Algal Endosymbiosis in Brown Hydra: Host/Symbiont Specificity

J. Cell Sci. 86, 273-286 (1986) 273 Printed in Great Britain © The Company of Biologists Limited 1986 ALGAL ENDOSYMBIOSIS IN BROWN HYDRA: HOST/SYMBIONT SPECIFICITY M. RAHAT AND V. REICH Department of Zoology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel SUMMARY Host/symbiont specificity has been investigated in non-symbiotic and aposymbiotic brown and green hydra infected with various free-living and symbiotic species and strains of Chlorella and Chlorococcum. Morphology and infrastructure of the symbioses obtained have been compared. Aposymbiotic Swiss Hydra viridis and Japanese H. magnipapillata served as controls. In two strains of H. attenuata stable hereditary symbioses were obtained with Chlorococcum isolated from H. magnipapillata. In one strain of H. vulgaris, in H, oligactis and in aposymbiotic H. viridis chlorococci persisted for more than a week. Eight species of free-living Chlorococcum, 10 symbiotic and 10 free-living strains of Chlorella disappeared from the brown hydra within 1-2 days. In H. magnipapillata there was a graded distribution of chlorococci along the polyps. In hypostomal cells there were >30 algae/cell while in endodermal cells of the mid-section or peduncle <10 algae/cell were found. In H. attenuata the algal distribution was irregular, there were up to five chlorocci/cell, and up to 20 cells/hydra hosted algae. In the dark most cells of Chlorococcum disappeared from H. magnipapillata and aposymbiotic hydra were obtained. Chlorococcum is thus an obligate phototroph, and host-dependent hetero- trophy is not required for the preservation of a symbiosis. The few chlorococci that survived in the dark seem to belong to a less-demanding physiological strain. In variance with known ChlorellaJH. viridis endosymbioses the chlorococci in H. magnipapillata and H. attenuata were tightly enveloped in the vacuolar membrane of the hosting cells with no visible perialgal space. Chlorococcum reproduced in these vacuoles and up to eight daughter cells were found within the same vacuole. We suggest that the graded or scant distribution of chlorococci in the various brown hydra, their inability to live in H. viridis and the inability of the various chlorellae to live in brown hydra are the result of differences in nutrients available to the algae in the respective hosting cells. We conclude that host/symbiont specificity and the various forms of interrelations we show in green and brown hydra with chlorococci and chlorellae are based on nutritional-ecological factors. These interrelations demonstrate successive stages in the evolution of stable obligatoric symbioses from chance encounters of preadapted potential cosymbionts. INTRODUCTION Two major groups have been recognized in the genus Hydra. One group comprises the green 'viridissima' hydra that host endosymbiotic algae in their digestive cells, and the second group are the 'brown' non-symbiotic hydra such as H. vulgaris or H. attenuata that were known to be found in nature without algal endosymbionts (Campbell, 1983). Goetsch (1924, 1926, cited by Kanaev, 1952), claimed that he found in his laboratory H. attenuata infected with a free-living 'Chlorella magna', and that he Key words: algae, Chlorella, Chlorococcum, Hydra, symbiosis. 274 M. Rahat and V. Reich could infect these hydra with other chlorellae. Such infected brown hydra have not been reported since, and later attempts to repeat these experiments and infect brown hydra with chlorellae have failed (Pardy & Muscatine, 1973; Muscatine et al. 1975; Jolley & Smith, 1980; Rahat, 1985a). To date, only Chlorella spp., of symbiotic or free-living origin, have been reported to form stable symbioses in the green hydra (Park et al. 1967; Muscatine et al. 1975; Jolley & Smith, 1980; Rahat & Reich, 1984, 1985a). It has been claimed for the brown hydra that they cannot phagocytose green algae or host them to form stable symbioses (Pardy & Muscatine, 1973; Jolley & Smith, 1980; Rahat, 1985a). The Japanese//, magnipapillata has been classified with the 'vulgaris' group of the non-symbiotic brown hydra (Campbell, 1983), but has recently been shown by us to host in its endodermal cells unicellular green algae of the genus Chlorococcum (Rahat & Reich, 19856). Extensive data have been acquired on the Chlorella/H. viridis endosymbioses (e.g. see Parker al 1967; Pool, 1979; Jolley & Smith, 1980; Meints & Pardy, 1980; McNeil ef al. 1981; McAuley & Smith, 1982; Rahat, 19856; Rahat & Reich, 1985a). It is thus of special interest to compare the specificity and interrelations of the 'old' and 'new' cosymbionts. We describe here the infection of green and brown aposymbiotic and non- symbiotic hydra with chlorellae and chlorococci, various degrees of prolonged persistence of algae in some aposymbiotic and non-symbiotic species of hydra and the consequent formation of stable algal endosymbioses in H. attenuata. We describe in detail some of the host/symbiont nutritional and ultrastructural spatial interrelations. MATERIALS AND METHODS Solutions used M solution: a buffered salt solution resembling pond water (Lenhoff & Brown, 1970), used for growing hydra. We added Phenol Red to this medium to facilitate the visual monitoring of the pH in this medium (Rahat & Reich, 1983a). BBM+: Bolds Basal Medium (Bischoff & Bold, 1963), with an addition of organic nutrients (Rahat & Reich, 1985a), used to grow the algae in vitro. Antibiotics mixture: lOO^gml"1 of each of penicillin, streptomycin, neomycin and rifampicin (Rahat & Reich, 19836), used to obtain axenic hydra. Hydra used The Japanese H. magnipapillata (Sugiyama& Fujisawa, 1977; Sugiyama, 1983; Rahat & Reich, 19856), a South African and an Australian strain of H. attenuata were used together with several strains of European hydra. The latter were H. oligactis, two strains of H. vulgaris and aposymbiotic hydra of a Swiss strain of H. viridis (Rahat et al. 1979). The European and Australian hydra were originally obtained from Professor P. Tardent, Institute of Zoology, University of Zurich, Switzerland, and have been cultured in our laboratory for several years. Three strains of green H. viridis were used: Swiss, European and a Jerusalem strain isolated here from an aquarium. The hydra were grown in M solution at 20 (±2)°C, under continuous illumination of 2500 lux (6500lux for H. magnipapillata), and fed three times a week with freshly hatched larvae of Artemia sp. Algal endosymbiosis in brown hydra 275 Preparation of aposymbiotic H. magnipapillata One single bud was cut from a dark-grown polyp and verified by light and ultraviolet microscopy to be aposymbiotic. This bud and its descendants were fed six times a week to repletion. Within 3 weeks a clone of about 100 aposymbiotic H. magnipapillata was obtained. Isolation of Chlorococcum for infection About 50 green H. magnipapillata were incubated for 48 h in a mixture of antibiotics to render them bacteria-free (Rahat & Reich, 19836). The hydra were then washed out of the antibiotics and placed separately into sterile test tubes containing 5 ml of BBM+ solution, one hydra in each test tube. In this medium Chlorococcum slowly 'leaked' out of the hydra to the bottom of the test tube. After a few days the algae were collected with a pipette, washed by repeated centrifugation and resuspended in M solution. These algae were used to infect the various species of hydra. Similarly isolated Chlorococcum were used to initiate in vitro cultures of these algae, to be described separately. In later experiments, in vitro cultured Chlorococcum were successfully used for infections. Infection of hydra with Chlorococcum and Chlorella Larvae of Artemia spp. were used as a vector to infect the various hydra, respectively, with symbiotic Chlorococcum isolated from H. magnipapillata, with eight species of free-living Chlorococcum, with 16 strains of in vitro cultured symbiotic and non-symbiotic chlorellae (Rahat & Reich, 1985a), and with symbiotic chlorellae freshly isolated by homogenization of three strains of green H. viridis, i.e. Swiss, European and Jerusalem strains. Four- to five-day-old larvae were mixed in M solution with a suspension of the respective algae, and incubated for 20—30 min. When the gut of the Artemia turned green, they were fed to the hydra. The algae were thus phagocytosed together with food particles. Compared to in vitro cultured Chlorococcum, chlorococci separated from freshly homogenized H. magnipapillata gave less satisfactory infections, as nematocytes present in such preparations prevented the Artemia from ingesting many algae. The infected hydra were fed two to three times a week. Examination of the hydra for infecting algae was best done after several days of starvation. Light and electron microscopy For microscopic examination of their cells, hydra were macerated according to David (1973). Photomicrography (Figs 1, 2) was done by Nomarski Differential Interference Microscopy on Technical Pan film. For electron microscopy (Figs 3, 4, 5), hydra were starved for 8 days to eliminate disturbing lipid globules from their tissue. The hydra were then fixed for 1 h in a solution containing 4% glutaraldehyde in 0-1 M-cacodylate buffer at pH 7-4, rinsed in the same buffer and postfixed for 1 h in 1 % OsO^ and 0-1 M-cacodylate buffer, rinsed again and left overnight in cacodylate buffer at 4°C. The hydra were then stained for 30 min in the dark with 2 % uranyl acetate, rinsed in distilled water, dehydrated through a graded ethanol series and embedded in epoxy resin (Spuir, 1969). Some sections were stained again with uranyl acetate. RESULTS Infection experiments As a control for all infection experiments, aposymbiotic H. viridis and H. magnipapillata were reinfected, respectively, with Chlorella and Chlorococcum originally isolated from these hydra, and algal persistence was verified. Chlorellae freshly isolated from three strains of green H. viridis and 16 strains of chlorellae grown in vitro in our laboratory disappeared from H. magnipapillata and the other brown hydra within 1-2 days, although eight of the strains grown in vitiv form stable symbioses with H. viridis (Rahat & Reich, 1985a). 276 M.

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