Massive prevalence of viral dna in ectocarpus (phaeophyceae, ectocarpales) from two habitats in the north atlantic and south pacific

Botanica Marina Vol. 43, 2000, pp. 157Ϫ159 Ą 2000 by Walter de Gruyter · Berlin · New York Massive Prevalence of Viral DNA in Ectocarpus (Phaeophyceae,
Ectocarpales) from Two Habitats in the North Atlantic and South Pacific

D. G. Müllera,*, R. Westermeierb, J. Moralesb, G. Garcia Reinac, E. del Campoc, J. A. Corread
and E. Rometscha

a Fakultät für Biologie, Universität Konstanz, D-78457 Konstanz, Federal Republic of Germanyb Facultad de Pesquerı´as, Campus Puerto Montt, Universidad Austral, Puerto Montt, Chilec Instituto de Algologı´a Aplicada, Universidad de Las Palmas de Gran Canaria, Telde, Gran Canaria, Spaind Departamento de Ecologı´a, Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Santiago, Chile Ectocarpus, a cosmopolitan genus of filamentous marine brown algae, contains two species, E. siliculosus andE. fasciculatus. Both species are subject to virus infections, which either destroy the host’s sporangia or persistin a latent state without visible symptoms. We used PCR amplification of a viral gene fragment to monitorthe infection status of Ectocarpus samples from Gran Canaria Island, North Atlantic, and southern Chileover 26 months.
At both sites, we found persistently high levels of pathogen prevalence: 40Ϫ100% of the Ectocarpus specimens from Chile and 55Ϫ100% from Gran Canaria contained viral DNA. No evidence for seasonalvariation could be detected. We conclude that vertical transmission of viral DNA through mitoticzoospores of the host is the key mechanism for the persistence of the pathogen. The PCR amplificationcharacteristics of samples from Gran Canaria indicate that two different virus genotypes coexist in thesame host population.
Materials and Methods
The two brown algal species Ectocarpus siliculosus Two habitats with perennial populations of Ectocar- (Dillwyn) Lyngbye and E. fasciculatus Harvey are pus were selected as collection sites. 1) A platform of well known cosmopolitan inhabitants of coasts in volcanic rock 100 ϫ 20 m at mid-intertidal level out- cold and warm temperate climate zones. Since the side of Muelle de Taliarte, Telde, Gran Canaria, end of the last century numerous reports described Spain (28Њ00Ј N, 15Њ22Ј W). In this habitat Ectocar- specimens with abnormal sterile sporangia from pus grows as an epiphyte on Fucus spiralis Linnaeus, many parts of the world (Sauvageau 1896, Cardinal and on rocks, shells or limpets in tide pools. 2) An 1964, Asensi 1974). Silva (1957) established the new experimental mariculture area located in the estuary species Ectocarpus dimorphus Silva to accomodate of the Maullı´n river near Puerto Montt, Chile such individuals. In 1974 Clitheroe and Evans de- (41Њ40Ј S, 73Њ45Ј W). In this sandy habitat Ectocarpus tected virus like particles in malfunctioning sporangia grows epiphytically on Gracilaria chilensis Bird, of Ectocarpus. Subsequently Müller et al. (1990) iso- McLachlan et Oliveira and other substrata in the lated an icosahedric DNA virus from E. siliculosus and demonstrated that free virions are able to infect At each site 30 tufts of Ectocarpus (approx. 5 mg spores or gametes of their host. Bräutigam et al.
dw each) were collected at bi-monthly intervals from (1995) developed a polymerase chain reaction (PCR) August 1995 to October 1997. Samples were blotted protocol, which allows the detection of viral DNA in dry with filter paper and sealed in polyethylene bags Ectocarpus. With this technique Sengco et al. (1996) with 1 g of dehydrated silica gel (Fluka blue 85340).
showed, that in a sample of 98 isolates from our Ec- Subsamples were fixed in acetocarmine and mounted tocarpus culture collection nearly 50% contained lat- in Karoă syrup as permanent slides for microscopic ent virus DNA. This result suggests that natural Ec- tocarpus populations are subject to much higher virus The DNA was extracted from 0.5Ϫ1 mg of the dry prevalence than hitherto judged from morphologi- samples and subjected to PCR amplification and de- tection of a fragment of the viral gene gp-1, following We describe here the level and temporal fluctua- the protocol described by Sengco et al. (1996). In tion of virus prevalence in two natural Ectocarpus some analyses the stringency of the PCR reaction was populations determined by PCR detection of viral reduced by lowering the annealing temperature from 64 ЊC (standard condition) to 55 ЊC.
Due to the great variability of Ectocarpus (Russell1966) identification of our field material to specieslevel was not possible. All Ectocarpus specimens ex-amined from both study sites were sporophytes withplurilocular sporangia. Unilocular sporangia werepresent in 3 out of 91 samples from the Chilean site,and were completely absent in samples from GranCanaria. Specimens with visible symptoms of virusinfection were rather rare in the Gran Canaria Ecto-carpus (1 out of 123 samples), and more frequent inthe Chilean habitat (15 out of 91 samples).
Our standard PCR amplification method revealed that between 40 and 100% of the Ectocarpus samplesfrom the Chilean study site contained viral DNA.
Temporal fluctuations in the infection level were ir- Fig. 2. PCR detection of viral DNA in Ectocarpus from the regular and showed no evidence for seasonality study site at Gran Canaria over 26 months. Total length of (Fig. 1). The detection of viral DNA in Ectocarpus bars represents 100% of sample size. Black and stippledareas represent percentages of samples containing viral samples from the Gran Canaria site proved to be DNA detected at two different stringency levels (PCR an- more complex. Analyses with the standard PCR pro- tocol gave low infection levels ranging from zero to20% (Fig. 2). However, under less stringent condi-tions the proportion of samples with viral DNA in gp-1. Now our study confirms and extends the find- the Canarian Ectocarpus increased strongly to values ings of Sengco et al. (1996) to natural populations of between 55 and 100%. Fluctuations were irregular, Ectocarpus: 1) The prevalence of viral DNA in Ecto- and a clear seasonal trend could not be detected carpus is exceedingly high and ranges between 40 and 100%. 2) In contrast, between less than 1 up to a fewpercent of Ectocarpus specimens exhibit morphologi-cal infection symptoms with the full expression of vi- Discussion
rion formation. This discrepancy can be explained by either a high degree of latency in the Ectocarpus Ectocarpus siliculosus and E.
virus, or the inactivation of the pathogen by partial fasciculatus are subject to infections by two different virus types (Müller et al. 1996). These two pathogens Unilocular sporangia are the site of meiosis in are species-specific, but closely related. Sengco et al.
brown algae, and laboratory cultures of infected (1996) confirmed that the viruses of both species respond in the same positive manner to PCR tocarpus sporophytes can produce virus free progenyby meiotic segregation (Müller 1991, Bräutigam amplification of the virus-specific gene fragment 1995). The relevance of his mechanism is negligible,since unilocular sporangia are completely lacking inthe Canarian, and only rarely found in the ChileanEctocarpus. Likewise, infections of new hosts must berare events, since only low numbers of virions can beexpected from the few individuals with visible symp-toms in both study sites. Consequently, vertical trans-mission via mitotic spores from plurilocular sporan-gia of the hosts remains the only plausible mecha-nism to maintain the high prevalence of virus infec-tions in the field populations of Ectocarpus. Samplesfrom the Gran Canaria site responded differentiallyto reduction of the annealing temperature from 64 to55 ЊC in our PCR protocol. This implies the coexist-ence of virus genotypes with nucleotide sequence dif-ferences in their gp-1 genes.
Presently, all attempts to explain the emergence and persistence of a viral pandemy system in a ma- Fig. 1. PCR detection of viral DNA in Ectocarpus from thestudy site at Maullı´n, Chile over 26 months. Total length rine cosmopolitan host like Ectocarpus must remain of bars represents 100% of sample size. Black areas show speculative. Since the Ectocarpus virus DNA is inte- percentage of samples containing viral DNA obtained un- grated in the nuclear genome of its host (Delaroque et al. 1999) it can be expected to interfere with mei- otic pairing of chromosomes. This fits with the ex- sive virus pandemy in Ectocarpus and related brown perience that laboratory cultures of Ectocarpus, Hincksia and Feldmannia rarely form unilocular mei-otic sporangia, which in addition tend to abort orproduce moribund progeny. This effect can lead to Acknowledgements
the elimination of sexuality, which is replaced by veg-etative reproduction with mitotic spores, providing a We thank C. Schmid and I. Maier for methodological favourable survival strategy for opportunistic colo- contributions and critical discussions. Financial sup- nists like Ectocarpus and related genera. Further port was provided by The European Commission studies on the interaction between host and virus ge- nomes and effects of viral latency on the physiologi- cal performance and fitness of the hosts are necessarybefore a comprehensive understanding of the impres- References
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