HELGOLANDER MEERESUNTERSUCHUNGEN Helgol~inder Meeresunters. 46, I-8 (1992)

W o r l d w i d e o c c u r r e n c e of v i r u s - i n f e c t i o n s in f i l a m e n t o u s m a r i n e b r o w n a l g a e D. G. Miiller & B. S t a c h e Fakult~t ffir Biologie der Universit~t; D-W-7750 Konstanz, Federal Repubh'c of G e r m a n y

ABSTRACT: Virus infections were detected in Ectocaz'pus sih'culosus and Ectocarpus [asciculatus on the coasts of Ireland, California, Peru, southern South America, Australia and New Zealand; in three Feldmannia species on the coasts of Ireland, continental Chile and Archipelago Juan Fernandez (Chile); and in Leptonematella from Antarctica. Natural populations on the Irish coast contained 3 % infected plants in E. fasciculatus, and less than 1% in Feldmannia simplex. On the Californian coast, 15 to 25 % of Ectocarpus isolates were infected. Virus symptoms were absent in E. siliculosus from Peru, but appeared after meiosis in laboratory cultures. The virus particles in E. fasciculatus are identical in size and capsid structure to those reported for E. sificulosus, while the virus in F. simplex is smaller and has a different envelope. Our findings suggest that virus infections are a common and worldwide phenomenon in filamentous brown algae.

INTRODUCTION While f u n g a l parasites in m a r i n e b r o w n a l g a e are well k n o w n an d systematically d o c u m e n t e d (Andrews, 1976), e v i d e n c e for virus-infections has b e e n e n c o u n t e r e d only sporadically in s e v e r a l EM studies. The few findings of "virus-like particles" to date w e r e listed by U s h i y a m a (1985). In 1988, a population of Ectocarpus siliculosus in N e w Z e a l a n d w as found to be i n f e c t e d with a d o u b l e - s t r a n d e d D N A virus, w h i c h is e x p r e s s e d in abortive g a m e t a n g i a or sporangia (Mfiller et al., 1990). Culture e x p e r i m e n t s with this material d e m o n s t r a t e d that the virus g e n o m e is closely associated with that of the host. N e w infections o c c u r w h e n the virus enters the unicellular motile spores or gametes. Reco m b i n at i o n during meiosis on an i n f e c t e d host plant can p r o d u c e healthy, symptom-free p r o g e n y (Mfiller, 1991a). The fact that virus-infected plants in the natural habitat an d in culture m a y be vital an d at least partly reproductive demonstrates that host a n d virus h a v e a c q u i r e d the potential to co-exist in a non-lethal and p e r h a p s p a n d e m i c manner. We p r e s e n t h e r e observations on the g e o g r a p h i c distribution and the p r e s e n c e of virus infections in field populations from various coasts of the world.

MATERIALS A N D M E T H O D S Infection symptoms in Ectocarpus and r e l at ed g e n e r a can be easily d e t e c t e d by light microscopy b e c a u s e they are e x p r e s s e d in a b n o r m a l reproductive structures. S a m p l e s of ectocarpoid a l g a e collected in the field habitat w e r e e x a m i n e d u n d e r the light micro9 Biologische Anstalt Helgoland, Hamburg

D. G. Mfiller & B. Stache scope with 10• a n d 4 0 x objectives. S p e c i m e n s fixed a n d s t a i n e d with c a r m i n e in 45 % acetic acid w e r e m o u n t e d in Karo syrup. The hyaline content of host cells c o n t a i n i n g virus particles w a s found to stain intensely With acetocarmine. Unialgal cultures of h e a l t h y a n d virus-infected plants w e r e d e r i v e d from filament fragments of field m a t e r i a l . Culture m e d i u m (Provasoli-ES; Start & Zeikus, 1987) was r e n e w e d at one- or t w o - w e e k intervals. Cultures w e r e k e p t at 12 + 1 ~ a n d i l l u m i n a t e d with white fluorescent l a m p s at 10 ~tmol m - 2 s -1 for 14 h p e r day. For electron microscopy, cultured p l a n t s w e r e fixed a n d e m b e d d e d in Spurr's resin following t e c h n i q u e s d e s c r i b e d p r e v i o u s l y (Mfiller et al., 1990). RESULTS

Ectocarpus fasciculatus H a r v e y f r o m I r e l a n d This s p e c i e s w a s f o u n d epiphytic on Scytosiphon 1omentaria (Lyngb.) Link at Killeany Bay, A r a n Islands, in S e p t e m b e r 1990. A m o n g a total of 91 s p e c i m e n s e x a m i n e d , 2 p l a n t s s h o w e d virus symptoms; while some plurilocular z o i d a n g i a a p p e a r e d normal, others s h o w e d a mosaic a r r a n g e m e n t of h e a l t h y sections a n d v i r u s - c o m p a r t m e n t s (Fig. 1). Clonal cultures d e r i v e d from these two plants m a i n t a i n e d this m o s a i c character. Electron m i c r o s c o p y r e v e a l e d virus particles in the h y a l i n e sections of the z o i d a n g i a (Fig. 2). T h e s e particles h a v e the s a m e structural characteristics as those found in Ectocarpus sih'culosus from N e w Z e a l a n d (Mfiller et ai., 1990). T h e y are polyhedral, 131-135 n m in diameter, a n d h a v e a l a m i n a t e d e n v e l o p e (Fig. 3).

Ectocarpus fasciculatus H a r v e y f r o m C a l i f o r n i a Since no m a t u r e e c t o c a r p o i d plants w e r e found at various locations of t h e Californian coast in O c t o b e r 1989, small inoculates of scrapings from Nereocystis luetkeana (Mert.) Post. & Rupr. a n d Laminaria sp. originating from Carmel, California, w e r e i n c u b a t e d . From the resulting r a w cultures, 26 u n i a l g a l isolates of E. [asciculatus w e r e obtained. Four of t h e s e s h o w e d virus symptoms (Fig. 4). As in the Irish material, the virus particles are polyhedral, with a d i a m e t e r of 138-144 nm a n d with a l a m i n a t e d e n v e l o p e (Figs 5, 6). Four u n i a l g a l Ectocarpus isolates w e r e m a d e from a r a w culture of Sphacelaria rigidula Kfitzing collected at M o n t a n a de Oro (Morro Bay). O n e culture d e v e l o p e d virus s y m p toms with the s a m e characteristics as the materials from Carmel, a n d a v i r u s particle size of 131-141 n m (Figs 7-9). A single Ectocarpus p l a n t isolated as an e p i p h y t e from Egregia menziesii (Turn.) Aresch. collected at D a v e n p o r t L a n d i n g (Santa Cruz) also s h o w e d virus symptoms.

Ectocarpus fasciculatus H a r v e y f r o m s o u t h e r n S o u t h A m e r i c a In F e b r u a r y 1989, collections at the following locations c o n t a i n e d E. [asciculatus s p e c i m e n s with virus s y m p t o m s in plurilocular zoidangia: Estancia M o a t (Ushuaia, Argentina, Fig. 10), C a m e r o n (Bahia Inutil, Tierra d e l Fuego, Chile, Fig. 11), a n d Puerto D e s e a d o (Patagonia, Argentina, Fig. 12). The virus s y m p t o m s in all t h e s e isolates p e r s i s t e d in clonal cultures.

M a r i n e a l g a l virus

3

Figs 1-3. Ectocarpus fasciculatus from Ireland. Fig. 1: Permanent mount of a wiid specimen with a normal (above} and a virus-producing plurilocular zoidangium with a normal section (below, arrow). Scale bar: 100 ~m, apphes to all light microscopic illustrations. Fig. 2: Longitudinal EM section through plurilocular zoidangium with virus formation. Scale bar: 5 ~m. Fig. 3: Higher magnification showing polyhedric virus particles with laminated envelope {arrows, apply also to Figs 6 and 9). Scale bar: 0.5 ~m Figs 4-6. Unialgal culture of Ectocarpus fasciculatus from Carmel, California, with virus symptoms in plurflocular zoidangia in hght microscopic appearance (Fig. 4) and EM sections (Fig. 5, scale bar: 5 ~n; Fig. 6, scale bar: 0.5 ~tm) Figs 7-9. Unialgal culture of Ectocarpus fasciculatus from Morro Bay, California; light microscopic appearance (Fig. 7) and EM sections {Fig. 8, scale bar: 5 ~tm; Fig. 9, scale bar: 0.5 ~tm)

D. G. Mfiller & B. Stache

Marine algal virus

5

E c t o c a r p u s siliculosus (Dillw.) L y n g b . f r o m A u s t r a l i a Field material collected i n the intertidal zone of Hinders, Victoria in S e p t e m b e r 1988 consisted p r e d o m i n a n t l y of normal E. sificulosus gametophytes. However, i n two specim e n s all plurilocular reproductive structures were diverted to virus formation (Fig. 13). Unialgal cultures m a i n t a i n e d this character, b u t occasionally some functional g a m e t a n gia were formed.

P,ctocarpus siliculosus (Dillw.) L y n g b . f r o m P e r u In a raw culture of Desmarestia peruviana Mont. collected in N o v e m b e r 1988 at Bahia San Juan, Nazca, a healthy looking Ectocarpus sporophyte appeared. Meiospores from unllocular sporangia of this p l a n t produced normal gametophytes a n d some plants with mosaic a r r a n g e m e n t s of normal a n d virus-forming sections i n their plurilocular zoidangia (Fig. 14).

E c t o c a r p u s siliculosus (Dillw.) L y n g b . f r o m N o r w a y In October 1977, 43 gametophytes were collected at Bergen a n d u s e d to estabhsh unialgal cultures for a study on sexual compatibility (Mffiler, 1979). O n e male gametophyte showed symptoms of virus-infection, w h e n re-activated from stock cultures in 1990. Inspection of a p e r m a n e n t shde m a d e in April 1978 from the same isolate (Fig. 15) showed that virus symptoms were aIready present at this early stage of the culture.

F e l d m a n n i a s i m p l e x ( C r o u a n ) H a m e l ,from I r e l a n d This species was f o u n d epiphytic on Codium fragile {Sur.} Hariot in the intertidal zones of Killeany Bay a n d Brannock Island (Aran Islands, Ireland) in S e p t e m b e r 1990. O n one Codium plant, 270 Feldmannia specimens had normal plurilocular zoidangia, while 5

Figs 10-12. Ectocarpus fasciculatus. Unialgal cultures derived from plants collected in southern South America. Fig. 10: Ushuaia, Beagle Canal, Argentina; plant shows normal, virus-forming and mixed plurilocular zoidangia (arrow}. Scale bar: 100 ~tm, apphes to all light microscopic illustrations. Fig. 11: Cameron, Tierra del Fuego, Chile, normal and virus-producing plurilocular zoidangia on same plant. Fig. 12: Puerto Deseado, Argentina, plant with normal and virus-producing plurilocular zoidangium Fig. 13. Ectocarpus sificulosus. Permanent mount of a virus-infected specimen collected in Hinders, Victoria, Australia in Sept. 1988 Fig. 14. Ectocarpus siliculosus from Peru. Virus-symptoms in plurilocular zoidangia of a plant derived from a unilocular sporangium in culture. Arrow: normal section Fig. 15. Permanent mount of a male gametophyte of Ectocaqaus sfliculosus from Bergen, Norway, made in April 1978 shows one normal gametangium (n) and one with virus production (v) Figs 16-19. Feldmannia simplex from Ireland. Fig. 16: Basal part of normal plant with functional pluri- (p) and unilocular sporangia (u). Fig. 17: Basal part of virus-infected plant with hyaline vesicular structures containing virus particles. Fig. 18: Low magnification EM section through a vesicular compartment of an infected plant. The entire cellular content is converted to virus production, with few remnants of host cell organelles persisting. Scale bar: 5 ~tm. Fig. 19: Higher magnification: polyhedral virus particles in Feldmannia cell with a simple envelope Scale bar: 0.5 ~tm

D. G. Mfiller & B. Stache plants s h o w e d virus symptoms. A n additional 855 Feldmannia plants from 15 Codium hosts w e r e all normal. Clonal cultures w e r e initiated from h e a l t h y and virus-infected Feldmannia plants. H e a l t h y plants f o r m e d functional uni- a n d plurilocular s p o r a n g i a (Fig. 16). Spores from both t y p e s of s p o r a n g i a d e v e l o p e d in an i d e n t i c a l manner, without e v i d e n c e of sexuality. In contrast, the v i r e s - i n f e c t e d isolates w e r e sterile. Virus particles w e r e f o r m e d in clusters of h y a l i n e vesicular cells. Their position on the b a s e of erect filaments i n d i c a t e s that t h e y correspond to m o d i f i e d z o i d a n g i a (Fig. 17). Virus-infected Feldmannia c u l t u r e s can be p r o p a g a t e d b y f r a g m e n t a t i o n a n d r e g e n e r a t i o n of filaments. Occasionally, s o m e of these r e g e n e r a t e s a p p e a r e d as h e a l t h y plants a n d h a d normal functional z o i d a n g i a . Virus particles are l i b e r a t e d from t h e vesicles of infected Feldmannia p l a n t s in the s a m e m a n n e r as d e s c r i b e d for Ectocarpus siliculosus (Mfiller, 1991b). W h e n motile zoids of h e a l t h y Feldmannia p l a n t s w e r e e x p o s e d to virus particles, 20 to 30 % o f the resulting d a u g h t e r p l a n t s e x p r e s s e d virus symptoms. Electron microscopic e x a m i n a t i o n of virusinfected Feldmannia p l a n t s r e v e a l e d d e n s e a g g r e g a t e s of virus particles a n d r e m n a n t s of cell o r g a n e l l e s within the h y a l i n e vesicular structures (Pig. 18). The Feldmannia virus is a p o l y h e d r a l particle, 112-130 nm in diameter. Its envelope s e e m s to consist of a simple e l e c t r o n - d e n s e layer (Pig. 19), in contrast to the Ectocarpus virus w i t h a l a m i n a t e d e n v e l o p e (Pigs 3, 6, 9).

F e l d m a n n i a globifer (Kuetz.) H a m e l f r o m o c e a n i c C h i l e A s p e c i m e n of Codium fernandezianum Setchell collected at S a n t a C l a r a Island, A r c h i p e l a g o J u a n F e r n a n d e z in F e b r u a r y 1991 was d e n s e l y c o v e r e d w i t h e c t o c a r p o i d epiphytes. 12 p l a n t s of Feldmannia globiferfrom this s6urce w e r e u s e d to e s t a b l i s h clonal cultures. A m o n g these, 11 w e r e normal a n d healthy, while one isolate s h o w e d h y a l i n e vesicles similar to those d e s c r i b e d a b o v e for Feldmannia simplex. Virus s y m p t o m s persist in this culture in a cryptic m a n n e r , while m a n y r e g e n e r a t e s p r o d u c e d from filament fragments are fertile a n d a p p e a r to b e c o m p l e t e l y healthy.

F e l d m a n n i a irregulans (Kuetz.) H a m e l f r o m c o n t i n e n t a l C h i l e A n e p i p h y t e on Scytothamnus austrafis (J. Ag.) H o o k e r & H a r v e y collected in J a n u a r y 1984 n e a r Puerto Montt, X. Region, g a v e rise to a clonal culture of F. irregularis. In addition to n o r m a l plurilocular z o i d a n g i a this culture s h o w e d c o n s p i c u o u s abnormalities, w h i c h p e r s i s t e d in culture a n d are now r e c o g n i z e d as s y m p t o m s of virus infection.

L e p t o n e m a t e l l a fasciculata ( R e i n k e ) S i l v a f r o m A n t a r c t i c a In J a n u a r y 1988, an a b n o r m a l s p e c i m e n of L. fasciculata was c o l l e c t e d at Bahia Fildes, King G e o r g e Island, a n d p r e s e r v e d as a p e r m a n e n t slide. E x a m i n a t i o n of this material shows that apical portions of m a t u r e filaments consist of series of h y a l i n e cells, the contents of w h i c h stain i n t e n s e l y with acetocarmine. Some cells w i t h functional plurilocular z o i d a n g i a are i n t e r s p e r s e d in a mosaic manner, thus confirming the characteristic features of virus s y m p t o m s for Leptonematella.

M a r i n e algal virus

7

DISCUSSION Virus infections in c o m p a c t b r o w n algal thalli can b e e x p e c t e d to b e difficult to identify, b e c a u s e s y m p t o m s s e e m to b e restricted to reproductive s t a g e s of the host. Thus, the only k n o w n case to d a t e is Chorda tomentosa Lyngb. (Laminariales), w h e r e viruslike particles h a v e b e e n d e t e c t e d in m o r i b u n d meiospores (Toth & Wilce, 1972). For this reason, w e c o n c e n t r a t e d our search on filamentous brown algal taxa, w h i c h p e r m i t the detection of virus s y m p t o m s more easily. The findings of virus infections in Ectocarpus siliculosus a n d Ectocarpus fasciculatus r e p o r t e d h e r e r e v e a l a surprisingly large a n d possibly global r a n g e , i n c l u d i n g Atlantic a n d Pacific o c e a n s with subarctic, t e m p e r a t e a n d subantarctic coasts. Various c o m m e n t s a n d illustrations in floristic hterature indicate that, in the past, virus-infected a l g a e h a v e b e e n e n c o u n t e r e d without k n o w l e d g e of the correct diagnosis. C a r d i n a l (1964) r e c o r d e d the occurrence of Ectocarpus dimorphus Silva for the English C h a n n e l area, a n d cites older reports from Pacific North America, Algeria, n o r t h e r n France a n d G r e e n l a n d . The illustrations and their interpretation b y C a r d i n a l indicate convincingly that this taxon r e p r e s e n t s virus-infected s p e c i m e n s of Ectocarpus. Similar findings w e r e r e p o r t e d b y Ardr6 (1969) for the coast of Portugal, a n d b y J a a s u n d (1965) for northern Norway. Culture e x p e r i m e n t s with clonal isolates of E. siliculosus a n d Feldmannia simplex show that the virus infection induces a stable transformation of the host plants. In the case of the N o r w e g i a n isolate of E. sfliculosus d e s c r i b e d above, the virus is still p r e s e n t after 14 y e a r s in culture. This fits with g e n e t i c a l studies in N e w Z e a l a n d isolates of E. siliculosus which indicate that the virus g e n o m e associates closely with the g e n o m e of the host. It passes t h r o u g h mitosis a n d meiosis in a highly c o o r d i n a t e a m a n n e r (Mfiller, 1991a). Virus-infected Ectocarpus plants in field collections and l a b o r a t o r y cultures are often s e e n with m o s a i c - s t r u c t u r e d plurilocular zoidangia. These p r o d u c e functional zoids w h i c h are able, at least partially, to restore a n d m a i n t a i n normal d e v e l o p m e n t of the host. This feature is subject to g r e a t variability. In e x t r e m e cases, plants h k e the P e r u v i a n E. sfliculosus can b e found that a p p e a r c o m p l e t e l y healthy. The existence of its virus infection b e c a m e a p p a r e n t only after meiosis u n d e r culture conditions. A n e x t r a p o l a t i o n of this t e n d e n c y suggests that Ectocarpus plants can be found w h i c h rarely or n e v e r express their virus infection.. Likewise, cultures of Feldmannia simplex a n d Feldmannia globifer p r o d u c e r e g e n e r a t e s that cannot be d i s t i n g u i s h e d from normal, h e a l t h y plants. It r e m a i n s a n o p e n question as to whether, in these cases, the virus g e n o m e has b e e n e h m i n a t e d or effectively s u p p r e s s e d . It seems possible that the entire Feldmannia p o p u l a t i o n of a g i v e n n a t u r a l h a b i t a t has b e e n in contact with the virus g e n o m e in the past. Virus-specific m o l e c u l a r m a r k e r s a p p l i e d to a l a r g e n u m b e r of individual plants will b e n e c e s s a r y to e v a l u a t e this assumption. Additional b i o c h e m i c a l studies a n d infection e x p e r i m e n t s are n e e d e d to e v a l u a t e the specificity of host-virus interactions in the g e n e r a Feldmannia a n d

Ectocarpus. Nucleic acid composition a n d g e n o m e size of the F. simplex virus is still u n k n o w n . A l t h o u g h there are m a n y similarities with the Ectocarpus system, the differences in e n v e l o p e structure s u g g e s t that the two viruses are distinct entities. Ectocarpus a n d Feldmannia are p l a c e d in the order Ectocarpales. The finding of

D. G. M f i l l e r & B. S t a c h e v i r u s s y m p t o m s i n f i e l d s p e c i m e n s of Leptonematella, w h i c h b e l o n g s to t h e o r d e r Chordariales, suggests that virus infections may be a general feature in the entire class Phaeophyceae. R e a n n e y (1974) p r o p o s e d a n i m p o r t a n t role of v i r u s e s i n e u k a r y o t e e v o l u t i o n a s v e c t o r s for n o n - s e x u a l g e n e t r a n s f e r . T h i s h y p o t h e s i s r e q u i r e s a w e l l - b a l a n c e d coe x i s t e n c e b e t w e e n D N A - v i r u s e s a n d t h e i r h o s t s , as w e l l a s p a n d e m i c o r a t l e a s t w i d e s p r e a d o c c u r r e n c e . B o t h p r e r e q u i s i t e s a r e fuifilled i n Ectocarpus a n d p o s s i b l y also i n Feldmannia. B i o c h e m i c a l s t u d i e s o n t h e m o l e c u l a r g e n e t i c s , a n d b i o l o g i c a l e x p e r i m e n t s o n t h e s p e c i f i c i t y of t h e s e s y s t e m s will b e n e c e s s a r y to d e t e r m i n e if, a n d t o w h a t e x t e n t , v i r u s e s c a n a c t a s v e c t o r s for h o r i z o n t a l g e n e t r a n s f e r i n Ectocarpus a n d Feldmannia.

Acknowledgements. Parts of this study were supported by the Deutsche Forschungsgemeinschaft. The Chilean institutions INACH a n d CONAF provided access a n d logistic support for field work in Antarctica a n d J u a n F e r n a n d e z Archipelago. Thanks are due to Dr. R. Westerme!er, for organizing a phycological expedition to subantarctic South America, and to Dr. A. Peters for collecting material from Peru. Our work in Ireland was done aboard the G e r m a n RV "Heincke" (Helgoland). LITERATURE CITED Andrews, J. H., 1976. The pathology of marine algae. - Biol. Rev. 51, 211-253. Ardr~, F., 1969. Contribution & l'dtude des algues marines du Portugal. I. La Flore. - Acta Biol. 10, 137-555. Cardinal, A., 1964. Etude sur les Ectocarpacdes de la Manche. - Nova Hedwigia (Beih.) 15, 1-86. Jaasund, E., 1965. Aspects of the marine algal vegetation of North Norway. - Botanica gothoburg. 4, 1-174. MOiler, D. G., 1979. Genetic affinity of Ectocarpus siliculosus (Dillw.) Lyngb. from the Mediterran e a n , North America a n d Austraha. - Phycologia 18, 312~-318. M~fller, D. G., 1991a. M e n d e h a n segregation of a virus genome during host meiosis in the marine brown alga Ectocarpus siliculosus. - J. PI. Physiol. 137, 739-743. M~iller, D. O., 1991b. Marine virioplankton produced by infected Ectocarpus siliculosus {Phaeophyceae). - Mar. Ecol. Prog. Ser. 76, 101-102. Miiller, D, G., Kawai, H., Stache, B. & Lanka, S., 1990. A virus infection in the m a r i n e b r o w n alga Ectocarpus sih'culosus (Phaeophyceae). - Botanica Acta I03, 72-82. Reanney, D. C., 1974. Viruses a n d evolution. - Int. Rev. Cytol. 37, 21-52. Starr, R. C. & Zeikus, J. A., 1987. UTEX - The culture collection of algae at the University of Texas at Austin. - J . Phycol. 23 (Suppl.), 1--47. Toth, R. & Wilce, R. T., 1972. Virushke particles in the marine alga Chorda tomentosa Lyngbye (Phaeophyceae) - J. Phycol. 8, 126-130. Ushiyama, R., 1985. Viruses in fungi and eukaryotic algae: Their possible origins a n d evolution. Microbiol. Sci. 2, 181-184.