Membrane details of resting trichocysts under the freeze fracture. The trichocyst tip tt and body tb are covered by the same membrane. The A-face of this membrane A-tin possesses randomly distributed particles whereas the B-face B-tin shows corresponding depressions. Picture from [ 9 ]. The trichocysts discharged by a cell of Paramecium tetraurelia exposed to picric acid solution.
Maupas, one of the pioneers of protozoology, first proposed the defensive function of trichocysts in Paramecium in , observing its morphological features and judging it as self-evident [ 38 ]; however, this point was questioned for years.
The main controversy was due to the fact that Paramecium species are easily preyed upon by Didinium in spite of massive trichocyst discharge by paramecia. Pollack reported that Didinium preys on wild-type cells as easily as trichocyst-defective mutants in P. However, further studies have unequivocally indicated that trichocysts in Paramecium exert an effective defensive function against unicellular predators, including the raptorial protists Dileptus margaritifer , Monodinium balbiani , Climacostomum virens , Echinosphaerium akamae, and E.
In addition, a more recent paper also analyzed the defensive function of trichocysts in P. The results of this study show the success in the defensive function of trichocysts against the rotifer and the ostracod while the mechanism seems ineffective against the flatworm. The authors speculate that the efficiency of the defense by means of trichocysts depends essentially on the kind of prey-capture behavior displayed by the predators.
In particular, the success of the defense mediated by trichocysts appears positively related to the time that the predator requires to capture and manipulate the prey before ingestion.
Consequently, and different from the turbellarian flatworm that directly swallows paramecia, predators such as the rotifer and the ostracod that, prior to ingesting paramecia, contact it with a ciliated corona or articulated appendices, give the prey sufficient time to activate the trichocysts discharge that allows it to escape [ 44 ].
Essentially this looks like the same phenomenon observed during the interaction between Paramecium and the predatory ciliate Dileptus margaritifer , that attempts to paralyze its prey with the toxicysts on its proboscis before ingestion, thereby inducing an explosive extrusion of trichocysts by Paramecium , which then swims away [ 44 ].
In this regard, another interesting observation was made when Paramecium was placed in a cell-free fluid containing the toxic material derived from the toxicysts from Dileptus [ 45 ] Miyake A. In this reaction, sometimes a single specimen cell of Paramecium was completely surrounded by its discharged trichocysts.
When this occurred, the Paramecium survived long after other cells were killed, moving slowly in the narrow space in the capsule of discharged trichocysts. But when it happened that one of these encapsulated cells managed to squeeze out of the capsule, it was quickly killed. This observation suggests that discharged trichocysts of Paramecium function as a barrier against the Dileptus toxins and hence the locally discharged trichocysts in the Paramecium-Dileptus interaction function as an instant shield against Dileptus.
However, especially in ciliates and flagellates, other kinds of extrusomes used for defense were found, ones that, unlike trichocysts, are capable of discharging toxic materials in response to predatory behavior. Pigment granules also called pigmentocysts and cortical granules are extrusive organelles containing pigmented or colorless toxic material, respectively, and they were originally classified as a special type of mucocysts [ 9 ]. Pigment and cortical granules are mainly present in heterotrich and karyorelictean ciliates, such as Blepharisma , Stentor , Loxodes, and Trachelonema, but they may also exist in other groups of ciliates.
They are usually present in great numbers throughout the cell cortex, sometimes providing bright colors to their bearers. Examples are Stentor coeruleus , whose coloration is due to the pigment called stentorin, and several red species of Blepharisma , whose coloration is due to blepharismins, formerly overall called zoopurpurin by Giese [ 46 ].
The coloration of these common heterotrichs has long attracted attention and most studies on pigment granules have been carried out using S. These granules have been shown to contain a mixture of five compounds called blepharismins that are multifunctional quinone derivatives structurally related to hypericin, a photodynamic toxin of Hypericum perforatum St. To date, two primary functions of blepharismins have been demonstrated: light perception and defense function against predators [ 47 , 48 , 49 , 50 , 51 , 52 ].
With regard to light perception, B. The step-up photophobic response helps the cells avoid strongly illuminated regions and lethal damage due to the photodynamic action of blepharismins [ 53 ]. In addition to light perception, blepharismins were found to act as chemical weapons via their light-independent cytotoxic effect against predatory protozoans and methicillin-resistant Gram-positive bacteria [ 49 , 50 , 54 ]. A possible explanation for this cytotoxicity can be found in the capability of blepharismins to form cation-selective channels in planar phospholipid bilayers [ 51 ], a phenomenon also expected to occur in the cell membranes of microorganisms exposed to toxic concentrations of ciliate pigments.
The defensive function of blepharismins was initially proposed by Giese in who found that crude extracts of Blepharisma were toxic to various ciliates but not to Blepharisma itself [ 55 ].
Unfortunately, however, his preliminary tests did not support this assumption, that is, Blepharisma was easily eaten by predators such as the heliozoan Actinospherium eichhorni and small crustaceans [ 46 , 55 ].
Some predators, Didinium nasutum , Woodruffia metabolica, and Podophrya fixa , did not eat Blepharisma , but they also ignored some other ciliates including uncolored ones. In the absence of further evidence, Giese was skeptical about the assumption [ 46 ].
This hypothesis was further unequivocally demonstrated by Miyake, Harumoto, and collaborators, comparing normally pigmented red cells of B. As a response to the attack by D. The discharge take place within a second and it is able to repel the predator, while the albino and light-bleached cells are much more sensitive to the attacks of D. Recently the defensive function of blepharismins was also investigated in two additional species of Blepharisma, B.
The results indicate that the chemical defense mechanism present in B. Authors speculate that the conservation of this panel of toxic secondary metabolites suggests that distinct roles for these molecules are likely required at least for the fine control of photophobic reactions, as initially proposed by Matsuoka et al.
Summarizing, the Blepharisma species studied are able to defend themselves against C. Additional toxic pigments, structurally related to hypericin, were found in other heterotrich ciliate species, such as stentorin in S. External morphology of a living cell of Blepharisma japonicum. Main secondary metabolites produced by ciliated protists. A Blepharisma being attacked by Dileptus. Arrow indicates the site of the damage inflicted by the proboscis of the Dileptus.
The rupture runs across the adoral zone of membranelles of the Blepharisma. B Enlargement of the region near the rupture in A. C The rupture magnification in B, showing the surface of Blepharisma peppered with spherules discharged from pigment granules. The surface is also pitted with small depressions presumably formed at the spots where the spherules have passed through the cell membrane. D Enlargement of a part of C. Pictures from [ 50 ].
Karyorelictean ciliates also possess pigment granules which are similar in size, structure, and distribution to those in the heterotrichs, but principally due to the difficulties to the growing species of karyorelictid in the laboratory, the chemical nature of their pigments is still unknown. The most studied species is freshwater Loxodes striatus , which presents yellow-brown pigment granules previously examined as photoreceptors [ 61 ].
More recently it has been proved that the pigment granules in L. Loxodes are able to discharge the toxic pigment as response to attacks of the ciliate D.
Intriguingly Finlay and Fenchel already proposed a defensive function for the pigment granules in Loxodes L. They assumed that this reaction may serve to localize Loxodes in regions of low oxygen tension where predators, such as planktonic metazoan, are rare and therefore the pigment may function as a predator-avoidance strategy.
If this is the case, pigment granules of Loxodes participate in two very different kinds of defense, chemical defense and the behavior-based predator-avoidance, conferring to the ciliate an ability to defend itself against a wider range of predators [ 62 ].
Predator-prey interaction between Dileptus margaritifer and Loxodes striatus. A Dileptus the slender cell at the left starts swimming backward after hitting a Loxodes with its proboscis. B The same cells as in A, about a second later, showing the retreated Dileptus and a mass of brownish material arrow near the Loxodes. Pictures from [ 62 ]. Pigmented granules are found also in other groups of ciliates as the Spirotrichea, and mainly in the genus Pseudokeronopsis, which shows species equipped with reddish-brown pigment granules morphologically similar to those in heterotrichs [ 63 ].
Particularly in P. New secondary metabolites, keronopsins and keronopsamides, respectively, produced by P. In the case of P. For these reasons a defensive function for these secondary metabolites has been proposed; however, no data relative to their cellular localization and mechanism of action are available to date.
On the other hand, in the case of P. The most extensively studied species is P. As the content of pigment granules, three new secondary metabolites have recently been characterized and named erythrolactones A2, B2, and C2.
These molecules were detected in the crude extract of whole cells together with their respective sulfate esters, erythrolactones A1, B1, and C1 Figure After the application of the cold-shock method on massive cell cultures of P. The mixture of these three molecules has been proven to repel some predators, such as the ciliate C. Erythrolactones A2, B2, and C2 are the only toxins present in the extrusome discharge of P. It is known that the process of sulfonation of endogenous molecules is a major metabolic reaction in eukaryotes that can increase water solubility and influence conformational changes but can also lead to the activation or inactivation of a biological effect see [ 70 ] for a review.
Buonanno and collaborators [ 64 ] speculate that the exclusive maintenance of the sulfate esters of the erythrolactones inside the P. External morphology of a living cell of Pseudokeronopsis erythrina. Other organelles strictly related to pigment granules are the colorless cortical granules in the heterotrich, sometimes reported as granulocysts to underline their extrusive nature.
These organelles show a greatest morphological similarity to pigment granules, as in the case of the cortical granules of Climacostomum virens [ 71 ] and Blepharisma hyalinum [ 72 ]. The function and biological activity of the secondary metabolites contained in the cortical granules seem to be primarily related to chemical defense or offense, and the cortical granules in C.
This freshwater heterotrich ciliate, if properly stimulated, is able to repel predators by discharging the colorless toxin climacostol Figure 14 and some related analogues. This toxin may be chemically classified within a large group of natural compounds known as resorcinolic lipids also called alkylresorcinols or 5-alkylresorcinols , widely detected in prokaryotes and eukaryotes [ 73 ] and with reported antimicrobial, antiparasitic, antitumoral, and genotoxic activities see [ 74 ] for a review.
A typical defensive behavior of C. Sometimes, together with the discharged material from C. Interestingly, the chemical defense adopted by C. In both cases, carbon-specific concentrations of the fatty acids obtained from the diet should increase in the studied ciliates as actually found.
In addition, both ciliates may have compensated for low fatty acid concentrations in their diet simply by exhibiting high ingestion rates. In this case, the cellular amount of fatty acids in the ciliates could have originated only from the diet. Alternatively, but implying high energetic costs, our ciliates may have been able to synthesize certain fatty acids, probably through elongation and desaturation of a precursor molecule.
Moreover, this ciliate is known to synthesize unsaturated fatty acids by two distinct pathways from palmitic acid [ 29 ]. However, synthesis of unsaturated fatty acids may be inefficient in the studied heterotrophic protists, as already shown for some zooplankton species [ 30 , 31 ].
For instance, DHA was found in Cryptomonas , but not in our ciliates. In light of feeding, such mechanisms involving antagonistic and synergistic interactions among biochemical molecules may influence the nutritional quality of a species. The SDA provided evidence for the polyunsaturated fatty acid composition as a major parameter separating Balanion and Urotricha from their algal diet Cryptomonas , with differences originating from a broader palette of polyunsaturated fatty acids than observed for monounsaturated or saturated fatty acids Table 2.
Although SAFAs and MUFAs were able to significantly discriminate between ciliates and diet, the PUFA composition and concentration seems to be rather diverse and perhaps more dependent on metabolic characteristics of the studied ciliates. This is not surprising, since energetic costs of building polyunsaturated fatty acids are much higher than those for monounsaturated or saturated ones.
Also the presence of a broader number of accessory enzymatic systems is necessary to insert double bounds into the hydrocarbon chain [ 34 , 35 ]. Furthermore, the ciliated protozoa were only separated at the second root of the SDA based on saturated and monounsaturated fatty acids, whereas a strong separation of the ciliates was already evident at the first root of the SDA on polyunsaturated fatty acids.
This suggests a rather similar composition of saturated and monounsaturated fatty acids in the investigated ciliate species, which could partially arise from the diet or from similar features in metabolism and synthesis of these fatty acid classes by both ciliates. On the other hand, large differences in the polyunsaturated fraction between Balanion and Urotricha suggest differences concerning metabolic abilities and requirements for polyunsaturated fatty acids in these species.
As observed for fatty acids, the carbon-specific amino acid concentrations were higher in the ciliates than in Cryptomonas. Again, ingestion and assimilation efficiencies may be the underlying mechanisms explaining the accumulation of amino acids in the studied ciliates. Amino acid requirement and assimilation in Tetrahymena depends on the concentrations of nutrients in the culture medium such as sodium [ 15 ].
This ciliate is able to recover changes in cell volume resulting from changes in extracellular osmolarity, partially by adjusting the intracellular concentration of free amino acids [ 37 ]. Moreover, since different biosynthetic pathways for single amino acids can differ among species [ 36 ], this may have contributed to the observed differences between Balanion and Urotricha. In this case, the dietary biochemical composition should have only a limited influence on the biochemical composition of the studied ciliates.
Hence, metabolic abilities of our ciliates, i. Moreover, metabolic features regulating assimilation and synthesis of complex biochemical compounds, such as PUFAs, may differ between both ciliate species. When considering overall energy uptake by predators, data on carbon-specific biochemical concentrations are important, because carbon provides a good biomass and energy estimate.
However, in light of the entire feeding process, which includes capture of prey, handling time and ingestion of prey, the prey size [ 38 ] and thus the cell-specific biochemical content is crucial. There is a trade-off between the effort of prey handling and the nutritional quality of the single prey. In the present study, the ciliated protozoan matter exhibited higher concentrations of various chemical compounds than the algal matter.
Regarding the aspect of biochemical accumulation, our findings support the assumption that ciliates may be viewed as prey of upgraded quality at an early stage in aquatic food webs. We thank D. Pflanz for helping with sample processing and maintenance of the ciliates and algal cultures; J.
Ogunji and M. Wirth for their support in the biochemical analyses. We also thank R. Sanders, B. Poynton for the English editing. Weisse is acknowledged for providing cultures of B. This work was supported by a Ph. D Grant to I. DeBiase A. Sanders R. Porter K. Google Scholar. Williamson C. Stutzman P. Moeller R. Goulden C. Aoki-Goldsmith R.
Adrian R. Wickham S. Butler N. Mohr S. Freshwater Biol. Ederington M. McManus G. Harvey H. Labarta U. Hydrobiologia , 73 — Stampfl P. Weers P. Siewertsen K. Gulati R. Desvilettes C. Bourdier G. Amblard C. Barth B. Klein Breteler W.
Schogt N. Baas M. Schouten S. Kraay G. Frolov A. Pankov S. Geradze K. Pankova S. Spektorova L. Aquaculture 97 , — Guisande C. Maneiro I. Riveiro I.
Helland S. Nejstgaard J. Humlen R. Fyhn H. Holz G. The nutrition of Tetrahymena : essential nutrients, feeding and digestion A. Pennsylvania 89 Guillard R. Lorenzen C. Foissner W. Berger H. Schaumburg J. Informationsberichte des bayer. Google Preview. Feig Y. Montagnes D. By comparing both ciliated protozoa we intended to identify species-specific differences in the metabolic features of these ciliates.
Carbon- and cell-specific concentrations of fatty acids and essential amino acids were investigated for the ciliates Balanion planctonicum and Urotricha farcta grown on the cryptomonad Cryptomonas phaseolus. Stepwise discriminant analyses SDA indicated differences in the biochemical composition between ciliates and their diet and between the two ciliated protozoa.
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