is light to deep burgundy in color, J anczewskia ; Laurencia ; life history, J anczewskia; hemispherical to glob ose, up to 1,5 em diam a nd parasitic algae; parasite, Janczewskia; Rhodophyta can be a solid tubercle in late su m me r or have nu­ merous radiating free branch es in winter and Sin ce [ anc zeioskia was first described by Solms­ sp rIng . Laubach (1877 ), its characterization as a parasitic red alga has remained uncertain because of a lack MATERIALS AND M ETHO DS of understanding of its physiology, d evelopment Between March and July 1976 , plants of Laurencia nipponica and relati onship to its hosts. Eddelbuttel (19 10) con­ fre e of [ anczru.skia morimotoi and mos t epiphytes were collected sidered only Choreocolax and H arueyella to be defi­ fr om lower int ert idal roc ks at Hokk aido University Marine Lab­ nitely parasitic becau se evidence from cystoca rp de­ o ra tories at Mu ro ran an d Osh oro , J ap an. j. morimo toi does not ve lo p men t clea r ly d emarcates th ese gene l-a as occu r at Oshoro. and L. nipponica was collected fo r cu lture here se para te taxonomic entities from th eir hosts, a nd as well as at Mu ro ran to d eterrnin e whether d iffe renl host pop­ lack of pigm entat ion and reduced th allu s size indi­ ulat ions re act differently to t he parasite. Between J u ne and Au­ g USl 1976, tetrasporo phytic and carpospo rophytic j. morimotoi cate nutrition al dependence on their hosts. All the gro wing on L. nipponu a were colle cted at Mu roran. Voucher other red algal parasites known at th e time of Ed­ sp ecimens fro m these collections wer e p ressed o r preserved in delbuttel's publication possess plastids and some de­ 5% formalin. Plants for culture were wash ed vigorou sly under gree of pigm entation and, thus, were considered running seawater or in buckets and transpo rted in plastic bags herniparasitic. On the basis of pigme nt ati on, Setch­ for 2-4 h to Hok kaid o U niversity in Sappo ro , Japan. ell (1914) stated th at janczewskia species co uld vary All host and parasi te thall i used fo r cultu re wer e subjected to the following co nd itions: plants were washed in 0.1% sodi um fr om hernipar asitic to holoparasitic. Ph ysiological hypochl or ite in seawater for 30 s and rinsed several lim es in investigati ons u sin g radioacti ve carbo n tracers ste rilized seawater (steamed 0 0' a u roc laved) . Fo r t he first mo nth , isolates we re gr o wn in 0.25-0.50 strength Provasoli's en riche d

I A ccepted: 2 / October t 978. seawater medium (McLach lan 1973) at 30-33'1,0 salin ity. Plants

154 j A NCZ E WS KIA iVlOR IMOTOJ 155

No te : Sca le = J m rn in Frcs. 5 & 6 , and 100}.Lm in other figures.

FIGs. 1- 8. Attac hment ofjanrU lOShw. morimo loi to La ure ncia nipponica and su bse q ue n t ea rly d e velo p me nt o f th e pa ras ite . FtG. I. [aruznoskia init ial att ach me nt to Lau renrin. FIG. 2. No npigmented co rt ical skirt at th e base (If a pigmented kn o b. FIG. 3. Well d e veloped no n pigmented co rtex with kn ob. FIG. 4 . In itiation of trichob last fo rmation with pale pigm e nta tion. FIG. 5. B ran ch initiat ion. FIG. 6 . Mature thallus with fuJI pigmentatio n. F IG. 7. L auren cia spo reling wit h [ aruze uskia in fect io n at its base (a rro w). FtG. 8 . H ost ge rm ling with an infection (ar row) .

were grown at 15 C, 16:8 LD p hotoperiod, a nd 100- 200 lx cool­ Tissue grafts fr om J. mori motoi "ere made o nto L. nipponira in whi te fiu oresce ru illuminati on. Three to five d ro ps of C eO , ( I two ways : a p ica l seg me n ts of j. mo,.imoloi bra nc hes were placed mg/m l) were added to 200 ml cu ltu res as need ed (Mc Lac h lan o n sc ra pe d or una braded host plants; o r , medu lla r )' and co rtica l 1973). During th e second month o f g ro wth , th e isolates were tissu e fr om j. morimotoi was g ra fte d int o inci sions bel ow th e host tra ns ferred to full strength Provasoli's e nriched seawater, 500­ epidermis. 1000 Ix cool-white Ruoresce nt illumination , a nd othe r cu lture For ligh t m in osco p )', seg me n ts of L. nipbonica with J. ", ulimului cond itio ns as given . Cu ltu re media were ch an ged every 3 wk . were fixed in Ka r rio vskv's ( 1965) fixative adj usted to 890 mos M Wh en mature, female tha lli were mix ed with male thalli at a ratio at room tempe ratu re for 2 h, and we re post-fixed in cacod yla te o f ca. 1:4 in 500 m l crystallizing di sh es o n table shakers at 100 buffered J% O sO. at 4 C for 12 h. T hey were dehydrat ed in rpm for 1 wk . A ll oth er cu ltu res we re grown in 200 ml to 400 ml ace to ne a nd embedded in Epon-Araldite m ixtu re No.2 (Mo llen ­ p yrex g lass sto rage vesse ls. ha ue r 1964). Se ctio ns were cut 1.5 }.Lm th ick a nd stained with T o isolate L. nipponu .a in to unialgal cu ltu re, J- 3 rnrn a pica l Aniline Blue-Bl a ck (AB B) a nd pe riod ic acid- Schiff's rea gen t seg me nts fr om teuasporo p hy tes we re excised a nd grown. After (PAS) (Fishe r 1968). In ad di tion to m icroto me sec tio ns , sq uash 2 1110, spores we re relea sed and collected by micr opipette and preparations we re used . These were h and sectio ned in Karno v­ grown in separate cu ltu re vessels. C a me to ph yre a nd tetrasp o ro­ sky 's fixati ve, washed in d istilled wate r with a final rinse in 5% phyte isolates wer e rhus available for in fection stu d ies. ace tic acid, placed in 1% ABB in [l% aceti c acid for J0 s, a nd For [anczeu.skia iso la tio ns, spore transfers and tissue grafts ont o quickly rinsed in 5% ac etic acid in wate r before squashing. uni algal Lourencu i cu ltu res were attem p ted to isolate the par asite Photographs o f sections and squash es we re taken on a Zeis s o n to its host. Fo r spore tra ns fe rs , with in 2 h of release, carpo­ p hotomicr osco pe wit h a poch romat ic ph ase co n trast obj ectives. spo res were co llected singl), b ), rnicro p ipeue a nd ri ns ed 5 x in Pict u re s of wh ol e mou nts we r e ta ken o n a Le itz m ic roscop e with seawater. The carpo spores we re pl aced on sub me rged host iso­ a 6.5 x e p i-illu minator o bje ct ive. Host seg m e nts with the parasit e lat es 3- 6 ern lon g. C u ltu res were not m oved fo r 36 h to p re vent we re placed in se awa te r in d eep co ncave slides with black ve lvet spores fr o m roll ing off the cylindrical h ost th a lli. Approxim at el y back ing to red uce re Aect ion . JOOO cat'pospore t rans fers were required fo r five successfu l u ni­ algal infections. T he res u ltin g tet rasporo phyre isola tes "ere used RESULT S to reinfect a new se ries o f host thalli by placin g the spore-releas­ Host-free spore gu mination. Within 4 h of release, in g thalli o ver unialgal h ost plants for 24 h . Subsequent h ost the spherical red tetraspores of[anczeioskia morimotoi ge nera tions we re infected b), putting fertile ca rpospo ro p hyt ic o r enlarged fro m 40 p,m to ca . 55 p,m, developing a tet ra spo rophyric j. morimoioi with L. ni pponica plants in 200 ml ve sse ls o n sha ker tables at 100 r pm for -1 h o r more. Over JOOO clear gelatin ous coat, and adhering to any solid J. monmotoi th alli spa n n ing eig ht generations were cultured o n surface. Spores elongated to 60 x 30 p,m ovoids . L. nippornca b), mea ns o f spo re transfers d u ring an 18 mo per iod . They ofte n showe d signs o f polarity with clea r gra n­ 156 ARTHUR M. NONOMURA

@

@

Not e : Scale = 1 mm in FIC. 22; 10 fLm in Frc s. 10-1 3, 19, 21 ; and 100 fLm in the others.

FIG. 9. [ anczeioskia spo reling attac hment to I.our encia. FIG. 10. Initial penetration betwee n and th rough host co rt ical cells. FtG. 1I. Sporeling pen etrating ho st outer wall (host wall is thi n ner at poi nt of rhizoid co ntact, arrow). FIG. 12. Initial pe netration be tween host cells (anow). FIG. 13. Rhizoids pe netrating through a seco nd layer of co n ical cells. FIG. 14. Pro liferatio n of rhizoids near host ap ex and spora ngiu m a nd in itiation of co rtex (e). FIG. J5. Expansion of cortex on host su rface. FIG. 16. Pad of cortical cells a nd rh izoids su rround ing host cells. Ft c . 17. A cortical layer is well dev eloped and host ce lls (H ) a re isolated and u plifted by parasite r hizoid s (hos t cells in contact with r hizoids have thi ck walls). FIG. 18. Trichoblast formation ((mow ). FIG. 19. En largeme nt of trichoblast area. FIG. 20. The knob and main body of parasite show mcristemati c ac tivity (arrow) . FIG. 2 1. Enlargement of trich oblasts in kn ob . FIG. 22. Re pro­ ductively mature I mortmo toi with new spore in fectio n nearby (arr ow). j ANCZt:IVSKI/l lvlORIM OTOI 157 ular cyto plasm occu pying the lower 10 p,m of the teristic ap ical meristems (Fig . 5). U nd er th e light cell , this e nd giving rise to th e rhizoid. Within 24­ microscope, internal parasit e tissu e generally was 36 h , th e spores di vided tr an sversely o nce o r twice , not di stin guish abl e fr o m incorporated host cells immediately followed by the formation of 1-3 co l­ (Fig. 20). Tetrasporophytes began to rel ease sp ores orless rhizoid cells. During the next 5 days, the rhi­ soon afte r br anch es developed at ca. 20 days a nd at zoids elongated and often di vided and branched, a size of 2 mm di arn. Individual branches of the reaching a maximum len gth of 800 p,m with 10-12 tetrasp orophyte could co ntinue to elongat e up to I colorless cells. Simulta neo usly. the pigmented par­ cm and often release d spores for 3 mo. Peak spo re asit e cells divided, producing a maximum o f 12 ce lls production fo r tetrasporophytes occurred betw een from tetrasp ores. Ge nera lly, ca r pospo res (50-55 30-40 days. Male thalli reach ed reproducti ve ma­ p,m ) developed similarl y to tetraspores bu t grew to turity after 25 days and tended to be more compact ca. 24 cells. The spo re lings showed a pol ysiphon ous th an tetr asporophytes. th e whole th allus not exceed­ structure, and after 10 days, re ached a maximum ing 1 em diarn after 3 mo. Female thalli were re­ length of I mm . Further devel opment did not occur productivel y mature a fter 30 da ys. These plants in the abse nce of suitable ho st mat erial, and the no rmally did not exc eed J.5 em di arn if exposed to sporelings lost pigmentation a nd di ed within 20 males. but if not crossed with mal es. they became da ys. H ost-free spore growth ofte n sto pped at any 2.5 ern dia m with loose branching. After crossing, of th e intermediate stages described above. fe males developed carpos po ro p hytes and rel eased Growth o] parasite on host. In the presence of the spores within 18 days. As compared with cultured host, spore germination in j. morimotoi was similar m a terial, field fem ale gametophytes and tetraspo­ to host-free development throu gh th e first ce ll di ­ rophytes appeare d as solid sphercs, o fte n having vision. A seco nd cell divisi on often occurred before numerous tightly a p p re ssed branches. C u ltured th e rhizoid penetrated the ho st (Figs . 1, 9). At th e ma le plants resembled th eir field co u nte rpa rts. T he point of co ntac t, th e rhizoid cr eated a n indentation reproductivel y mature J. morimotoi (Fig . 6) was ge n­ in th e ho st cuticle (Fig. II). The rhizoid elongated , erally as darkly pigmented as its hos t. pushing betw een o r directl y through host cells (Figs. Cytulogy. During th e seco nd wee k of growth, wh en 10, 12). T he host did not need to be ab raded o r the parasite was not pigmented and had carried up othe rwise damag ed fo r spore entry a nd su bseq ue nt a layer o f ho st co n ical ce lls, interesting cytolog ical growth of the parasite. The parasite penetrated sev­ events occurred that strongly suggested a reaction e ra l cells deep into th e host (Fig. 13) and the rhi­ of the host to th e parasite. Host ce lls in co ntac t with zoids occasion ally br anched several times. Simu lta­ pa rasite cells cha r acte ristically devel oped thicker cell ne ou sly with o r soon after rhi zoid proliferati on , a walls, and co ntents of th e large ce ntra l vacu ole were co rtex developed fr om th e basal upright cell of the stain ed more darkly than normal host cells (Figs. 28, parasite (Fig . 14). The co rt ex took the form of a 29). Often vesicles a nd starch grains (red with PAS) skirt of nonpigm ented cells lyin g o n th e host su r face were abundant in infected host cells (Fig. 28), al­ (Fig. 15). Meanwhile, the upright portion developed th ough starch grains were often seen in host me ri­ into a slightly pigmented knob, at which stage the ste rna tic cells. At thi s stag e. host and parasite ce lls parasite appeare d as a white pad 100 p,m di am (Fig. were readily di stinguishable by means of the differ­ 2) . T h ercafter, with ex pa nsio n of th e co rtex o n th e ential staining re action to ABB a nd their relati vely ho st su rface, th e rhizoids con tinued to proliferat e, different cell sizes. At thi s ea rly stage of g rowth, the often completel y surrounding host cells (Fig . 16). parasite was m eriste rnaticall y more active than the Aft er 10 days, the rhizoidal cells multiplied to the host. The hi gh protein co ntent a nd the co ncentr a­ extent th at a layer of host cells was ca rried up into tion of th e organelles in the parasite cells ca used a the body of the parasite ("H " in Fig . 17). So me of d eep blu e sta ining reacti on with ABB through out these ho st cells were com p letely se para ted fro m one th e cell, whereas th e host cells that were not in co n­ ano the r, but th ey remain ed pigmented. The para­ tact with parasite cells were lightly sta ined o r stained site had a co mpletely differentiated co rt ical layer at only aro u nd th e pe riphery of th e vacuole where the this stage (Fig. 17), appearing as a white hemisphere cytoplasm and organe lles were concentra ted . H ost 200 - 300 p,m diam with a slightly pigmented kn ob co rt ical cells te nd ed to be very large , (60- 100 p,m ), (Fig . 3). Usually, as a seco nd or third layer of host whereas parasite cells were sm aller (20- 30 p,m) . ce lls was di spl aced upward by th e proliferation and Interconnections between host and parasite ce lls expa nsion of p arasitic rhizoids, a shallow cup of co r­ were ob served only du ring ea rl y non-pigmented tical celJ s with very sho rt tri ch oblasts cou ld be o b­ periods of g ro wth . Pit co nnections between host and serve d (Figs. 18, 19). By 16 days, a fte r three o r four parasite cells were rare, an examination of 500 ran­ layers of host ce lls had been uplifted, tri ch obl asts dom sections of j uvenile stages rev ealin g o nly one could often be see n in th e kn ob (Figs. 20, 2 J). At (Fig. 26 ). Parasite rh izoid cell s were fr equently this stage, the parasite appeared white to pale p ink found abutting pit co nnections between host ce lls with a dark kn ob (Fig. 4). During th e third week of (Fig. 30). Direct penetration s through host cell walls growth, th e parasite was still slightly less pigmented by the parasite a lso occu rre d (Figs. 27-29), if infec­ than the host, a nd it formed bran ch es with cha rac­ tion s were near tetrasporangia, th e parasitic rhizoids 158 A RTH UR M. NONOMURA

No te: Sca le = 10 J.l. m in FIGs . 24-30 , 10 0 J.l.m in FIG. 23 .

F IG. 23. Laurent ia epiphytes (f,') o n Lau rencia (H) . FI G. 24 .Enlargemen t of epiphyte-h ost inte rfac e. F IG. 25. Parasite (P) pe netratio n th ro ugh host tet raspo ran gium ( T ). F IG . 26. Parasite-h ost pit co nnectio n (P it) . F I G. 27 . Pen etrati on th rough ho st cell wall ( WP): note da rkly stained chlorop lasts (Ch ). F I G. 28. Host co rt ical cells sur rounded by parasite cells with thick cell walls (T W) , n um erous vesicles (V), a nd darkened centra l vacuole ch a racteri zin g host response to contac t with par asite : no te starch grains (SG) and host wall penetration ( WP) . FI G. 29. Pe netratio n through host cell by intact parasite r hizoid (P ). FIG. 30. Pa rasite rhizoid "bu ll ing host ( ell pit co nnection (H P) .

occasionally penetr ated through th e sporangiu m br an ching, [he a bove described co nne ctions were (Fig. 25). Nu me r ous vesicles ofte n aggregated near less frequently observed. Host a nd pa rasite tissu e th e sites of pen et rati on (Figs. 27, 28 ). I n late r stages, became ind isti nguis hable by th e time the parasit e as th e pa r asit e becam e pi gmented a nd initiated had reached re prod uctive m a turity (Fig. 2 2) . JANCZ1:W SK IA M ORIMOTOI 159

5

... Z 2

5 10 15 20 23

LENGTH of INFECTED SEGMENT AFTER 5 MONTHS ( , m)

FIG. 3 1. Effects of [ anczeioskia infections on g row th of Laurencia host segment s of vari ou s lengths over 5 mo . Growth of infected h ost seg me nts correlates to initi al length at time of in fection . Co rrelatio n coe fficient is 0.716. a value > 0 at 0.05 level of sig nificance . 0 = infected ho st segment s that cea sed growth within J mo ; + = infected ho st segments that sh owed continuous grow'lh.

Groioth. rail's, parasite mfectitnt», Spores o f j. mo­ in length grew a t least 45 em in total length in 5 m o . rimotoi sh ow ed an ability to infect all re p rod uc tive When spores o f L. nipponica were g rown epiphyt­ phases and all naturally occ u r ring growth forms o f icall y on mature h ealthy thalli of L. nipponica (Fig. L. nipponica. All reproductiv e phases of j. morimoloi 23), no penetration by the sporeling was observed developed as d escribed ab ov e fr om spores regard­ and no cytoplasmic or pit connections were seen. A less of the reproductive phase of the host. In no distinct interface between the two pl ants was alw a ys instance did tissue grafts sh ow significant growth or maintained (Fig . 24), a nd the plants remained nor­ reach reproductive maturity. mall y pigmented thro ugh out. I ne ver sa w L. nippo­ Growth o f th e parasite tended to be much fast er nica e p ip hy tic o n L. nipponica in th e field. than th at o f the host. j. morimotoi reached repro­ Infection of j. morimotoi by j. morimotoi occurred ductive maturity in 3-5 wk from spore germination occasio nally. Initial penetration a nd rhizoid el o n­ when infecting mature h ost th alli, while L. nipponica ga tion of the sp ore followed ex actl y the same pat­ required a t least 8 wk to reach reproduct ive matu­ tern o f developm ent as spores th at parasitized L. rity from spore ge r m in a tio n. Pa rasite sp ores infect­ nipponica. Such infecti ons rarel y d ev eloped to ma­ ed 2 wk old h ost sporelings a nd grew to reproduc­ turity. tive maturity in 4 mo. Penetrati on occurred most o ften through the basal di sc o f the host (Fig . 7), but D ISC USSIO N infection has be en observed on all erect portions of In this paper, I have reported the culture of J an­ the host sporeling (Fig. 8) exce p t the apical meri­ czeioskia morimotoi on the host Laurencui nipponica ste m . The parasite neither o utg rew nor killed the fr om spo re infecti on to spore producti on, the first host germling , but developed more slo wly th an nor­ suc h cu ltu re of a parasitic red a lga . Suc cessive in­ mal. If a host sporeling was infected by the parasite fecti ons co m p le ted a triphasic life cycle. Discussion during th e fir st m on th of gro wth, it was stu n ted , of as pects of development, cytology, a n d the effects and it did not exceed a fin al length of 3 cm in 4 mo. of th e parasite on growth o f the host foll ow. Such a stunted host plant, how ever, often reached Tetraspores and ca r pospores of j. mori motoi are reproductive maturity within 2 wk after the parasite able to infect and gro w to reproductive matu rity on matured a nd released spores th at again gav e rise to all for ms of L. nipponica, includ ing ga metop hy te and normal host plants. Uninfected host plants re ached tetrasp orophyte sporelings, seg me n ts, a nd full y ma­ a length o f 8- 12 em in 4 mo fr om th e time o f spore ture th alli . ln the lab orat ory, J. morimotoi ca n infect germination. strains of L. nipponica which are normally not in­ j. morimotoi g re w on cuttings o f L. nipponica with fected by the parasite in th e field. Sai to et al. (1977) elongation of parasitized segments correlated with investigat ed field coll ected j. iokidae o n L. nipponica length at th e time of infection. The correlatio n coef­ and found no correl ati on between reproductive ficient was 0.716, a value >0 a t 0.05 level of sig nif­ phase of the parasite a nd th at o f th e host. The oc­ icance (Fig . 3 1). Most segments 3 cm o r less in length currence o f Janczewskia in the field, therefore , prob­ when initiall y infected ceased growing within 1 m o abl y is lim ited by environ mental factors rather than after infection , whereas segments more th an 3 cm by strain differences or reproductive phas es of the long when initially infected co n tin ued growin g vig­ host. orously. FCluivalent uninfeeted host segments 1 em j. morirnotoi is able to grow to a limited ex ten t in 160 A RTHUR M . NONOMU RA

host-free cu ltu re . It so mewhat resembles j. uerru­ bu t could no t find an endophy tic stage; th ey d e­ caejormis (Fe ld ma n n an d Fel d mann 1958) and j. lo­ scribed only the thallus on th e su r face o f th e host. kidae (Sai to et al. 1977 ) in th at polarity is q uickly T he ir illustrat ions o f th e initial su rface d evelopmen t es tablished, and a lon g colorless r hizoid and pi g­ o f j. verrucaeformis, however, are simila r to my ob­ men ted a pical cells develop. The exte n t o f host-free se rva tio ns ofJ. morimotoi. One would anticip at e sim­ d evel opment appears to be so mew hat different for ilar d evelopment in sp ecies of th e sa me ge n us, p ro b­ each species , with j. morimotoi showing the most ex­ ab ly th e d evelopment seen in j. morimotoi occurs in tensive de velopment. Although diffe re nces in ex­ all species o f [anczeioskia . tent of unialgal spore growth ma y be geneticall y d e­ For C. pol)lsiphomaf, Rich ards (189 J) descr ibed a termined, mos t of th e g row th d ifferences o bse rved patte rn o f d evelopment similar to j. monmotoi whe re probably can be att ributed to culture co nd itio ns. It the su pe rficial and endophytic systems develop si­ is d ear, however , th a t in th e pr esent cu lture co n­ multa neously. Stu reh (1926), however. reponed a d itio ns, [an czeioskia can initiat e limited growth in th e filam entous endophy tic system wh ich gave rise to an co mplete absence o f its h ost , but it requires so me ex ternal syste m d istan t from the po int o f infecti on growth fac to rs of the host to reach matu rit y. fo r Choreocolax. Stu rc h (1899) d escribed a similar The nature of penetration int o the host is impor­ initial period of e ndophy tic growth for H. mirabilis tant since it is th e fir st ste p towards th e establ ish ­ where in te rn al filam ents g ro w toward s the h ost su r­ ment of th e parasitic relationship. Am ong the para­ face , the filaments proliferat ing between host pe ri­ sitic red algae, penetrati on has been d escribed for ce ntral a nd cortical cells. As it g ives rise to a closely Erythrocystis (R icardia), Ha rueyella and [ an czeu.skia. packed layer of cells, it br ea ks th rough the externa l Pen etrati on through apical pit me riste rns of host me mbrane of th e host. H ost cells are carried up branch es has been de scribed for Erythrocystis (Killia n within the body o f th e pa rasite by the proliferating 1914 ). H aro eyella mirabilis requires wound sites for filam ents. Go ff ( 1976) also shows uplifting of ho st spore pe netrati on a nd subseq ue nt d e vel o pme nt ce lls b)' H . mirabilis, an d d escribes a pustule ruptu r­ (Go ff a nd Cole 1976). Feldmann an d Feldmann ing th rough th e host co rtex , but photogra phs (Goff ( 1958) ha ve found th at j. verrucaeformis ca n infect an d Co le 197 6) on ly show ev idence th at th e host a ny part of L. obtusa without any a p paren t require­ o uter wall is ruptured. ments fo r wound sites. and m y investi gati ons of j. Gen erally, in previous inv estigati ons of th e devel­ morim otoi ag re e with the ir observations . opment o f parasitic algae, field material was st ud ied As the rh izo id of j. monmotoi penetrat es th e host , or cultured directly. Although d evelopmental pat­ a n ind entation of the o ute r cut icle is ev ident whic h terns ma y diffe r fro m gen us to ge n us, a nd endo­ con forms to the con to u r of the r hizoid foot. The ph ytic modes of in fection probably occur, endophy­ relatively abru pt decrease in thi ckness of th e cu ticle tic systems wh ich ha ve been presumed to p recede a t this point suggests a d iges tive ra ther than a com­ fo rmation of e ru ptive shoot systems co u ld have plete ly mech anical mod e of penetration. be en re m na n ts o f previous in fec tions and may be Among th e pa rasit ic red algae, th e on ly species questionab le. As has bee n d emonstrat ed by thi s in­ fo r whi ch d evel o pment is described in su fficient d e­ vestigation, a stu dy of cu ltu re iso lates may clarify tai l fo r compa riso n a re Kvaleya epilaeoe (Adey a nd q uestio ns pertainin g to th e mo de of infection by Spera pa ni 1971 ), j. uerru caelormis (Feldmann an d pa rasitic red algae. Feldmann 195 8) Choreocolax polysiphoniae (Stu rch The occurrence of isola ted host cells among par­ 1926, Ku g rens and West 1973), an d H. mirabilis as ite tissue was menti oned by Sohns-Laubach ( 1877) (Stu rc h 1899, Goff J976, Goff and Cole J976) . K. in his o riginal d escription of janczewskia. This has epilaeue develops su perficial and endophy tic systems been o bse rve d in o ther ge ne ra whe n cross sec tions sim u lta neous ly, a nd although the rhizoidal fila­ o f whole thalli were taken or electro n micr oscopy ments d o no t penetrat e d eeply, direct pe netrations was e m ploye d-C. polysiphoniae (Stu rch 1926, Ku ­ . into host cell walls are made. In ea rly in vestigations g rens and Wes t J97 3), C. albus (Kuckuck J894), H . o f j. uerrucaejo rmis , d evelo pment of a n e ru ptive mirabilis (S tu rch 1899, Go ff 197 6), Callocolax japon­ shoot system was described (So lrns -Lau bac h 1877 , icus (T sugi 1963), and Gra cilariophila gardneri (Daw­ Falk enberg J90 I). The endophytic stage d epicted son 1949), among others. (Falken be rg 190 I) ma y ha ve be en based o n a sec tion Thick walls in h ost cells th at h a ve been co n tac te d taken at an oblique an gle near a young thallus there­ by pa rasite rhizoids have be en obse rve d previously by missin g th e ce n tra l body o f th e parasite. A sim ilar in othe r red alga l pa rasites (Dawso n 1949 , Kugrens sectioning problem may be seen in an illustra tion o f a nd West 1963, Go ff 1976), and the reaction of iso­ a matu re [a nczeioskia (B.0rgesen J930), wh ere th e lat ed L. nip poni ca cells to co ntact with j. mo ri motoi rhizoids d o not ap pear to penetrat e as deeply or as r hizoids as see n with th e light mic ro sco pe resembles prolifically as depicted by others (Solms-Laubach that of P olysiphonia to Choreocolax (Ku gre ns a nd J877 , Falkenberg J90 J). It is probabl e th at th e sec­ West J963) a nd Odo nt ho lia to H arveyella (Go ff tion used by B.0rges en was not of th e ap prop riate J97 6), in which ultrastructure work has been d one. thi ckness or posit ion to show th e ext en t o f penetra­ The restorat ion of most host cells to normaJ cell tio n . Feldmann and Feldmann (1958) Jater so ugh t, conten t a nd wall thi ckness a fter the o nset of parasite j ANCZF. WSKVJ k IORU"IOTOI 161

pigmentation has not been ob served previ ously. essarily serves as a basis for predi cting di sposition That th e cellular phenomena asso ciated with host towa rd s cellular co n nectio ns. reaction to parasite co ntact appear only du ring th e In the evo lution of ad e lp ho paras ites, Fan (l 96 1) nonpigmented stage o f development of J. monmotoi believed th at th e ability to establish pit co n ne ctions suggests that its holoparasitic nature may be limited with host cells may have been the key adaptatio n. to its juvenile stages. It is to be not ed th at intercon­ Feldmann a nd Feldmann (1958) theorized th at ne ction with host cells is only observed during th e ad elphoparasites would not occ ur in families th at do nonpigmented stage, a nd the unusuall y thi ck walls not no rmall y form seco nda ry pit co nnections , with in host cells th at have been co ntac ted may be a te m­ th e exception of th e Co ra llinac eae . T he ability of J. porary re sponse to dir ect penetrations. mori motoi to penetrate host cell walls plus the rarity To my kn owled ge , direct pla smi c co n nections by o f pit co n nections during th e juven ile period sug­ parasitic red algae with th eir hosts has been d e­ gests that pit connections are not as important as scri bed previou sly o nly for Gracilariophila gardneri Fan ( 196 1) suggests. Several nonpigmented pa ra ­ (Da wson 1949). T he level of understanding of gen­ sitic red alg ae have been observed to penetrate host eral host-parasite intracellular relatio ns has in­ cell walls , e.g. H. mirabilis (Go ff 1976), K. epilaeoe creased considera bly since Da wson did his work. (Adey and Spera pa ni 1971). a nd G. gardneri (Da w­ however, a nd ultrastructural investigation is neces­ so n 1949). (K. epilaeoe may be an exceptiona l case sary to ve rify th e cyto plasmic co ntinuity obse rved by since non-p arasitic members o f Melobes ioid eae nor­ lighT micr oscopy (Friend and ThrefalJ 1976). mally show cell fu sions betw een cell rows within sin­ Penetrati on of host cells by intact rhizoids o f Ja n­ gle th alli ; however, cell wall pe netrati ons o f the host czeioskia has not been observed previously. The for­ have no t been r eported for a ny other species of mation of numerous pit connections be twee n ho st parasitic Mel obesioideae.) These obser vatio ns sug­ a nd parasite cells, however, had been reported for gest th at th e ability o f th e ep ip hyte to penetrat e host [a nczeioskia by Feldmann and Feldmann ( I958) and cell walls for nutrient tra nsfe r , whi ch would thus was the bas is for th eir h ypothesis regarding bio­ create a haustorium (Kuij t 1969), is of primary im­ chemical similarities of parasite s that a re closely re­ portance. It follows th at pit co n nec tio ns may func­ lated to their hosts (adelphopa ras ites). Feldmann tion structurally and not haustorially in some o f the a nd Feld mann ( 1958) postulated th at parasites th at parasitic red algae, o r th at insufficie nt tr an slocat ion a re within the sa me fa mily as their hosts would occurs across pit co n nec tio ns d uring nonpigmented readil y form pit co nnec tions with th eir hosts an d stages of parasitism . th at cytoplasmic mixing would occu r with m inimal There are no pr evious qu antitative in vestigations antagonism and no subseque nt destruction of host of th e effects of parasitic red algae on th e gro wth ce lls due to the ir bioch emical similarities. They also of th ei r hosts. Martin a nd Pocock (1953) co ncluded postulated that parasites th at do not occur within that th e effects of nine parasitic re d algal species on th e sa me fa mily as th eir hosts (allopa rasites) would their hosts in th e field was insignificant. Rich ards kill a nd d igest host cells as a co nseq ue nce of th e (189 l ) found clearly deleterious effects of C. /}oly­ mode of penetration involving enzymes alie n to th e siphonuie on its host , a nd he described parasitized host system. Feldmann and Feldmann ( 1958) also plants as generally less vigo rous th an uninfected stated th at th e formation of pit co nnec tio ns with plants. On the basis of depth and geograph ic dis­ host cells was not limited to adelpho pa ra sites , spe­ tribution. Ad ey a nd Sp erap ani ( 197 1) hyp othesized cifically citing obs ervatio ns of fr equent pit co nnec­ that th e abundance of the adelphoparasite K. epi­ tions between th e cells of so me allo pa rasites and laeoe increases with th e metab olic efficiency of th e th eir hosts, e.g. H. mirabilis a nd C. polysiphoniae. The host at temperatures below 3-5 C. In th e field, near simil arity of tissu e in mature [anczeroskia a nd its ho st the peak of the gro wing seaso n fo r L. nipponica, I made it di fficult to dete rmine the fin al ex tent of have freq ue ntly seen ap parently healthy and repro- . host-parasite cellular interco nne ctions, but follow­ d uctive plants with more th an 100 thalli o f j. mo­ ing Feldmann an d Feldmann's (1958) h ypothesis, rimoun growing o n individu al plants. Although th e one would expec t pit co nnectio n fo r mation between parasite seemingly does not aff ect host growth in host and parasite cells during th e early growth of the field , it does affect host gro wth in culture. th e parasite , and no evidence of host cell a ntago ­ My obse rva tion that th e growth of ho st seg me nts nism . The rarity of parasite-host pit connections, th e less th an 3 em lon g can be stopped by th e parasite occ u rre nce o f host ce ll penetration s by pa ras ite rhi­ with in o ne month of infection sho ws that th e parasite zoid s, and the cha nge s in host cells in co ntact with does a ffect host growth soo n afte r penetration. T h e J. monmotoi cells (d iscussed ea rlie r) obse rved in thi s failu re of man y of the sho rt segme nts to g ro w afte r in vestigat ion do not sup port Feldmann and Feld­ infection may indicate that th eir rneristematic re­ mann's (1958) h ypothesis . Additionally, the a ppar­ gio ns are destroyed by the proliferating rh izoids of e nt abs en ce of pit co nnections bet ween ce lls o f epi­ th e parasit e. o r th at th e parasite dep letes gro wth phyte and substrate when o ne plant of L. nipponica fac to rs and nutrients in the host. The fact that in­ grows on a no the r of the same spec ies sho ws th at fected ge rmlings, in which th e apical meristem is neither taxon o mic nor bioch em ical proximity ne e­ closer to th e parasite th an in short segments, co n­ 162 ARTHUR M. NONOMURA tinue to gr ow, albeit slowly, su ppo rts th e latter ex­ Dawson, E. Y. 194 9 . Studi es o f no rtheast Pacific Gracila ria ceae planation. (pl ates 1-25). Allan H ancork Foun dation Pub lication' of the Uni­ Althou gh th e cessatio n of gro wth in sho rt host vcrsit), ojSouthem Cahforn ia, Occasional Papers 7 : 1-105. Eddclb u ttel . H . 19 IO. U be r di e Ken ntn is des parasiia re n C ha­ segments but continued growth in infec ted host rakters del' als " Pa ra siren " be ka n nten Flo ri d een , in sbeso n­ germlings would see m to be inconsistent, the di ffer­ d ere der Gattunge n Choreocolax Re ins ch . und H arueyella en ce be twee n the grow th rates of th e parasite in Schm . et Rk e. Botanische Zeitung 68 :22 6- 32. each case may offe r an explanation. Since [a nczeios­ Eva ns , L. V., Ca llow .J. A. 8.: Ca llo w. M. E. 1973. Structural a nd kia shows a normal growth rate on excised segme n ts, physiol ogical st udies o n the pa rasi tic red a lga H olmsrlla. N ew P hytol. 72: 39 3-402. it can presumably exploit th e entire host syste m bet­ Falkenberg , P. 190 1. Die Rhod ornelacce n des Gol fes von N eap el ter than whe n it is growi ng on host germlings . Also, u nd der angrenzenden Mee resa bsch ni tte . Fauna und Flora reduced grow th rates of par asites on host germlings des Goljrs vun Neapel, 25 . 754 p p . may be caused by in su fficient quantities of essential Fa n , K. 196 1. Studies o n H vpneocclax, wit h a dis cussion on the origin o f parasiti c red a lgae. Nov a H eduneio 3 : J [9-30. nutrients or hormon es present du rin g th e ea rly Feldmann , j.. & Feldmann, G. 1958. Re cherches sur q ue lques stages of developmen t. Moreover, becau se th e host Floridees pa rasit es. Rev . Gen. Bot. 6 5 :49 - 124 . germling is not mu ch bigger than the parasite germ­ Fis her, D. B. 19 68 . Prot ein sta in ing o f ribbo ne d epon sections lin g, the two could be competing for nutrients fro m fo r lig h t mi cr oscopy H istochrmir 16 :92-6. the su rro und ing medium. Fr iend, .J. & T h re fal l. D. R. J97 6. Biochemical Aspects oj P la nt Parasite R elationships. Academic Press. l nc ., Lo nd on. 354 pp. Fr om the growth ex pe rime nts and cytological ob­ Goff, L. J. 1976. The biolog y of H aru ryrlla mirabilis (Cryp to n e­ servations of this inv estigation , it is evident th at j. miales, Rhod o phyceae). V. Host respon ses to pa ra site infec­ morim otoi shows a mode of infection a nd d egree of tion. I Phvtol, 12:3 18-28. parasitism d uring early stages of grolVth th at pre­ - - - & Co le, K. 1976. T he biolog y of H aru ryella mirabilis (C rypto nemia les. Rhodo ph yceae). l l l . Spore germin atio n viou sly had not been known. The grad ual de vel­ and subseq uent d e velopment with in th e host Odonthalia floc ­ opment of pigmentation obser ved may be indicative cosa (Ce ra rrual es, Rh od o p h yce ae). Can . I B ol. 54 :268 - 80. o f th e ra te of development of chloro plast pig ments Karnov sky. M. J. 1965. A formaldehyde-gl utaraldehyde fixative and/or the increase in chlo ropl ast concentration. o f high osm o la lity for us e in e lectron micros co p y. j. Cell. Bioi. 27 :J37 A-8A. The presence of chloroplasts in matu re [nn czeioskia Killi an . C. 1914 . Ub e r di e En twicklung ei nige r Flo rid cen. Z. Bot. an d the ir development in juvenil e th alli will req uire 6 :209-78. further investigat ion with electron micro scop y. Re­ Kuckuck , P. 18 94 . Chorrocolax albus n . sp ., e in ec h te r Schrna­ cent physiological investigation s would tend to con­ ro tze r u nte r den Florid cen . S itzu ngsber. Akad. Wiss. Berlin fir m th e hypothesis th at non-pigmen ted form s of 38: 983- 7 Kug re ns, P. & West. J. A. 1973. The u lt rastructure of a n allo ­ parasitic red algae inco rp orat e host ph otosynt hat es pa rasitic red a lga Choieocolax polysiphoniae. Phscologia 12: 175­ (Evans et al. 1973, Go ff 1976), whereas pig mented 86 forms do not utiliz e them (Evans et al. 1973). j. Kuijt. J J969 . The Bio logy ofParasitic Flowering Pl anls. U niversity morim otoi, which is deepl y pigm ented at maturity, of Ca lifo rn ia Press , Ber ke ley. 24 6 pp. Marti n . M . T. & POLOLk, M. A . 19 53 . So u th African p arasit ic a ppears to be holoparasitic du ring its ea rly non pig­ Flo rideae and th eir h os ts. 2. So m e So ut h Africa n parasi tic mented stages of growth. Ph ysiological tests of this Florideae. I Linn. Soc. Bal. 55:48-64. j uven ile state mu st be undertaken before a com p lete Mc Lachlan,j. 1973 . Growth me d ia-ma rine . In Stc iri.}. R· lE d .] asses sment of the parasitic nature of the species ca n H andbook uJ Ph)'<·ololiiraI IHe/h orl.\--Culture M e/hods a nd Growth be ma de . M easurem ents, Ca m br id ge U n iversity Press, New Yo rk , 25­ 51. This investigation wa s supported by a n exchange fe llowship fro m Moll e nh a uer. H . H . 1964. Plas tic embed d in g m ixtu res for use th e J a pan ese Ministry of Education . 1975-1 9 77 . I a m m ost g ra te­ in e lect ro n mi croscopy . Stai n T erhnol. 39: I I 1-4. fu l to Professor M. Ku rogi a nd th e mem be rs of hi s laboratory in Rich a rd s, H . M . 18 91. On the structure and d eve lo p me nt o f Choreocolax PolY.lipholriae Reinsch. Proc. Am. Ph ilos. Soc. 26 :46 ­ Sappo ro for th e ir u n bo unded patie nce a nd gu id a nce . [ als o wish 63 . to th a nk D rs. J. A. West, G. F. Papenfuss, a n d P. C. Silva fo r Sa ito , Y., Yone ta, T. . Yoshikawa. M. 1977. The rel a tionships o f cr itica lly reading thi s manuscript. T han ks a lso go to D. Kaplan, parasite a nd host in the red a lga e [anc zeu.skia tokidae a nd \N. J e nsen , and M. Ashton for the use of their microscopes. l. aurencia nipponica Bull. [ap. Soc. Ph)'rol. 2S(s uppl.):3 l l- 7 . T hi s pape r is d edicated to Professor Ra lp h Emerso n. H is te ach­ Seich ell , W. A, 1914. Parasitic Flo ri d eae, I. Un iv . CaliI Pub l. ing, p ursu it o f know ledge , a nd o pe n-m inded co nsideration of all Bot. 6: 1-34 . th ings a re the foundation of my understanding of science . So hn s-Lau bac h . H . [8 77 . Note sur Ie [ ancz ru-shia nouve lle Flo­ ridee parasite d u Chondria obtuse. M rm. Soc. Sc. Na t. Chcrbourg Ad ey, W. H & Sperapani, C. P. 197 1. The bio log y of Kvalfya 21 :209- 24. epilaeoe, a new pa rasitic ge n us a nd species o f Coral lin ac eae. S tu rc h, H . H . J899. H a rvc setla mira bilis (Schmitz e t Rei n ke). Ann . Phyw logia 10:29-42. Bat. 13: 83-1 00. . Be rgeseu. F. 1930. Marine algae fr om th e Canar y Isla nd s . . . --- 19 2 6. Cho rroro lax polysiph on iae , Reinsch . Alln. B ul. [ [ I. Rhodo phyceae Pa rt III Cera miales. K. Da n. Vidcnsk, 40 :585-60 5. s.e« B ioI. Mer/rl. 9(1 ):1-159. T o kid a , j. 1947. Notes o n some new or littl e kno wn marine Court. G . J 1977 . The sy m bio tic ass ociatio n o f th e red a lga e a lgae. ( I}.j.jap Bol. 2 1: [27-30 [a nczeioskia ga rdnni and Lou rencia sprctal nlis (Ceram ia les, T su gi, Y. 1963 . Nippon sh insa n Callot olax oyobi shinshu Callo­ Rhodophyceae). I Ph yrul. 13(su ppl.): J4. t otaxjaponicus ni tsu ite H okkaid o Shi gaku Kcnkvu. 1: 1- 7.