So that the software could visually separate Paramecium from artefacts, the video recordings were converted to 8-bit format, and a size threshold of 10— pixel lengths was specified for Paramecium. Videos in which Paramecium ceased swimming, swam vertically or exited the field of view were discarded. ImageJ's Particle Analyzer and Particle Tracker returned x , y coordinates pixel locations within the field of view for cells within each frame of each video — along with estimates of length and width in pixels, aspect ratio dimensionless ratio of length to width , and cross-sectional area in square pixels.
Mobility was quantified in terms of the frequency of turns made by Paramecium and the average swimming speed of Paramecium in each video. Turns were defined as movements that caused Paramecium to deviate at least 45 degrees from its original trajectory. Swimming speed was defined as spatial displacement over time. This was calculated by taking the square root of the sum of the squared change in position along the X -axis and the squared change in position along the Y -axis Pythagorean Theorem.
The resulting measure was converted from pixels per frame to millimetres per second and then averaged for each Paramecium cell. To ensure that this measure of swimming speed was not confounded by how often Paramecium made turns and how much it slowed down or sped up at the beginning or end of a turn, only linear parts of trajectories were used to make the calculation. Also omitted were videos in which Paramecium was undetected or misidentified by ImageJ's Particle Tracker e.
Estimates of cross-sectional length, width, area and aspect ratio were calculated for these same remaining videos by taking the mean of the output of ImageJ's Particle Analyzer across frames for each cell. Effects of infection level on swimming speed, aspect ratio, total per capita turns and cross-sectional area were assessed using one-way analysis of variance anova. Details regarding the terms used in these equations are given in Appendix S2 Supporting information.
Despite the addition of methyl cellulose to the growth medium, the population dynamics of Didinium and Paramecium did not exhibit sustained oscillations in any of the replicates. In one replicate of the SPDH treatment, Didinium went extinct immediately after its introduction to the system.
This replicate was therefore excluded from analyses. After c. Abundances in the predator- and parasite-free control populations - - remained high for c. Initially, predation by Didinium led to similar rates of decline in Paramecium abundance in both infected and uninfected populations.
After Day 22, the decline stabilized in two uninfected SPD- populations, while the third went extinct. In contrast, at the same time, abundance began to increase in the two infected SPDH populations to levels observed in corresponding uninfected populations with the predator, and even exceeding abundance levels of infected SP-H populations in the predator-free treatment Fig.
Population growth curves showing abundances N of Didinium , Paramecium and Serratia in each microcosm. N was measured as no. Didinium abundance increased initially, reaching peak levels 10—12 days after introduction. However, all populations went extinct over the following 20 days. This difference was particularly pronounced during the initial period after introduction Days 11— This reduction was almost fold, when Paramecium populations were at their peak density Fig.
However, this difference disappeared c. In other words, the combined action of parasite infection Holospora and predation Didinium on the abundance of the intermediate consumer Paramecium , as well as that of its resource Serratia , was non-additive. Specifically, predation by Didinium reduced the negative effect of Holospora infection not only on Paramecium abundance, but also on Serratia abundance — such that the presence of the predator removed the population size advantage observed in uninfected Paramecium populations.
Maximum-likelihood estimates of parameters and initial state variables used in the model. Best fits of the model to the population dynamics observed in all microcosms containing Didinium treatment codes and symbols same as in Fig. Dotted lines denote model predictions.
Swimming speed was higher in covertly infected Paramecium cells than overtly or uninfected cells. Aspect ratio was lowest in overtly infected Paramecium , moderate among covertly infected Paramecium and highest among uninfected Paramecium — meaning that — as is consistent with the literature Fokin , Paramecium cells became shorter and fatter with increasing parasite load Fig.
Differences in behaviour and morphology among Paramecium with differing levels of Holospora infection. Given the differences in swimming speed and cell length among infected and uninfected Paramecium , both Fenchel's method and Verity's method of estimation predict higher encounter rates with Serratia for infected Paramecium than for uninfected Paramecium Fig.
S2 , Supporting information. We investigated the mechanisms and consequences of parasite-mediated effects in experimental food webs, containing two protozoans predator and prey , a bacterial parasite and a bacterial prey species.
The observed population dynamics of the different protagonists revealed a mix of direct and indirect effects along the food web, including a modulation of parasitic effects in the presence of a predator. Below, we explore some potential explanations for and implications of the observed results, involving density-mediated and trait-mediated mechanisms. As expected, both parasitic infection by Holospora and predation by Didinium had a strong negative impact on Paramecium abundance.
Initially, the introduction of the predator led to a massive decrease in abundance of both infected and uninfected Paramecium , and a concomitant increase in Didinium.
Didinium grew to lower peak density and tended to die out earlier when preying on infected Paramecium. Since Didinium does not feed on Holospora independently of Paramecium , and Holospora cannot infect Didinium , one can infer that Holospora's effect on Didinium abundance was mediated via Paramecium.
Most likely, this was due to the abundance of infected Paramecium being generally lower than that of uninfected Paramecium. However, the methods employed in our study do not entirely rule out the possibility of infection reducing the nutritional quality of Paramecium.
They found that Didinium exhibits decreased fission rates, abnormal cell formation and inability to encyst when fed Paramecium aurelia that have been progressively starved or malnourished.
Future studies should explore the likelihood of Holospora -infected Paramecium being less nutritious to Didinium than uninfected Paramecium and of this being the basis of Holospora's effects on Didinium population dynamics. There were signs of synergism between the effects of parasitism and predation during the decline phase of the Didinium.
In particular, the infected Paramecium populations recovered and achieved the same if not higher abundance than their uninfected counterparts. This suggests some kind of predator-related buffering or overcompensation for parasitic effects on the part of Paramecium. A similar effect has been observed in another study involving Paramecium and Holospora , wherein a certain type of stochastic environmental fluctuation allowed infected populations to maintain the same density as uninfected populations Duncan et al.
This phenomenon may be generalizable and therefore warrants further investigation. Image analysis revealed significant effects of infection on Paramecium aspect ratio and swimming speed. Cell length decreased with increasing parasite load, meaning that heavily infested Paramecium cells become smaller and fatter. Theoretically, this could increase vulnerability to predation by Didinium , given that Didinium feeds more readily on smaller cells Hewett On the other hand, we found that infection also tended to increase swimming speed, at least in covertly infected individuals with more moderate parasite loads.
This enhanced activity is intriguing in that it contrasts with previous findings of reduced short-distance dispersal of infected Paramecium Fellous et al. Moreover, the modified aspect ratio it is associated with should make the energetic cost of locomotion higher for infected Paramecium Roberts , which in combination with infected Paramecium having higher rates of encounter with Serratia could explain Paramecium's enhanced negative effect on Serratia abundance in the presence of Holospora discussed further below.
In a follow-up experiment, we evaluated the potential net effect of these parasite-induced modifications on predation risk. To this end, the feeding rate of individual Didinium cells facing 10 infected or uninfected Paramecium cells was measured over the course of several hours further details provided in Fig. S3 caption, Supporting information. This experiment revealed no significant effects of infection status on Didinium feeding rate Fig. S3 , Supporting information , suggesting that the above trait modifications either played no role in terms of predation risk or cancelled each other out.
Future studies might re-evaluate this conclusion based on results of a rigorous functional response experiment that systematically varies the level of Paramecium abundance to which Didinium is exposed. The modelling results were in line with the above observations.
We found that Holospora's effects on Didinium abundance and on Didinium and Paramecium's predator—prey dynamics were more likely due to density-mediated indirect effects than to a combination of density- and trait-mediated indirect effects.
Altogether, reducing Paramecium's growth rate and carrying capacity was sufficient to produce a close fit of the model to the dynamics of Didinium and Holospora -infected Paramecium , implying that, under these circumstances, Holospora's density-mediated indirect effects were the most important. While Serratia abundance was indeed highest in the complete absence of Paramecium , it was lower in the presence of infected Paramecium than in the presence of uninfected Paramecium. This suggests that infection with Holospora may increase Paramecium's per capita feeding rate, despite Holospora's generally negative effect on Paramecium fitness.
Similar to what it did in the case of Paramecium abundance, the presence of the predator also reduced the negative effect of infection on Serratia abundance.
Indeed, a week after introduction of the Didinium , Serratia abundances in infected Paramecium populations had caught up with those in uninfected populations, which was not the case in predator—free populations. The reduction in abundance due to Didinium may have had greater weight in the case of the infected Paramecium populations due to Holospora's enhancement of Paramecium's reduction of Serratia.
One important finding regarding parasite Holospora dynamics is the buffering effect of predation. Predation by Didinium ultimately had a net beneficial effect on infected Paramecium populations, allowing them to recover from critically low abundances reached towards the end of the experiment.
The present experiment did not address the consequences for parasite horizontal transmission and epidemic spread. However, it is clear that maintaining relatively higher population density may further increase the force of infection, as the number of infected hosts is directly related to the frequency of new infections. Moreover, a second follow-up experiment provided evidence for a direct impact of predation on Holospora transmission.
S4 , Supporting information; further details provided in Fig. S4 caption, Supporting information. A possible explanation is that these parasite transmission stages are released while Didinium devours and digests infected Paramecium.
This may directly enhance the chance of transmission in a population under predator attack. Our study illustrates how the addition of a single additional antagonist here Didinium can have complex, and partly unexpected, demographic feedbacks on host—parasite interactions and vice versa.
We also found that such effects can be mirrored at lower levels of a food chain. The broader significance of these results is that they point to possible complications for pest management in agriculture and conservation, when additional players in the natural community are certain to come into play.
For example, the use of certain parasites as biological control agents may be inadvisable, if another species here Didinium interacting with the target obstructs the expected effects due to the target's behavioural and physiological responses.
Here, we described the impact of a single episode of proliferation and prey reduction by a predator which subsequently went extinct. Building on this simple framework, future work may address more complex scenarios, such as the spread and maintenance of an epidemic or the occurrence of co-evolution, when all interacting species are maintained over longer time-scales.
Additional Supporting Information may be found in the online version of this article. National Center for Biotechnology Information , U. The Journal of Animal Ecology. J Anim Ecol. Published online Dec Alison Dunn, Handling Editor.
Author information Article notes Copyright and License information Disclaimer. E-mail: ten. Received May 15; Accepted Oct This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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. Predator-prey interaction between Dileptus margaritifer and Climacostomum virens. A Dileptus the slender cell at the center starts swimming backward after hitting with the proboscis Climacostomum. A small bulge arrow is developing on the surface of the Climacostomum at the site where the proboscis has just hit. B The same cells as in A, about a second later, show the retreated Dileptus and a small cloud arrow near the Climacostomum.
Dark field micrographs of living cells. Pictures from [ 75 ]. Hazy cloud consisting of needle-like structures discharged from the toxicysts of Dileptus margaritifer. If the defensive function of cortical granules in C. Differently from the Paramecium species which do not have trichocysts exclusively for defense localized in the oral apparatus, C.
These prey are sucked up into the buccal cavity of C. A cell of P. Perhaps, as in the case of contact with the toxicysts of the raptorial ciliate D.
A similar phenomenon also occurs with different preys which possess chemical extrusomes for defense such as the ciliate S. In this case, after a cell-cell contact, the S. If this is the case, it is likely that the cortical granules of C. Besides the natural role of climacostol and thanks to the availability of a straightforward method for its chemical synthesis [ 78 ], other bioactivities of the toxin and its potential application to human health are, to date, investigated in various biological systems.
The toxicity of climacostol proves very effective against pathogenic Gram-positive bacteria such as Staphylococcus aureus or S.
In addition, on the basis of the anticancer properties of other resorcinolic lipids, the toxic potential of climacostol is also studied against cancerous and non-cancerous mammalian cells, including human cell lines. The results show that climacostol effectively inhibits the growth of some tumor cell lines in a dose-dependent manner by inducing programmed cell death, with non-tumor cells proving significantly to be more resistant to the toxin [ 73 , 80 ]. More recently the anti-tumor therapeutic activity of this toxin was also proved in vivo , using a melanoma allograft model in mice [ 81 ].
These results are quite interesting also in light of the fact that different molecules produced by other ciliate species show some particular pharmacological properties such as the sesquiterpenoid euplotin C or the cell type-specific signaling protein pheromone Er-1 from Euplotes species see [ 82 ] for a review. Returning to the topic of this chapter, different secondary metabolites have been also isolated and characterized from other heterotrics, such as Spirostomum ambiguum, and S.
The species is very common in the sludge-water contact zone of wells, ponds, sewage ponds, lakes, oxbows, ditches, and in the sediments of alpha- to beta-mesosaprobien rivers [ 77 ]. The defensive function of its cortical granules was recently demonstrated against different predators and the toxicity of its content was tested on a panel of freshwater ciliates [ 77 , 83 ].
The cold-shock method was applied to S. Both untreated and cortical granule-deficient cells were exposed to the attack of C. Similarly to untreated cells, cortical granule-deficient cells of S. The toxin involved in this interaction was purified by reversed phase high-performance liquid chromatography RP-HPLC , and its structural characterization was carried out through nuclear magnetic resonance spectroscopy NMR and mass spectrometry MS measurements and revealed as 2- 3-methylbutenyl benzene-1,4-diol mono-prenyl hydroquinone Figure Prenylated-hydroquinone derivatives are metabolites of abundant occurrence and have been isolated from fungi, algae, plants, animals, and bacteria [ 77 ].
In this case the involvement of this molecule in predator-prey interaction is clear. Interestingly, another freshwater species of the genus Spirostomum, S. It is no novelty that closely related organisms can produce different or even biogenetically distant specific secondary metabolites [ 77 ], and it is very common for ciliates [ 56 ].
To date, the only reported exception to this phenomenon is related to the genus Blepharisma in which the three species B. External morphology of living cells of Spirostomum ambiguum. Reduction in the number of extrusomes cortical granules in Spirostomum ambiguum obtained by cold-shock treatment.
A Extrusomes in an untreated cell. B Extrusome-deprived cell after cold shock. Pictures from [ 77 ]. Predator-prey interaction between Climacostomum virens and Spirostomum ambiguum. A 1: Cell of C. B Predator-prey interaction between C.
Micrographs extracted from a film clip. Another peculiar defensive mechanism, reported as inducible defense, has been described for some Euplotes species as the response to the presence of some predators, such as microturbellarians, ciliates, or amoebas.
These predators can release active substances, called kairomones, which induce some behavioral and morphological changes such as the formation of spines in Euplotes as a defensive mechanism in response to the presence of the predator [ 85 , 86 , 87 , 88 ] for a review.
It could be interesting to study the efficiency of the inducible defenses, if compared to mechanical and chemical defense by means of extrusomes.
In this regard, a first study was performed to compare the efficiency of the defense mediated by trichocysts in P. The authors reported that the mechanical defense in Paramecium against metazoan predators appears to be equally effective as the chemical one, but can be successfully activated only during the very early interactions with the predator, whereas it is ineffective after the ingestion of the ciliate.
These cilia are arranged in bands around the body. One is located around the middle, and the other at the front. The front ends in a pointed snout cytosome. Didinium are unicellular and have an oval shape.
Didinium are heterotrophic organisms. They only have one type of prey; the much larger cilate Paramecium. When a Didinium finds a Paramecium , it ejects poison darts trichocysts and attachment lines.
The Didinium then proceeds to engulf its prey. Althoug h Paramecium are large, Didinium are voracious eaters and will be ready to hunt for another meal after only a few hours. If Paramecium become depleted, the Didinium encyst themselves until its food source becomes replenished. Didinium , like all members of Ciliophora , can reproduce sexually or asexually. Nucleoli degranulate before binary fission; the bands are dispersed throughout the macronucleus. These bands are segregated during division, but then re-form granular parts.
The nucleoli are separated into granular and fibrillar parts during conjugation. The fibrillar parts are usually eliminated.
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