Ultrastructures of endosymbiotic Chlorella vulgaris under the influence of cinoxacin

The toxic effect of antibiotic cinoxacin on endosymbiotic green alga (Chlorella vulgaris) from green hydra (Hydra viridissima Pallas) was studied. It is known that some antibiotics have antichloroplastal and antimitochondrial effect on Euglena and green hydra1, 4, what points out the connection of chloroplasts and mitochondria with eubacteria2. The individuals of unicellular green algae are placed in gastrodermal myoepithellial cells of green hydra as endosymbionts, at most 20 algae in one gastrodermal myoepithellial cell (Fig. 1). Endosymbiosis is possible because of specific preadaptations of both endosymbiont and its host. Green algae contain the characteristic huge kidney-like chloroplast with dense thylakoides that form numerous grana. This organelle is, as well as mitochondria, sensitive to antibiotic treatment. Individuals of green hydra were treated for 72 hs in glass dishes under laboratory conditions (room temperature 21oC, 14 hs daylight, and 10 hs dark) with 62.5 mg/L and 117.6 mg/L of water solution of cinoxacin, using aquarium water. They were compared to control group of organisms. For conventional transmission electron microscopy standard preparation methods were used. Treated as well as control endosymbiotic organisms were fixed with 1% glutaraldehyde pH 6.9, buffered with sodium-cacodylate and postfixed with 1% osmium-tetroxide buffered with the same buffer. Afterwards, material was dehydrated in the ethanol-series, immersed in araldite and cut with the glass knife on the ultramicrotome. Preparations were dyed with 4% Pb-citrate and uranil-acetate. Micrographs were made using electron microscope Zeiss EM10A. Endosymbiotic green algae of Chlorella vulgaris species were damaged. Changes in algal structure and ultrastructure were observed. It was remarked that cinoxacin had the strongest effect on endosymbiotic alga performing 72 hs treatment especially higher concentration concerned. Antichloroplastal and antimitochondrial effect was the most expressed. In some algae irregularities in chloroplastal thylakoide system were observed. Thylakoides changed their shape or they were swollen. Mitochondria increased in size. They were also swollen (Fig. 2). Cinoxacin belongs to quinolone and fluoroquinolone group of antibiotics. It blocks the synthesis of bacterial DNA by inhibition of DNA gyrase enzyme. Since it is assumed that chloroplasts and mitochondria originated from eubacteria, it was expected to trace damages on these organelles. Except these strongest damages on chloroplasts and mitochondria, changes were observed also on other cell-components of green algae. Ribosomes decreased in number. Plastoglobules increased in size and number. Different kinds of membrane structures were formed inside the vacuole in the shape of osmiophyllic and myelin figures. These manifestations are known response to the antibiotic treatment3. Tough cell wall was also damaged. Less damaged organisms regenerated after a period of time and made normal endosymbiotic interaction again. From the results it could be concluded that cinoxacin showed the toxic effect and that toxicity resulted in structural and numerous ultrastructural changes of green alga. Results of research on the field of antichloroplastal and antimitochondrial effect of antibiotics point out the evolutionary connection of chloroplasts and mitochondria with eubacteria and their prokaryotic origin.