Viral control of Emiliania huxleyi blooms?

Abstract Virus and virus-like prrrticles (VLP)

have been observed in all major algal classes. Few host-virus systems of microalguc

have until now been brorlght into culture and extensively studied. For Emiliania

hdeyi

we have been able to describe viral

infection during blooms in mesocosms and in landlocked fjords. Evidence of viral lysis of E.

Irrrr/eyi

during blooms in the

also beerti obtained. We have also developed a plaque assay for E. hmleyi virus with which we have been able to isotzte a virus tha!. could be propagated in the laboratory. This virus isolate lost its virulence possibly due to defective

Ejorth

,‘?a has

interfering particles (DI,,. The sizes of viruses related to E. huxleyi

indicate two major groups, one with a particle diamctcr

of 180 nm and one with C,head diameter of I40 nm.

1. introduction

phyte Mb-wwt~us pusilln (Mayer and Taylor, Waters and Ghan. 1982). Viruses

Virus-like-particles (VLPs) have been observed in phytoplankton cells of all major algal classes (Van Etten et al., 1991; Reisser, 1993). Most of these observations have not been the result of any deliberate search fcr VLF!s but have been incidental to other ,studies. The information available in the literature on VLPs is largely concerned with the morphology, structure and.size of the VLPs, their location in cells and tissue, and with their effect on cell ultrastructure. Very little is known about the biology, life cycle and lytic activity of algal viruses as only a few have been cultured in the laboratory. The exceptions among the tnicroalgae include the viruses infecting an endosymbiotic form of Chforcflu isolated from Purunwcium (e.g. Van Etten et al., 1985; Reisser et al., 1988; Van Etten et al., 19911, and the viruses of the prasino-

infecting

exsymbiolic

strains

IO30:

of

;I

Ci?lorctllu-like algae (strain NCd4A, Pbi etc.) appertr to be widely distributed in fresh water (Van Etfcn et al., 1991; Yamada ct al., 1991). Virus tilres are typically in the range of I - IO0 PFlJ ml ^;’, but values as high as 4 X I@ ml - ’ have been recorded (Van Etten et al., 1985). Several clones of viruses which infect CCflorellu have been isolated from natural waters. Restriction enzyme analyses of these clones indicates that their diversity is high (Van Etten et al., 1991 and references therein). In cultures of Chlorellu (strain NC64A) the CXlorellu virus strain PBCV-I has a lytic cycle of 3-4 h and a burst size of 200-300 infective particles (Van Etten et al.. 1983). Van Etten et al. ( 1991) reported that the NC64A strain may harbour the PBCV-1 virus in a carrier or pseudolysogenic state. Cottrell and Suttle (1991) have investigated the occurrence and clonal variation of viruses infecting

09247963/96/$ I5.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. P/I SO924-7963(96)000 18-8

the mafine prasinophyte Micrononas pusilla. Ljtic viruses were found in estuarine, coastal and oceanic habitats and in concentrations ranging from unde-

tectable to 4600 ml - ’. The examination of the 7 viral clones that were isolated showed that all were different with respect to EcoRI restriction fragment banding patterns, that 4 phenotypes could be identified on the basis of the molecular weight of the viral proteins, and that no morphological differences could be observed among the clones. The lytic cycle of the virus infection in Mi~~*omonas pusilla is about 14 h and the burst size of the cells is about 70 infective particles (Waters and Ghan, 1982). Two new host-virus systems with eucaryotic pb.yropiankters have recently been reported to be in culture. A virus capable of lysing the chrysophyte Aureococcrrs anophagefferens, which has caused devastating brown tides on the west coast of USA, has been isolated by Milligan and Cosper (1994). The viruses found ill the culture lysates were described as phage-like with a polygonal head approximately SO-70 nm in diameter and with a 80- 100 nm long tail, Suttle and Chan (1995) have isolated a virus infecting the haptophytes Chrysochromulina breuij?/rcnr and C, strobihrs. This double stranded DNA virus has icosahedral symmetry and is about

145- 170 nm in diameter. The presence of virus-likc,particles (VLPs) in phytoplankton cells collected in natural waters suggests that viral mortality may play a significant role in the population dynamics for some species (Piennur, 1976:Mayer and ‘Taylor, 197% Johnson and Sieburth, IYSZ; Sicburth et al., 198X; Proctor and Fuhrman,

1991). Suttle and coworkers have found that viruses concentrated from natural sea water may infect a variety of important marine primary producers, including diatoms, cryptophytes and prasinophytes (Suttle et &!!.,1990, 1991). Circumstantial evidence for viral termination of algal blooms has been obtained for two eucarytic phytoplank ters, the raphidophyte Heterosignta akdiwo (Nagasaki et al., 1994a,b), and the co~colithophorid Et,rilianicrhtrxleyi (Eratbak et al., 1993). CMls of H. aknshiwo, collected during red tides dominated by this species in Hiroshima Bay (Japan) in 1992 and 1993, were found to contain virus-likeparticles WLPs). At the end of the bloom in 1992 up to 5% of the cells in a natural water sampk con-

tained VLPs (Nagas‘aki et al., 1994a). In a water sample collected at the end of tile bloom in 1993 and incubated in the laboratory, it was observed that ca 60% of the cells disappeared within 26 h and thai 11.5% of the remaining cells contained VLPs (Nagasaki et al., 1994b). The evidence for viral interaction with E. huxleyi will be discussed in detail below.

2. Viruses and virus-like-particles of huxdeyi The first observation of virus-like-particles (VLPs) in Emiliania huxleyi (Lohmann) Hay and Mohler was published by Manton and Leadbeater (1974). The intracellular particles they observed in thin sections of nanoplankton collected from natural waters were approximately 200 nm in diameter (erroneously reported as 22 nm) and hexagonal in outline. Later, the relationship between VLPs and E. huxleyi was studied in a series of mesocosm experiments (Bratbak et al., 1993; Egge and Heimdal, 1994) and during blooms of E. huxleyi in Norwegian coastal waters (Bratbak et al., 1995) and the North Sea (Heldal and Rratbak, unpublished results). Viruses capable of lysing cultures of E. huxieyi have also been isolated and pmpag:~t.cdin the laboratory (Witson, unpublished result!;). The large virus-like-particles (LVLP) we have related to specific phytoplankton populations XL between 140 and 200 nm diameter and of uniform size ( f 5 nm) and morphology. They are thus easily distinguished from the majority of VLPs found in natural sea water which are 30-60 nm in diameter.

2. I. Mesocomsnr studies The results from mesocosm experiments, carried out on the west coast of Norway between 1988 and 1991 where viral activity was considered in relation to blooms of E. huxleyi, are published earlier (Bratbak et al., 1993). LVLPs observed in these studies

~were180 nm in diameter and similar to those observed by Manton and Leadbeater (19’743.The LVLPs

wrRrefound both as free particles and as intracellular particles in up to 3% of thin-sectioned cells, from which it was estimated that 20-40% of the cells

15

phate was no~-~~m~ti~g. found to restrict the pr~uctio~ of LVLPs, while nitrogen limitation appeared t The main conclusions fro were later confirmed in mesocos 1992 (Fig. 1) and in 1993 (Bratbak et al., 1995). The LVLPs observed in 1993 were .smaller thzn ,those nm in diameter) and they observed earlier (about 1 increased in abundance while the host population was relatively low and apparently without a simulta-

0

:,

10

15 I1

0.0 40

20

tklq

.5 Fig. 2. Dynamics of LVLPs

(N.P=l6:0.2)

the E. hcxkyi

1.2

lo9

LVLP

:6

20

L-’

and E. hrr.rk~i during one week in

bloom corresponded in time and space with an

increase in LVLP 20

0.a

May 1993 in Fauskangerpollen, western Norway. The collapse of

lL,

30

I

’

0.4

pbytoplankton

10

itbundancc. Simukancous chnngcs in other

populations were small (for

further details XC

Ftratbak et al.. 1995). 7 ii w = iii m

10

:,

0

0

t3ag 6 (N:P=16:1)

0 ;5

7

-1 E:

u. >-I _J 10 a

neous decrease in host abundance. This suggested that the LVLPs accum!rla,ted at the expense of the growing host cell populrrtion and prevented an E. huxleyi bloom from developing. These results await confirmation in subsequent studies.

30

15

20

10

2.2. Blooms in a landlocktx1fiord

1C

5

In 1993 we followed the collapse of an E. huxleyi bloom in the landlocked fjord Fauskangerpollen on the west coast of Norway (Bratbak et al., 1995) (Fig. 2). The collapse was accompanied by a simultaneous increase in abundance of LVLPs similar to those observed in the mesocosm experiments conducted this year, i.e. about 140 nm in diameter. The exchange of water in Fauskangerpollen is restricted by a narrow inlet and the simultaneous changes in the

0

10

15

20

Dote Moy Fig. I. Dynamics of LVLPs

25

r e

D

30

1992

and E. huxleyi

in mcsocosms with

high and low N:P nutrient loading. The increase in LVLP abundance is delayed and less in the enclosure with high N:P ratio (i.e. low P) as compared with the enclosure with low N:P ratio (i.e. high P; for further details see Egge and Heimdal.

1994).

abundance of other phytoplankton populations were small. Our conclusion therefore, is that the viruses in this case were the main cause for the temlination of the bloom. 2.3. Blootm in the Nortlt Sea A bloom of E. huxleyi in the North Sea was investigated on a cruise with R / V Ha&on Mosby between June 22 and July 5 1994. A small bloom area with E. fruxleyi abundance up to 7 x IO6 cells I-’ was located between 59’47’-59’55’ N and 0”36’-0’47’ E on June 27. The cell density decreased during the next few days as the bloom stretched out between 59-60”N and O-1°E, and on June 2-3 the bloom was observed to be was dying out and sinking. Samples collected at l-20 m depth along two cruise transects (st. no. 27-31 and st. no. 34-37)

A

00

0

0

lo3 Cells

0.95

1 7

0

1

.oo

@g Chl

Fig, 3, Correlation belwecn (A)

ml.

-I

1 35

1.50

a 1.-l

largevines-like-panirks (LVLPs) and chl ;L

and E. hudcyi(p= 0.666, p
(c = 0.849.F < 0.001)in mmples from tie North Sea in 1994.

EHUX cruise to the

acrossthe

bloom area, showed a signific‘ant correlation between the abundance of LVLPs and both E. fuccleyi and chl Q (Fig. 3). The LVLPs were about 140 nm in diameter (Fig. 4B) and similar in size and morphology to the particles observed in the mesocosms and in Fauskangerpollen in 1993 (Bratbak et al., 1995). In a sample collected at st. 29 (June 30) intracellular LVLPs were observed in ca. 10% of the cells (Fig. 4A). We interpret these results as indicating that viral infection may have played an important role in the control and termination of this bloom. 2.4. Isolatiotl and plaque formation of an E.huxleyi virus

During the mesocosm experiments carried out in 1993 (Bratbak et al., 1995). attempts were made to isolate and propagate a virus infecting E. huxfeyi. A sea water sample (60 I) was collected early in the experimental period from one of the control bags (Cl) and the cellular fraction was concentrated by continuous centrifugation at 5000 rpm with a flow rate of 350 ml min- ‘, The resulting cell concentrate was resuspended in 500 ml and contained approximutely 1 X lo6 E. huxleyi cells ml- ’ (determined by examination under the light microscope). In order to induce vi&ds production in E. huxleyi. 50 ml aliquots of the cell concentrate were placed in a glass petri dish (I5 cm in diameter) and exposed to UV light at various doses (254 nm wavelength, Philips fluorescent tube, type 57415 P/40 A6 T UV 15W, 40 cm distance, 15 s to 3 min exposure time). The cell suspensions were then incubated at 15°C in the light over-night, centrifuged at 5000 rpm to remove cell debris and viruses in the resulting supematant were enumerated by plaque assay as follows. Serial dilutions of the supematant were added to separate 0.5 ml volumes of an 80 X concentration of exponentially growing of E. huxleyi (strain SC44, graciously provided by E. Paasche) grown in f/2 medium. Supematant-host suspensions were incubated at 15°C for 1 hour, with occasional agitation to encourage virus adsorption, and then added to 2.5 ml of 0.4% (w/v) molten f/2 agar (42”C), mixed gently and poured evenly on to a solid 1% (w/v) f/2 agar plate. Agar used in this procedure was purified prior to use by washing in double distilled water,

Fig. 4. Large virus-like-particles(LVLPs) in samplescollec~cdduring the E. hrcdcyi bloom in the Nolzh Sea in 1994. (A) Thin-section of cell showing inlracellular LVLPs (arrowheads). (B) FIW LVLPs associatedwith the collapse ol’UIC bloum (anowhc;ltls). The pwtdc diameteris abultt I40 nm. The sample was collcctcd at st. 29, ccmctxWirlc~I, r:nhkbxi, dk-sectioned and inspctcd in lk trimmibbloil clectron microscopeas describedin Brathak et al. (I 993).

ethanol and acetone as previously described (Waterbury et al., 1986). The plates were incubated at 15°C under constant illumination and virus plaques were observed on E. huxleyi lawns after two weeks of incubation. Some of the plaques were resuspended in 0.5 ml volumes of sterile f/2 medium and 100 ~1 volumes were used to re-infect 100 ml cultures of exponentially growing E. huxleyi. Cultures were found to lyse completely after incubation for 4-7 days. It was also noticed that regrowth of E. huxleyi cells appeared approximately 7-10 days after lysis. The viruses in the lysed cultures of E. huxleyi and in the resuspended plaques were visible in the epifluorescence microscope as bright fluorescent spots after

staining with the DNA specific stain DAPI. From this we conclude it to be a ds DNA virus. On subsequent re-infections, the time taken for E. huxieyi cultures to lyse increased until, after 6-10 re-infection cycles, the E. huxleyi virus @h-V) became unable to lyse any E. huxleyi culture. 3. Discussion During our mvestigations we have associated two different large virus-like-particles with E. huxleyi, one with a diameter of 180 nm and one slightly smaller with a diameter of 140 nm. Our main argument for a host-virus relationship has been the co-

80

sionally arise in a host-cell infected by a normal (non-defective) virus. The DI particle will subsequently be replicated together with non-defective viruses until the DI particles are a predomi~~t part of the viral progeny, hence, interfering with normal viral processes and preventing lysis of host cells. A gradual loss of viral titre would be observed, as was the case when Eh-V preparations were propagated on E. huxleyi.

Although we demonstrated that E. huxleyi is susceptible to viral infection and that the LVLPs Fig. 5. Correlation between large virus-like-particles(LVLPs) and E. kuxkeyi (r = 0.177. n = 180. p = 0.018) in all sampleswhere countsof LVLPs has been relatedto E. h~leyi. Line is functional regression.

distribution and population dynamics of these LVLPs and of E. huxleyi. When all data are pooled there is a positive correlation between E. huxleyi and these VLPs(Fig. 51, This may, of course, be interpreted as yet another example of the general correlation between the abundance of VLPs and biological parameters such as chl a, primary production, bacterial direct count etc., that has been demonstrated earlier (Boehme et al,, 1993: Cochlan et al., 1993). However, during the collapse of E. huxkyi blooms, we have several times seen a negative correlation between the two parameters (Bratbuk et al., 1993. l995), suggesting a cause and effect relationship. Based on the general assumption that the LVLPs are viruses pracluced by E, hu.&yi we may interpret then correlations as follows: With an increasing abundanceof E, hu.rleyi,the number of virus producing cells will increase and result in an increase abundanceof free LVLPs in the water, However, for short periods of time as a bloom collapses due to viral lysis, the abundance of E, huxleyi will decrease while the abundance of LVLPs increases, Once we were successful in isolating a virus for Emilianiu huxleyi, but we failed to establish a stable lytic host-virus system that could be propagated in the laboratory for an extended period of time. One hypothesis that may explain this phenomenon is that /2 huxleyi, in addition to infective viruses,produces defective interfering (IX) viral particles.Such particles are known to interfere specifically with the intracellular replication of non-defective viruses (Hururg BIKE Baltimore, 19701,and they will occa-

have some of the properties we expect viruses to have, e.g. size, morphology and lysis of host cells, this does no! prove that the LVLPs indeed are viruses. We do, however, consider this as the most likely conclusion. Our general conclusion from the data on E. hualeyi

viruses and VLPs published earlier, and the new information presented here, is that viruses infecting E. hrcxleyi can control and terminate blooms of this organism. Available evidence on viruses and VLPs of other phytoplankton species suggests that viral infection, besides nutrient limitation and grazing, may be a third major factor controlling many phytoplankton species. Further progress in research on the ecological significance of phytoplankton viruses depends on the development and application of methods for detection and enumeration of specific viruses. The most straightforward approach will be to develop plaque or most-probable-number (MPN) assays which do not depend on the availability of a virus, only on the availability of a susceptible and culturable host strain. In addhion, this method yields isolates of any virus that may infect the host cells. A more sophisticated approach will be to use molecular techniques, DNA probes (e.g. Chen and Suttle, 1995) or antibodies raised against viral proteins, to tag both free viruses and viruses intracellularly in their respective host cells. This approach requires that the virus in question is available in culture, but it has the potential of being much more sensitive than any culture method. Acknowledgements

This work was supported by funding from The Research Council of Norway to the European Com-

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