Virus-like particles in an apochlorotic flagellate in Hiroshima Bay, Japan

Virus-like particles (VLPs) were detected in an apochlorotic flagellate (species unknown) in a water specimen obtained from a bloom of Prorocentrurn tnestinum (Dinophyceae) in northern Hiroshima Bay, Japan. The cells containing VLPs were discovered In a natural water specimen and were eshmated to be about 20 % of the apochlorotic cell population. The VLPs were pentagonal or hexagonal in cross section, indicating icosahedral symmetry, and were associated with the viroplasm (virogenic stroma) It is suggested that viral infection may be one of the important factors regulating the dynamics of heterotrophic flagellates in aquatic environments Recently, the concentration of virus particles in various aquatic environments was measured as 106 to l o g ml-l, much higher than had been estimated by plaque assay method (Bergh et al. 1989, Bratbak et al. 1990, Suttle et al. 1990, Hara et al. 1991). Therefore, it is of great interest to clarify their role in aquatic environments and the interaction between them and aquatic microorganisms. Studles on viruses infectious to bacteria (Hidaka 1972, Borsheim et al. 1990, Heldal & Bratbak 1991, Bratbak et al. 1992) and algae (Dodds 1979, Sieburth et al. 1988, Cottrell & Suttle 1991, Van Etten et al. 1991, Yamada et al. 1991) in aquatic environments have been carried out recently. On the basis of these reports, viruses have been recognized as active members of the microbial food web in aquatic ecosystems. However, as far as we know, information on viruses infectious to aquatic heterotrophic flagellates, the ecological importance of which as grazers of bacteria has been highlighted recently (Sherr et al. 1986), is scant except for virus-like particles (VLPs) in Aulacomonas Skuja cultured with Scenedesmus obliquus as prey (Swale & Belcher 1973). In the present paper, we describe VLPs observed in an apochlorotic flagellate (species unknown) in natural water of Hiroshima Bay, Japan. Materials and methods. During a bloon~ of Pi-orocentrum triestinum in Hiroshima Bay, Seto Inland Sea, Japan, on 19 June 1992, surface water (ca 2 1) was collected. Cells in the water specimen were prepared for electron microscopy to a great extent according to the method described by Hara & Chihara (1982). Thin sections were viewed with JEOL JEM-1010 and Hitachi H-800 transmission electron microscopes. Results and discussion. In the sections, a kind of flagellate, the form of which was roundish but changeable, ca 5 to 8 pni in diameter, was frequently observed (Fig. 1) as well as Prorocentrum triestinum. The flagellate was phagotrophic and each cell had several food vacuoles, some of which were packed with smaller prey being digested. As all the observed cells lacked chloroplasts, we regarded them as apochlorotic flagellates. Intracellular plate structure typically showed a cornice-like cross section, the pattern of which appeared subtly different in the sections (arrows in Fig. 1). An apochlorotic flagellate which was reported as a predator of the brown tide organism Aureococcus anophagefferens by Sieburth et al. (1988) also has a very similar structure. As little morphological information was obtained by light microscopy, we could not identify the flagellate. Some of the cells were found to harbor VLPs (Figs. 2 & 3). As 8 out of 42 cells in 2 different blocks contained VLPs, the frequency of the cells with VLPs was tentatively estimated to be about 20%, suggesting that nearly the whole population was at a lytic stage (Proctor & Fuhrn~an 1990). Except for those released to O Inter-Research 1993 308 Mar. Ecol. Prog. Ser 96: 307-310, 1993 the cytopiasm or exterior of the cell, the VLPs were associated with the viroplasm. The VLPs were usually pentagonal or hexagonal in cross section suggesting that they were icosahedral in 3-dimensional morphology. No tails could be observed. The coat measured ca 13 to 15 nm thick, around an osmiophilic-lucent fibrillar core (Fig. 4). Considering the previous reports on viroplasms, there seem to be at least 2 types of mechanism for their generation. In one case, the intact nucleus exists apart from the viroplasm (Prelsig & Hibberd 1984); in the other, the nucleus itself is degenerated to form viroplasm (Swale & Belcher 1973). It seems that the ultrastructural observation in the present study corresponds to the former, i.e. the nucleus remained apart from the viroplasm. Although these cells were probably moribund, mitochondria and food vacuoles also appeared to be intact. Further, in coll.apsing cells, the viroplasm separated from the cytoplasm (Figs. 2 & 3 ) , and apparent budding of VLPs from the periphery of the viroplasm was clearly observed (small arrows in Fig. 4) . The VLPs appeared to emanate from the viroplasm to the cytoplasm. As ribosomal aggrega.tes were also observed both inside and around the circumference of viroplasms (Fig 3 ) , it was s.uggested that proteins had been intensively synthes~zed for the construction of VLPs. The observations su.ggest that viral infect ~ o n may be one of the important factors regulating the dynamics of heterotrophic flagellates in aquatic environments, as is 4 Fig. 2. Electron micrograph of a cell contalning VLPs in a relativeIy early stage of viral replication. ViropIasm does not separate from the cytoplasm, and several VLPs are observed sporadically. Origin of 2 empty VLPs in the food vacuole is unknown. Scale bar = 1 pm. Abbreviations as in Fig. 1; VP: viroplasm 4 Fig. 1. Electron micrograph of an apochlorotic flagellate frequently observed in the red-tide specimen. Arrows indicate the plate structure showing a cornice-like cross section characteristic of t h ~ s flagellrlte, the pattern of which appears subtly different in the sections. N: nucleus; M: mitochondrion; FV: food vacuole. Scale bar = l urn