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Epulopiscium fishelsoni, Big Bug Baffles Biologists!

Presenters: Peggy E. Pollak, Ph.D and Dr. W. Linn Montgomery

Imagine the lessons to be learned if bacteriologists could work with pure cultures of a prokaryote that is: larger than many eukaryotic cells; structurally and ecologically complex; highly motile and covered with bacterial flagella; undergoing marked circadian cycles in physiological processes; and found only as a gut symbiont of a vertebrate host.

Epulopiscium fishelsoniThese traits are characteristic of gigantic (to at least 600 x 60 µm) eubacterial gut symbionts of tropical surgeonfishes (Family Acanthuridae; "tangs"). Although only one form has been named (Epulopiscium fishelsoni; Montgomery and Pollak 1988a), 10 structural "morphs" of these symbionts (differing in maximum size, shape and reproductive traits) have been recorded in surgeonfishes from Hawaii, Guam, Japan, French Polynesia, the Great Barrier Reef, Tuvalu, South Africa and the Caribbean (e.g., see Clements et al. 1989). We use the term “epulo” to distinguish these organisms from other types and taxa of microbes.

Epulos possess a distinct nucleoid separated from the cytoplasm by an unidentified, thin, membranous structure, and the cytoplasm is packed with an intricately-folded system of what appear to be membranes (Fishelson et al. 1985; Montgomery and Pollak 1988a; Clements et al. 1989; Bresler et al. 1998). Condensed DNA is arranged in elongate, chromosome-like structures with cross-striations similar to those seen in dinoflagellates, so-called "mesokaryotes". Cells are highly motile, driven by a covering of bacterial flagella (Fishelson et al. 1985; Clements and Bullivant 1991) yet they do not display the typical “run and tumble” behavior of most flagellate bacteria. Instead, they spiral back and forth, much like the single-celled protozoan, Paramecium.

Epulopiscium fishelsoniDuring the day, as the host fish feeds, Epulopiscium fishelsoni grows (size increases from 30-50 µm to > 200 µm; Fishelson et al. 1985, Montgomery and Pollak 1988a). At night, when fish are inactive, E. fishelsoni undergoes recurring reproductive events that return cell size to its initial small range. Lengths of E. fishelsoni span from (at least) 10-15 µm to > 500 µm. DNA quantity is proportional to cell volume over cell lengths of 30 µm to >500 µm (a >2000-fold difference in DNA quantity; Bresler et al. 1998). Cell division involves production of two nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and epulo cell growth and during nocturnal periods of host inactivity when mean cell size declines. Epulos also form some cells with unusually heavy cell walls (spores?) at night (Bresler et al. 1998).

Although we had originally suggested that epulos were protists due to their large size, structural complexity and unusual life cycle (Montgomery and Pollak 1988a), molecular work by Esther Angert (now at Cornell), Kendall Clements (University of Auckland) and Norman Pace (now at Colorado; Angert et al. 1993) identified epulos as the largest Eubacteria. Their paper also brought scientific attention to apparent violations by epulos of long-standing generalizations about cell size in bacteria, specifically to the structural and physiological phenomena previously thought to limit cell size, and to assignment of fossil unicells as prokaryotes or eukaryotes on the basis of size alone (Sogin 1993). These cells are large enough to allow use of many light microscopic techniques as well as manipulative and invasive techniques (e.g., microprobes, microinjection) to study the physiology of individual bacteria. Research on epulos could address: factors which may limit cell size and cell morphology, or support gigantism, in prokaryotes; subcellular compartmentalization in prokaryote cells; mechanisms of cell division, daughter cell formation and encystment; vertebrate host-symbiont interactions in an open system (the gut is continuous with the environment); and bacterial dispersal and evolution in widespread but patchily distributed habitats (surgeonfishes on tropical reefs throughout the world).

As noted, structural features such as size, shape, and mode of daughter cell formation distinguish a variety of epulo “morphs” (Clements et al. 1989), but 16s rRNA gene sequence divergence has apparently not paralleled structural divergence (Treutner, et al. in prep.). Angert et al. (1993) used 16S rRNA gene sequences to place epulos among the low G+C gram positive bacteria related to Clostridium spp., and later (Angert et al. 1996) identified Metabacterium polyspora from guinea pigs as a sister group of epulos. We have since examined epulos from 17 specimens of 5 host fishes from the Red Sea, Japan and the Hawaiian Archipelago (Treutner, et al. in prep). Epulos represent a distinct, monophyletic clade with two genetically distinct subdivisions within the epulo clade. The subdivisions do not appear to relate to epulo morph, locality of collection or identity of host species, and both subdivisions can be found in a single host fish.

At this time, we do not know how epulos are transmitted to the host. Epulopiscium fishelsoni occupies the mid-intestine of the fish during the day, follows food materials into the posterior intestine as these materials are evacuated at night, and remains in the posterior intestine overnight (Fishelson et al. 1985, Montgomery and Pollak 1988a). We found neither epulos nor clearly identifiable resting stages in the first fish feces of the day, on the algae that the fish eat, in the water, or in fish eggs. Fish that were starved to eliminate epulos, released back upon the reef, and recaptured in 10-12 days were not reinfected although the sample size was small (n = 2).

We also do not know the nature of the symbiosis between epulos and their host fishes. Anecdotal observations suggest a mutualism (two organisms living in a close, mutually beneficial relationship). A tang that became ill and died in an aquarium in Tel Aviv lacked epulos. Epulopiscium fishelsoni suppresses local gut pH by 1 pH unit during the day, but not at night (Montgomery and Pollak 1988b), reflecting a significant metabolic shift from day to night that parallels the host’s feeding activity. Epulopiscium fishelsoni from the Red Sea enhances lipid digestion by the host (Pollak and Montgomery 1994), but smaller morphs from Australian fish may enhance carbohydrate digestion (Clements 1997).

These unique bacteria remain an intellectual oddity rather than a laboratory tool for one primary reason: they cannot be maintained in a culture where they exhibit the complex life cycle and dynamic cell and population growth seen in nature. All knowledge about these unique bacteria comes from preserved cells or live cells within 1 hour of collection. This necessitates repeated travel and long stays at tropical reefs to collect the hosts and microbes.

Successful culture of epulos will require collaboration between ichthyologists trained to collect and dissect the fish and microbiologists skilled in handling and maintaining bacteria. In May 1999, a German graduate student and microbiologist (Silke Treutner) in our laboratory attempted culture of epulos from surgeonfishes collected in Hawaii. Previously, epulos had been kept alive for only a few hours (personal observation; K. Clements, pers. commun.). In contrast, our epulos remained alive for several months. Unfortunately, only small cells survived and neither individual cell growth nor significant population growth occurred. Thus, while the first and perhaps most significant step in culturing epulos has been accomplished (maintaining live cells for an extended period), we still cannot support the complex patterns of growth and reproduction seen in nature. Early in 2001, we will begin research to extend Treutner’s important work and, hopefully, find a way to culture epulos.

Post your question for Dr. Pollak and/or Dr. Montgomery.


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