Demographic and baseline smoking data for Respiragene trial.
\\n\\n
Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 179 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 252 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"4816",title:"Face Recognition",subtitle:null,isOpenForSubmission:!1,hash:"146063b5359146b7718ea86bad47c8eb",slug:"face_recognition",bookSignature:"Kresimir Delac and Mislav Grgic",coverURL:"https://cdn.intechopen.com/books/images_new/4816.jpg",editedByType:"Edited by",editors:[{id:"528",title:"Dr.",name:"Kresimir",surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"53900",title:"How Effective is Fear of Lung Cancer as a Smoking Cessation Motivator?",doi:"10.5772/67235",slug:"how-effective-is-fear-of-lung-cancer-as-a-smoking-cessation-motivator-",body:'\nThe reasons why smokers either continue to smoke or stop smoking are diverse and every case is probably unique. However, there is a basic truth in that there is a constant seesaw between smoking cessation motivators and the rather less well understood de‐motivators. The importance of de‐motivators is illustrated by the simple fact that over 60% of smokers say they would like to quit but most seem somehow unable to do so [1, 2]. The obvious explanation is that this is due to nicotine addiction but this may be only one of many de‐motivators.
\nIn 2014, my colleagues and I at the University of Surrey, United Kingdom (UK), carried out research into smoking cessation. We recruited 67 smokers who wanted to quit from a primary care database of 32,000 (Table 1) and randomized them to either a control group or a test group. The test group had an additional motivator to quit. This was the Respiragene risk score for lung cancer derived from a genetic test (19 single‐nucleotide polymorphisms (SNPs) and one deletion mutation) and clinical criteria including history of chronic obstructive pulmonary disease, family history of lung cancer, and age. Both groups attended 8 weekly smoking cessation clinics which took place at the same primary care venue but with test group and control group attended on different weekdays. We published our protocol and outcome measures a priori [3]. Primary outcome was smoking cessation after 6 months.
Demographic/smoking feature | Test group (n=36) | Control group (n=31) | p‐Values (test) |
---|---|---|---|
Gender: female | 55.6% | 53.3% | 0.747 (Chi‐square) |
Mean age (at start of study) | 49.7 | 49.0 | 0.812 (Unpaired t) |
Mean age at completion of education | 18.4 | 18.5 | 0.971 (Unpaired t) |
Years in education (excluding interruptions) | 22.8 | 26.2 | 0.517 (Unpaired t) |
Pack years | 32.0 | 28.9 | 0.396 (Unpaired t) |
Cigarettes/day at start | 18.1 | 18.1 | 0.993 (Unpaired t) |
Demographic and baseline smoking data for Respiragene trial.
During our research, we found that 36% of smokers had stopped smoking and were still not smoking after 6 months. Of the 64% who were still smoking at 6 months, all but two participants planned to stop smoking at some time in the future and 30% had cut down substantially (by 10 or more cigarettes/day) [4].So why don\'t they just quit?
\nOur hypothesis was that when participants were told their lung cancer risk, this would tend to outbalance any de‐motivators such as issues around nicotine addiction (Figure 1) and give a high 6‐month quit rate. However, we were probably underestimating the potency of known and unknown de‐motivators. It certainly cannot be entirely due to nicotine addiction because varenicline blocks the physical addiction and prevents most withdrawal symptoms and yet 50–75% of subjects taking varenicline will still be smoking 6 months later [5, 6]. There must, therefore, be more to it than nicotine addiction.
For smokers: the seesaw of destiny. The balance between motivators and de‐motivators determines success or failure for smokers trying to quit.
As already reported [4], the 6‐month quit rate in our Respiragene trial was more dependent on the risk score than we had expected. The laboratory reported the Respiragene risk score as three categories: average risk of lung cancer, high risk of lung cancer, and very high risk of lung cancer. Only non‐smokers and ex‐smokers can achieve the category “low risk”. We were also able to estimate lifetime risk as a percentage (i.e., a 50% lifetime risk meant that the risk of lung cancer was like tossing a life/death coin). The 6‐month quit rate results are summarized in Figure 2. However, assessing the balance of motivators and de‐motivators was included in secondary outcomes. The relative importance of ten smoking cessation motivators was estimated using a feedback questionnaire at 8 weeks and again at 6 months. The results show the perceived importance of these motivators (Figure 3).
The blue “glass ceiling” represents the quit rate for the control group. Quit rate at 6 months for controls was 57%. Subjects with an average risk score for lung cancer (only non‐smokers and ex‐smokers are assessed as “low risk score”) had a lower quit rate than controls, and a “moderately high” quit rate was no better but difficult to judge due to small numbers (only 4). However, all but one of the nine subjects with a “very high” risk score (equivalent to 50% lifetime risk of lung cancer) had quit at 6 months giving an 89% quit rate.
Mean values for motivators and influences that have helped to reduce or stop smoking: “Please score each of the items below according to how strong an influence they have been in helping you to quit smoking”. Scores for motivators for individual participants were calculated as percentages of the sum of total scores of the individual and mean values calculated from these percentage scores.
From taking notes on comments from patients during counselling and from responses to open‐ended questions in feedback questionnaires that participants completed, we were able to clarify the roles of some of these de‐motivating factors. Most smokers have two main de‐motivators:\n
Nicotine addiction and fear of withdrawal symptoms [7–9]. Nicotine has been shown to be as addictive as illegal drugs such as morphine and cocaine.
Optimism bias [10–13].Tendency to underestimate the health risks of smoking and the feeling “It\'ll never happen to me”.
We expected that our study would confirm the hypothesis that being told a risk score for lung cancer would cancel out both nicotine addiction and optimism bias in at least 50% of participants. An earlier study (n = 99) using the Respiragene risk score had shown that smokers were more likely to quit compared with a control group whatever risk score they were given [14]. However, these participants were recruited from a hospital in New Zealand. We carried out a similar trial in a UK primary care setting. A surprise finding from our trial was that although all but one of the participants with a very high risk score had stopped smoking at 6 months, participants with an average risk score were more likely to be smoking than controls (Figure 2).
\nThe test subjects with an average risk score demonstrated a quit rate that was significantly lower than the quit rate in the control group (p = 0.03) which suggests that they had been de‐motivated and encouraged to think that it was safe to carry on smoking because their lifetime risk was perceived as “not so bad”. Or to put it another way, their optimism bias has been reinforced by their average risk score! Psychologists refer to this phenomenon as (No. (iii)) confirmation bias [15] and explain that when a subject has two conflicting ideas in their head (i.e., smoking is too risky versus the risk of smoking is exaggerated), this is intolerable—a phenomenon known as cognitive dissonance [16]. This mental discomfort can only be solved by ditching one idea and giving undue prominence to the other. A classic example is the smoker who responds to a challenge about the risks of his habit by saying: “Uncle Charlie smoked like a trouper and lived to be 90”. Any evidence to the contrary, such as other smokers in the family who died young, is conveniently ignored.
\nOther possible factors that make it difficult to quit that we noted in our participants and which have been previously recognized by other researchers in this field were as follows:\n
Attentional bias [17, 18]. The smoker is plagued by recurrent thoughts about the pleasures of smoking that serve to increase craving for the next cigarette.
Post‐traumatic stress disorder (PTSD) [19–21]. This is a mental health condition caused by a traumatic event such as rape, warfare experiences, and other near death experiences such as road traffic accidents and industrial accidents. The subjects experience distressing dreams and flashbacks, and they are more likely to become heavily addicted smokers.
Anxieties about smoking cessation and weight gain [22, 23]. This is an issue for many female smokers who start smoking when they are relatively young to control their weight. Later in life, they may try to quit but revert to smoking when they put on weight.
Side effects of smoking cessation therapy [24–26]. Patients using pharmaceuticals such as nicotine replacement therapy (NRT) patches or nicotine blocking drugs frequently report side effects. Once they have experienced a side effect, they usually revert to smoking.
Fatalism [27, 28]. This is the attitude that “What will be will be”. These smokers either feel they have little or no control over outcomes such as lung cancer or they simply do not care if they are destined to develop a smoking‐related disease.
Peer pressure [29, 30]. The influence of fellow workers on smoking can be a decisive factor. All the emphasis has been on peer pressure in adolescence and initiation of smoking, but peer pressure can be equally important in the adult work force.
Lack of family cohesion [31, 32]. Research has shown that family cohesion is associated with concerns about passive smoking and smoking cessation. Conversely, lack of family cohesion is associated with a significantly higher incidence of persistent smoking.
Inadequacy of the risk score as a motivator. Our own research, as described above, suggests that a risk score for a single disease (lung cancer in this case) is not a powerful enough motivator to cancel out de‐motivators in 64% of smokers, especially if the risk score is “low average” when they may be falsely reassured and continue smoking.
To preserve confidentiality, the case histories I present here have been altered (age, gender, and circumstantial details) so that the participants in our research project are unrecognizable. However, basic clinical details have been preserved as far as possible. These cases help to demonstrate why some smokers cannot quit, despite stating that they would like to.
\nThis participant was a young housewife who was stressed by having to care for two mildly hyperactive small boys aged 3 and 5 and was still smoking 15 cigarettes/day at 6 months. She seemed falsely reassured by the 35% lifetime risk commenting: “only 35% that\'s not so bad”. When I gave her an analogy: “What if I told you that if you carried on living in your present house, you stood a 35% chance of being murdered in your bed, but if you moved to a house in the next road the risk would drop to 1%”. She hesitated a moment then said: “But doctor, that\'s completely different”.
\nA 35% lifetime risk is less than the risk of tossing a life/death coin but close enough to be worrying. So why wasn\'t this patient worried? Her hesitation suggests cognitive dissonance [16]. That is two competing ideas buzzing around in your head. For stability and well‐being, one of the competing ideas must give way. Her most comforting solution was to accept that the 35% risk of lung cancer was nothing like the risk of being murdered in your bed. Well, of course, it is a different scenario but the risk of death is identical. This is also a good example of confirmation bias [15]. She managed to confirm her feeling that a 35% risk was “not so bad” by rejecting my analogy.
This participant had been a mature medical student who qualified in his mid 30s. Soon after qualifying, he was at BMA House, Tavistock Square, in 2007, when the suicide bomber detonated on the top deck of a bus in Tavistock Square, and he was the first doctor on the scene. Although he was a non‐smoker at the time, he found the only way to cope with flash backs and other PTSD symptoms related to this horrific incident was to become a habitual smoker. He is now a part‐time psychiatrist near retirement and was still smoking 10 cigarettes/day at the 6‐ month follow‐up.
\nThis participant started smoking for the first time aged 40 years, which is unusual. However, the circumstances were also unusual. Although this is obviously linked to post‐traumatic stress disorder (PTSD), there may be less dramatic and less obvious versions of PTSD that fuel the smoking habit such as unreported domestic abuse.
This participant was a 23‐year‐old woman who worked as a stable maid. She had quit at 8 weeks and she had always seemed highly motivated. However, she sustained a compound fracture to her right tibia when she was kicked by a horse. As a result, she was stuck at home, off sick, with the injury for some time. All the pain and worry and the boredom of being at home all the time is what started her back on the cigarettes (10/day)—they made it all a bit more bearable.
\nAlthough this participant cites pain and boredom as the reasons for relapsing to smoking, there were also features of PTSD. Her failure to quit was surprising as she was the “leading light” of the test group. She was the first in her group to quit and gently encouraged other participants. Perhaps this case illustrates how PTSD acts as a very powerful de‐motivator.
This 58–year‐old woman in the control group did not seem to have much of a problem quitting after a lifetime as a smoker (started aged 14 year) but she told me, at the 6‐month follow‐up that it was far from easy and described it as being like bereavement. It is, quite literally, as difficult to deal with as the death of someone very close to you. On the other hand, her lead motivator was concern about the affects of side‐stream smoke on someone very dear to her—her new baby grandson.
\nThis participant was remarkably open and honest about her feelings, and it is certainly sobering to think that smoking cessation is as difficult to cope with as suffering a bereavement. However, the fact that her main motivator was concern for her grandchild is very significant. Researchers have shown that family cohesion is associated with a lower incidence of smoking and lack of family cohesion with a very high incidence (70%) of smoking [32]. Family cohesion and awareness and acceptance of the health hazards of side‐stream smoke correlated (p < 0.01) in a paper from Texas in 2010 [31]. Two other participants mentioned the influence of grandchildren in relation to passive smoking and their decision to quit. Altogether, 8/67 (12%) of our participants mentioned passive smoking and family as a key motivator without prompting (in response to an open‐ended question asking for “further comments“).
This 48‐year‐old woman, who had recently been through a stressful divorce, was unable to work due to the debilitating effects of Crohn\'s disease. She was well aware that smoking cessation would probably improve her Crohn\'s symptoms. She started on varenicline but had to stop taking it after 3 days due to an acute exacerbation of Crohn\'s symptoms. She never returned to the clinic and was still smoking 12/day at the 6‐month follow‐up saying: “This is my only way of coping with boredom”.
\nThe impression from this patient was that she had simply “given up”. There are varying degrees of fatalism like this exhibited by smokers [27]. She might have been able to fight back with the help of varenicline, unfortunately she had gastrointestinal side effects that she interpreted as an exacerbation of her Crohn\'s disease so stopped taking varenicline after 3 days. In her case, the varenicline side effects seemed to a significant de‐motivator.
This 35‐year‐old single man in a high‐powered office job had managed to stop smoking for 5 years when his younger brother died of lung cancer. This was the third 1st degree relative to die from lung cancer. However, his current work ethos was one in which “everyone smoked” and now he was back on 15 cigarettes/day. Despite the family history and a high risk score he was still smoking 15 cigarettes/day at the 6‐month follow‐up. He blamed “work stress and peer pressure” for his inability to quit.
\nThis subject\'s inability to quit was really puzzling, and he himself was puzzled by it. There may have been several de‐motivating issues but peer pressure at work was certainly very significant in his case.
Nicotine, cannabinoids, and cocaine act as insecticides to protect plants from insect attack. Mammals that eat plants have evolved to tolerate these chemicals but only humans have developed the habit of burning and inhaling plants containing these toxic chemicals. Archaeologists have found evidence for this habit going back into prehistory [33]. There is even evidence of genetic adaptions to nicotine specific to humans [34]. Edward Hagan, professor of Anthropology at Washington State University, argues that there is a balance of benefits and costs to smoking tobacco. Nicotine must have some advantages that outweigh the health costs in some circumstances. Our ancestors may have found the effects of nicotine on the brain beneficial in times of stress and hunger but Hagan argues that nicotine\'s greatest evolutionary advantage may have been efficacy as an anti‐helminth drug, especially in controlling those helminth parasites that migrate through the lungs [35].
\nIt is no surprise, therefore, that there are human genes that relate to smoking behaviour. A recent review estimated that, according to twin studies, 75% of behavioural variation (variation in smoking initiation, persistence, and cessation rates) is genetically determined [36]. However, only about 5% of this variation can currently be explained by known gene variants, mainly single‐nucleotide polymorphisms (SNPs) but 19% of the variation in smoking initiation can be explained by known SNPs. Research is ongoing in this area with the hope that identification of further SNPs and other gene variants will improve our understanding of smoking behaviour and smoking cessation de‐motivators leading to new effective treatments to aid smoking cessation [8, 36]. This research includes increasing our understanding of epigenetic off/on gene switching in determining various aspects of smoking behaviour and pathologies associated with smoking [8, 37].
\nAn understanding of the genetics and epigenetics of PTSD is very relevant to helping smokers to quit, especially those who seem to be hardened nicotine addicts. Twin studies have shown that only 20–30% of subjects exposed to severe trauma develop PTSD [38]. Less than 50% of women who experience violent rape develop overt PTSD. Genetic studies have shown that a combination of four or more high‐risk alleles (single gene variants) confer a sevenfold increase in the risk of PTSD following trauma [38]. However, lesser degrees of PTSD associated with cigarette smoking [19] may also have a genetic component. Further research in this area seems likely to overlap with research focused on the genetics of smoking behaviour and is likely to lead to new strategies in treatment of both PTSD and in helping to achieve smoking cessation.
I had been unaware of the possibility that PTSD could be a common barrier to smoking cessation. Beckham et al. [20] showed that there is a significant difference between PTSD and non‐PTSD smokers during attempts to quit with the PTSD subjects being more likely to lapse after 1 week (Figure 4). There is also a growing body of research that shows that we may only recognize the more obvious instances such as PTSD in war veterans and rape victims. The literature is unclear on the incidence of PTSD in the general population with reports ranging from 1 to 5%. If we take 3% as a median value, it is likely that a sample of lifelong non‐smokers would exhibit a lower incidence (approx. 2%) so that the incidence of overt PTSD is 3 times higher in smokers. A paper by Matthews et al. [19] showed that 6.7% of smokers are suffering from overt PTSD but also showed that another 73% of their study group of current smokers (n = 342) had symptom scores suggestive of some degree of stress or as they termed it: “sub‐threshold PTSD”. There was no correlation between smoking and anhedonia. Only 20% of their sample was completely negative for their PTSD score. Perhaps sub‐threshold PTSD includes unreported domestic abuse and bullying at work. Domestic violence has been recorded as a cause of PTSD‐related smoking [39]. There is evidence from research by neuropsychiatry that nicotine inhibits negative symptoms experienced in PTSD and that the positive “feel good” effects of nicotine is relatively insignificant [19, 21]. Further research is needed to clarify the differences in PTSD scoring between smokers and non‐smokers and to determine what can be done to help this category of refractory smokers.
Survival curves for smoking lapse in PTSD (n = 55) versus non‐PTSD (n = 52) in first week of a quit attempt showing that PTSD is associated with a higher smoking relapse rate (from Beckham et al. [16]).
Smokers who are concerned about weight gain will need special help but sometimes counselling and dietary advice are ineffective. Hurt et al. at The Mayo Clinic, USA, are currently researching the combined pharmacological approach of varenicline and lorcaserin (a new anti‐obesity drug) for overweight smokers who want to quit. Early results are encouraging (personal communication from Hurt).
\nDo smokers who experience side effects from smoking cessation drugs tend to give up trying to quit as seemed to be the case with case 5? The literature is unclear on this issue. However, the Respiragene project certainly showed a significant difference between those that had been able to persist with their original smoking cessation prescription (varenicline or nicotine replacement therapy) and those who had stopped due to side effects (Table 2) with quit rates at 6 months of 42.6 and 15.3%, respectively (p = 0.01).
Stopped smoking at 6‐month follow‐up | Total | |||
---|---|---|---|---|
Lost to follow‐up | Yes | No | ||
Prescription history unknown | 2 | 0 | 5 | 7 |
Persisted with first prescription | 3 | 20 | 24 | 47 |
Stopped first prescription | 0 | 2 | 11 | 13 |
Total | 5 | 22 | 40 | 67 |
Smoking cessation outcome for subjects who stopped smoking cessation therapy due to side effects compared with subjects who had persisted with smoking cessation therapy.
χ2 = 6.6, p = 0.01.
Studies linking work stress to smoking are equally balanced between those that do and do not show a link. One of the best studies, however, from Finland shows an odds ratio of 1.28 (p < 0.01) for smoking where there is a high imbalance between effort and reward consistent with work stress [40]. Concerns about passive smoking in the family home have received a good deal of publicity recently despite attempts by the tobacco industry to play down the risks [41]. Finding that 12% of our participants mentioned this as a key smoking cessation motivator was not, therefore, unexpected. Just as family cohesion is a factor here, conversely lack of family cohesion is emerging as a significant de‐motivator [31, 32].
\nFatalism and peer pressure are well known as factors that encourage smoking but the precise role of adult peer pressure in the workplace needs further research. A review of smoking cessation in the workplace has outlined strategies for influencing the workplace ethos to improve attitudes and introducing workplace smoking cessation programmes and smoking cessation inducements [42].
\nThe efficacy of a risk score such as the Respiragene risk score as a smoking cessation motivator could, perhaps, be improved if it included cardiovascular risk as well as lung cancer risk. A recent paper estimated that smokers double the risk of an early death from cardiovascular events but that risk reverts to normal after 2 years of smoking cessation [43]. Personalized data on cardiovascular risk could be included in the risk score in future. This might include genetic risk factors such as the apolipoprotein E4 gene but clinical factors such as family history, blood pressure, body mass index, lipid profile, and HbA1C would be equally important.
Fear of lung cancer can certainly act as a powerful motivator as demonstrated by the high quit rate for subjects with a very high Respiragene risk score. However, the problem with a personalized risk score is that if the risk is relatively low, it may act as a de‐motivator. Including a personalized risk score for life‐threatening cardiovascular events (stroke and myocardial infarction) might help to counter this problem, especially as most smokers will be given a risk score round about the mean of 100% increase in risk of a fatal event. However, even the most persuasive smoking cessation motivator is unlikely to overcome powerful de‐motivators such as PTSD or weight control issues in about 20% of smokers. If a smoker in this category who may have attempted to quit 2 or 3 times already is still determined to quit, the de‐motivator that is standing in the way of success must be addressed and this may need intense one to one counselling and/or a pharmacological intervention. New and better pharmacological approaches are likely to result from genetic studies on smoking behaviour.
I am indebted to my colleagues Paul Grob, Wendy Kite, Peter Williams, and Simon de Lusignan at the University of Surrey who helped me in the planning and implementation of the Respiragene project and were my co‐authors for the main paper reporting the results of this research [4]. I could not have completed this research without the help of: A Telaranta‐Keerie and the staff of Lab 21, Cambridge, who processed and analyzed the buccal swabs for genetic testing; A Roscoe and the staff of the Integrated Care Partnership, Epsom, for help with recruitment and premises; and Surrey Smoking Cessation Practitioners J Golding and H Phillips for their expertise. We are grateful for grants from Lab 21 and Synergenz Bioscience Ltd without which this research could not have been completed.
A common definition for predatory behavior describes it as the process through which one animal, the predator, captures and kills another animal, the prey, before eating it in part or entirely [1]; however, according to the opinion of a number of microbiologists and protistologists, this definition should be also extended to different organisms included in other life Kingdoms, with particular regard to microorganisms. Indeed, especially in the last 30 years, a lot of studies have been devoted to describing the predator-prey interactions among unicellular eukaryotic organisms like protists. Whittaker [2] originally defined protists as those “organisms which are unicellular or unicellular-colonial and which form no tissues,” and for this reason they must carry out at the cellular level all the basic functions which can be observed in multicellular eukaryotes. Among these functions, self-nonself recognition mechanisms are represented by a large repertoire in protists and can trigger either autocrine or paracrine processes in some ciliates (see [3] for a review), together with the capability to detect prey (food) or predators in others. In this regard, it is known that protists have developed a variety of strategies of feeding behaviors especially in response to different environmental factors, together with a diverse kind of food available in micro-habitats. Figure 1 shows a general scheme of predator-prey interactions, where the predator recognizes the presence of the prey (step 1) and can attack it (step 2). On the other hand the prey recognizes the presence of the predator (step 1′) and it can organize its defense mechanisms (step 2′) [4]. This scheme should be considered functional for both animals and protists, and indeed several studies have shown that the food recognition and the offense-defense mechanisms adopted by some groups of protists can be compared, in terms of complexity and variability, with those observed in animals.
General scheme of predator-prey interactions. Redrafted from [4].
In this context, a common feeding mechanism found in heterotrophic protists is phagocytosis, a process which requires specific organelles for food assimilation and which occurs in three steps: food capture, phagosome formation, and food digestion [5]. Different techniques of phagocytosis have been described in various protists, where they have especially been investigated in ciliates [5, 6, 7]. Verni and Gualtieri [5] describe three main phagocytotic processes in ciliates: filter feeding, suctorial feeding, and raptorial feeding. The authors compare them to the strategies used in fishing, like netting, trapping, and harpooning. In filter-feeding ciliates, the food, represented by small organisms or edible debris of various types, was pushed into the ciliate buccal cavity by the rhythmical beats of the cilia located in its adoral apparatus. Suctorial-feeding ciliates are represented by sessile or sedentary species that for most of their lives remain attached to other organisms or various substrates, intercepting the food particles with their specialized tentacles. Finally, raptorial ciliates are able to directly catch other organisms using peculiar organelles to paralyze and/or kill their prey, generally called extrusomes.
The term “extrusome” was proposed, for the first time, by Grell in 1973 for extrusive (ejectable) bodies, which occur widely in protists [8]. They are membrane-bound organelles usually located in the cell cortex, attached to the cell membrane. They can display differences in structure and morphology, but they share the common characteristic of discharging their contents to the outside of the cell in response to mechanical or chemical stimuli. Remarkably, when the extrusomes are discharged, the cell remains intact and functional. Studies on extrusomes and related organelles have been reviewed by Hausmann [9], Dragesco [10], Kugrens et al. [11], Hausmann and Hülsmann [12], and Rosati and Modeo [13]. Typical examples of these organelles include toxicysts, trichocysts, mucocysts, cortical, or pigment granules in ciliates and flagellates, haptocysts in suctorians, and kinetocysts in heliozoan actinopods. Some extrusomes are known to be related in predator-prey interactions, for example, to catch and kill the prey (such as toxicysts, haptocysts, kinetocysts, and some cortical granules), or used as defensive organelles (such as the trichocysts and various cortical or pigment granules), but the role of other kinds of extrusomes such as the mucocysts in Tetrahymena or the trichites in Strombidiidae [13] still remains obscure.
Offensive extrusomes generally possessed by raptorial protists and located usually at or near the feeding apparatus are discharged after contact with a possible prey, which is immobilized, damaged, or firmly bound to the predator. Among these, organelles, certainly the most widely studied, belong to the category of toxicysts (toxic extrusomes) and they play an essential role in capturing and killing prey [7, 13]. Toxicysts are synthesized in Golgi or ER vesicles and are usually localized in the cell cortex attached to the cell membrane. Most of them are observed in species belonging to the class Litostomatea and subclass Haptoria, but they are also present in other predatory ciliates. They are usually positioned in a specific region of the cell, near the oral apparatus, and generally in the first portion which contacts the prey during the raptorial feeding [13]. Independently of the specific differences in the morphology of the cytostome, the toxicysts are always present in an appreciable number, for example, in the genera Didinium, Dileptus, Prorodon, Litonotus, Colpes, Homalozoon, and many others. In resting position, the toxicysts appear generally as rod-like elements (Figure 2), and could be discharged in milliseconds, if exposed to an appropriate stimulus such as contact with a prey (Figure 3) [7]. In this case, the tubules of the toxicysts are suddenly introduced into the cytoplasm of the prey’s body, like hypodermic needles, to inject the toxic material. Hausmann [7] reports essentially two ways by which the toxicysts may be discharged: in the first case, there is a fusion of the toxicyst’s membrane with the plasma membrane, followed by the tubule discharge via evagination; in the second, observed in certain ciliate species, a telescopic discharge of the tubules was observed. During or near the end of the toxicysts’ discharge, the toxic secondary metabolites were secreted by the tubules. It is worth noting that this mechanism of discharging toxic substances shows the structural and functional similarities that can be found between the toxicysts in ciliated protists and nematocysts in Cnidaria, despite the substantial differences in size [7].
Transmission electron microscope (TEM) picture of the toxicysts in a dividing cell of a ciliate Didinium nasutum. Scale bar = 1 μm. Original picture by R. Allen from http://www.cellimagelibrary.org/images/10010.
Predatory behavior of Coleps hirtus on Pseudokeronopsis erythrina. The predator attacks the prey with its toxicysts (arrow). Micrograph extracted from a film clips. Scale bar = 200 μm.
In contrast with recent and less recent studies about the nature of the toxic secondary metabolites used by ciliates in chemical defense, no exhaustive data are yet available about the composition of the toxins stored in the toxicysts of predatory ciliates. This is essentially due to the difficulty in separating the content of extrusomes from other molecules produced by the ciliate, in order to purify them at homogeneity for subsequent chemical and structural analyses.
To date the presence of acid phosphatase has been demonstrated in the toxicysts of Didinium nasutum [14] and four other raptorial ciliates such as Enchelys mutans, Lacrymaria olor, Homalozoon vermiculare, and Pseudoprorodon niveus [15]. It has been supposed that this enzyme, generally present in lysosomes of animal cells, may probably be used by these ciliates to start the digestion of the prey.
The complete analysis of the content of the toxicysts, together with observations of the predatory behavior, was also performed on another species, Coleps hirtus, a freshwater protostomatid ciliate. C. hirtus (40–65 × 20–35 μm) has an oral apparatus placed at the anterior end of the cell and its barrel-shaped body is covered by calcified armor arranged in plates. This ciliate is able to feed off bacteria, algae, flagellates, and ciliates, but it is also histophagous, that is, it feeds on living plant and animal tissue such as rotifers, crustaceans, and fish [16, 17]. Coleps is also reported to show a scavenger feeding on tissues of dead metazoans, such as Daphnia, Diaphanosoma, and chironomid larvae [18], as well as toward dead ciliates and dead specimens of its own species. Coleps is usually equipped with toxicysts used by the ciliate to assist its carnivorous feeding, and its predatory behavior has recently been analyzed against another ciliate species used as prey, Euplotes aediculatus. Observations conducted on a mixture of predators and prey showed several contacts between the specimens of Colpes and Euplotes, but only after 5–10 min did interactions between the anterior section of a predator with a specimen of Euplotes become effective. This time was probably essential for prey detection and recognition, followed by prolonged contact between predator and prey, generally ending with the rapid backward swimming of the latter which separated the two organisms. When the attacks became numerous some individuals of Coleps remained attached to their prey (Figure 4), which decreased their swimming speed and gradually stopped swimming. After 20–30 min, the prey was fragmented and eaten by several specimens of Coleps, and a similar predatory behavior was also observed using different ciliate species as prey [19]. On the contrary the toxicysts-deficient specimens of Colpes (Figure 5) obtained by means of the application of the cold-shock method capable of inducing an exclusive massive discharge of extrusomes in ciliates [20] appear unable to catch and kill their prey [19].
Multiple attacks by different cells Coleps hirtus on a cell of Euplotes aediculatus. Micrograph extracted from a film clips. Scale bar = 200 μm.
(A) The toxicysts in Coleps hirtus appear as rod-shaped organelles (arrow) in the oral basket of a cell. (B) The photomicrograph shows the toxicysts discharged (arrow) into the medium, immediately after a cold-shock treatment. (C) No toxicysts are detected in a toxicyst-deprived cell. Photomicrographs of fixed specimens by protargol stain. Scale bar = 10 μm. Pictures from [19].
Unexpectedly, the analysis of the bioactive fraction of the toxicyst discharge of Coleps hirtus (performed by liquid chromatography-electro-spray-mass spectrometry and gas chromatography-mass spectrometry) showed the presence of a mixture of 19 saturated, monounsaturated and polyunsaturated free fatty acids (FFAs) with the addition of a minor amount of a diterpenoid (phytanic acid) but did not reveal the presence of enzymes, as reported for other predatory ciliates [19]. To date this is the only report on the presence of FFAs as toxic substances in the extrusomes of ciliated protists, but the use of this class of compounds as toxins by Coleps is shared with at least 15 freshwater, 13 marine, and 6 brackish water potentially harmful microalgae, as well with some multicellular organisms. For example, a chemical defense by a mixture of FFAs was studied and demonstrated for the harmful microalga Fibrocapsa japonica (Raphidophyceae) [21, 22, 23], and also in animals, a defensive strategy mediated by FFAs was recently described for the fish Barbus barbus which adopted it to protect its eggs from predators [24].
Very little is known about the role and source of phytanic acid in ciliates, this being the additional component detected in the toxicyst discharge of Coleps. Phytanic acid can be produced from the biodegradation of the side chain of chlorophyll [25], so one possible source arises from Coleps’ carnivorous feeding on photosynthetic microorganisms [19]. Some insects, such as the sumac flea beetle, accumulate chlorophyll-derived metabolites as a chemical deterrent in excrements [26]. Komen et al. [27] demonstrated the toxic effect of phytanic acid on human skin fibroblasts, where it impaired mitochondrial respiration through protonophoric action. Regarding the role of phytanic acid in Coleps, it is possible to hypothesize that it can be used as a weapon, deterrent, or, at least, it could be stored in toxicysts given its potential toxic activity. In addition, it is known that ciliates themselves are also able to synthesize a huge number of terpenoids [28, 29]. This is the case of Euplotes focardi [30] and Euplotes rariseta [31] where the production of new diterpenoids was demonstrated. Terpene compounds and FFAs may also act together to exert cytotoxic effects [19]. FFAs may serve as a matrix to deliver toxic compounds to prey or predators and also to create a perfect environment where toxic metabolites can exert their functions.
It has been demonstrated that the substances discharged from the toxicysts by Coleps are highly toxic for a number of ciliate species such as Euplotes aediculatus, Paramecium tetraurelia, Spirostomum teres, and S. ambiguum or Oxytricha sp. [19], and their action mechanism appears to be related to a necrotic process. The term necrosis refers to a rapid (unprogrammed) cell death, with plasmatic membrane rupture, often caused by external factors such as toxins. On the contrary, the apoptosis is programmed cell death characterized by nuclear condensation, cytoplasmic shrinkage, and disintegration of the cell into small, membrane-bounded fragments. As shown in Figures 6 and 7, the purified toxin from Coleps is able to induce rapid cell death in E. aediculatus and in S. ambiguum preceded by cell membrane fracture without any changes in the morphology of the macronucleus. An action mechanism of this type seems to be a “good choice” for Coleps as it induces paralysis and a very rapid death in the prey.
(A) Necrotic effects of the toxicyst discharge of Coleps hirtus on Euplotes aediculatus and (B) Spirostomum ambiguum. Arrows indicate the cell-membrane fractures. Scale bar = 100 μm. Pictures from [19].
(A, B) Effects of the toxicyst discharge of Coleps hirtus on the macronuclear morphology in specimens of Euplotes aediculatus and (C, D) Spirostomum ambiguum. Cells were stained with acridine orange and ethidium bromide and observed by fluorescent microscopy. Viable cells show intact, bright green nuclei, nonviable cells show red/orange nuclei. M = macronucleus, m = micronucleus. Scale bar = 100 μm. Pictures from [19].
Interestingly, the cells of Coleps can also be damaged if exposed, in vitro, to their own toxin discharge [19]. Nevertheless, this cannot occur in nature, because on the one hand, the toxins are stored in the toxicysts of the ciliate, thus avoiding autotoxicity and on the other hand, the accidental exposure of Coleps to the toxicyst discharge dissolved in the medium is also unlikely, due to the choice of the predator to directly inject the toxins into the prey [19]. In this context, it is worth remembering the peculiar predatory behavior of Coleps, which usually leads to the observation that the same prey undergoes multiple attacks by several raptorial specimens, a behavior also adopted against young larvae of zebrafish [17]. It is likely that this behavior has evolved to ensure a fast immobilization of the prey, that after simultaneous multiple attacks, it can easily accumulate lethal concentrations of toxins injected by numerous predators. Therefore, essentially for the “wolf-like” group hunting behavior of Coleps, the species that appeared relatively resistant to its toxicyst discharge may also be easily caught and killed.
Differently to Coleps, other ciliate species have specialized in hunting and catching a few preferential prey. This is, for example, the case of Didinium nasutum that is capable of capturing and killing several species of Paramecium and few other ciliates. Generally, Paramecium species are able to defend themselves by means of mechanical extrusomes like trichocysts (that will be discussed later on this chapter) but Didinium seems to overcome the defense of Paramecium by means of a highly specialized combination of extrusomes. Present on the proboscis of Didinium are several units of two different kinds of extrusomes: toxicysts, as in other Litostomatea, and pexicysts, another specialized offensive extrusome observed only in this species [32]. These authors describe the discharge of pexicysts as the first response after the prey recognition [14], which is typically followed by the discharge of toxicysts. At the same time, the prey (generally a Paramecium) discharges its trichocysts which separate the two organisms, but the proboscis of Didinium remains attached to the prey by a tiny connection probably composed of a bundle of discharged pexicysts and toxicysts (Figure 8). Subsequently, the Paramecium will be reached again and captured by the predator. In the light of this observation, the pexicysts seem to act most by a mechanical function (as harpoon-like organelles) rather than with a chemical offense. This assumption is supported by the fact that another species of predatory ciliate, Monodinium balbiani, which is morphologically similar and phylogenetically close to Didinium, but without the presence of the pexicysts on its proboscis, unlike the Didinium, is sensitive to the defense mechanism possessed by Paramecium, which is often able to avoid capture [33].
Scanning electron microscope (SEM) picture on the predator-prey interaction between a cell of Didinium nasutum and a cell of Paramecium multimicronucleatum. The bundle of toxicysts and pexicysts can be seen between the two organisms (arrow). Magnification ×50. Original picture by G. Antipa from http://www.cellimagelibrary.org/images/21991.
In this context it is also relevant to mention the subclass Suctoria, represented by ciliates which become sessile during development and consequently lose the ciliary structure. Suctorians are able to feed on other protists and frequently on other ciliates by means of specialized tentacles. The distal ends of these tentacles are often equipped by peculiar extrusomes called haptocysts that are involved in prey capture. When a tentacle touches a possible prey, the discharge of haptocysts is able to penetrate the prey’s membrane, forming a connection between the predator and the prey and injecting the extrusome content into the latter, which also concurs to the fusion of the membranes belonging to the two organisms [13, 34]. However, the fusion of the two membranes is not always immediate, for example, in Heliophrya erhardi, Spoon et al. [35] observed that many specimens of Paramecium contacting the tentacles of the suctorian escaped discharging trichocysts at the point of contact, suggesting that Paramecium is able to defend itself from the puncture of the haptocysts.
In addition to predatory behavior, ciliated protists have also evolved different defense strategies, many based on the discharge of extrusomes. Two different mechanisms involved in their defense behavior are essentially observed: the first is mediated by the mechanical actions of trichocysts as in Paramecium or Frontonia and the second is mediated by the toxic secondary metabolites of different kinds of chemical extrusomes.
Spindle trichocysts (or simply, trichocysts) are spindle-shaped organelles which discharge their content in the form of a thread. They are found in some ciliates and flagellates and are sometimes furnished with a specially constructed tip [9]. The best known and studied trichocysts are those in the genus Paramecium. Trichocysts in Paramecium are 3–4 μm long, carrot-shaped membrane-bounded organelles armed with a sharply pointed tip, and are present in thousands all over the cell surface, except at the oral apparatus (Figures 9 and 10). When paramecia are subjected to various stimuli, the membranes of the trichocysts and the cell membrane blend together, and the content of the extrusomes is immediately discharged to the outside of the cell, forming a spear-like structure in milliseconds (Figure 11) (see [13] for a review). Trichocyst discharge has therefore been extensively studied as a model system of exocytosis [36] (see [37] for a review). Synthesis, processing, and sorting of component proteins in trichocysts are also studied as model systems of protein biosynthesis [36] for a review.
Scheme of the ciliary structure and the trichocysts of Paramecium. Picture from http://biodidac.bio.uottawa.ca, redrafted by R. D’Arcangelo.
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. Scale bar = 1 μm. Picture from [9].
The trichocysts discharged by a cell of Paramecium tetraurelia exposed to picric acid solution. Scale bar = 100 μm.
Maupas, one of the pioneers of protozoology, first proposed the defensive function of trichocysts in Paramecium in 1883, 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. tetraurelia [39]. 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. nuceofilum [33, 40, 41, 42, 43]. In addition, a more recent paper also analyzed the defensive function of trichocysts in P. tetraurelia against some microinvertebrate predators, such as a rotifer (Cephalodella sp.), an ostracod (Eucypris sp.), and a turbellarian flatworm (Stenostomum sphagnetorum) [44]. 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. personal communication); indeed after contact with this toxic solution, Paramecium cells violently reacted by immediately discharging most of their trichocysts before being killed. 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.
To summarize, the mechanical defense by trichocysts and related extrusomes appear to be multiple, including quick physical displacement, the thrust into a predator, and protection against the predator’s toxins, increasing the chance for the prey to survive and escape. 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. coeruleus, and a few red species of Blepharisma. B. japonicum (Figure 12) is the best studied species of the genus Blepharisma and it presents pigment granules usually in a size of 0.3–0.6 μm diameter, arranged in stripes between the rows of cilia that confer a red-pink coloration to the ciliate (Figure 13). 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. John’s Wort), and stentorin, produced by the ciliate S. coeruleus [47, 48] (Figure 14). 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. japonicum shows a temporal backward swimming or rotating movement (step-up photophobic response) if exposed to a sudden increase in light intensity. 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 1949 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. japonicum, albino mutant cells, and light-bleached cells (a phenocopy of the albino mutant) as prey for the raptorial ciliate Dileptus margaritifer and evaluating the toxicity of purified blepharismins on various ciliate species [49, 50]. As a response to the attack by D. margaritifer versus one cell of B. japonicum, the latter releases the toxic blepharismins, visible as spherical bodies of 0.2–0.6 μm in diameter under scanning electron microscopy (Figure 15). 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. margaritifer [49, 50]. Recently the defensive function of blepharismins was also investigated in two additional species of Blepharisma, B. stoltei, and B. undulans against two predatory protists (C. hirtus and Stentor roeseli) and one metazoan, the turbellarian S. sphagnetorum [56]. The results indicate that the chemical defense mechanism present in B. stoltei and B. undulans is mediated by the same five blepharismins present in B. japonicum, although produced in different proportions [56]. 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. [48]. Summarizing, the Blepharisma species studied are able to defend themselves against C. hirtus, although S. sphagnetorum and S. roeseli seem to overcome Blepharisma’s chemical defense, but it was observed that after the ingestion of intact cells of the toxic ciliates these predators are not able to reproduce, suggesting the presence of the post-ingestion toxicity phenomena [56]. Additional toxic pigments, structurally related to hypericin, were found in other heterotrich ciliate species, such as stentorin in S. coeruleus (see [57] for a review), amethystin in S. amethystinus [58], and maristentorin in the marine ciliate Maristentor dinoferus [59], but the defensive function was experimentally proved only for S. coeruleus [60].
External morphology of a living cell of Blepharisma japonicum. Scale bar = 100 μm.
Extrusive pigment granules in Blepharisma japonicum (arrow) visible as red/pink dots under a vacuole. Scale bar = 100 μm.
Main secondary metabolites produced by ciliated protists. Erythrolactones: A1 (R1 = SO3−; R2 = C6H13 (n-hexyl)); B1 (R1 = SO3−; R2 = C7H15 (n-heptyl)); C1 (R1 = SO3−; R2 = C8H17 (n-octyl)); A2 (R1 = H; R2 = C6H13 (n-hexyl); B2 (R1 = H; R2 = C7H15 (n-heptyl)); C2 (R1 = H; R2 = C8H17 (n-octyl)).
SEM micrographs of the predator-prey interaction between a cell of Dileptus margaritifer (DI) and a cell of Blepharisma japonicum (BL). (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. Scale bar = 50 μm. (B) Enlargement of the region near the rupture in A. Scale bar = 5 μm. (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. Scale bar = 5 μm. (D) Enlargement of a part of C. Scale bar = 0.5 μm. 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. striatus are extrusive organelles which contain a toxic photodynamic pigment used for chemical defense against predators [62]. Loxodes are able to discharge the toxic pigment as response to attacks of the ciliate D. margaritifer (Figure 16) or of the turbellarian S. sphagnetorum repelling predators. Intriguingly Finlay and Fenchel already proposed a defensive function for the pigment granules in Loxodes (L. striatus and L. magnus) based on different evidences; specifically, they found that light induces in Loxodes a characteristic behavior to escape from toxic water and that the pigment granules are the photoreceptors for this reaction [61]. 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. Micrograph extracted from a film clips. Magnification ×70. 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. carnea [64] and in P. erythrina [65], these granules are reported as extrusive organelles. New secondary metabolites, keronopsins and keronopsamides, respectively, produced by P. rubra and P. riccii, were recently isolated together with their sulfate esters (Figure 14) [66, 67]. In the case of P. rubra, it was demonstrated that a crude extract of this organism containing keronopsins, A1 and A2, and their sulfate esters B1 and B2, is capable of paralyzing or even killing ciliates and flagellates [66]. 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. riccii, the function of the alkaloid secondary metabolite keronopsamide A and its sulfate esters B and C has not been investigated, and the possible localization of the pigments in the cortical granules is only presumed [67]. The most extensively studied species is P. erythrina; previously described as an estuarine one, it was successively found also in the freshwater environment and hence reported as a euryhaline organism [68]. This ciliate shows an elongated body (Figure 17) equipped with spherical, dark-reddish, brown, or brick red colored pigment granules of about 1 μm in diameter that are mainly arranged around ciliary organelles [69]. As the content of pigment granules, three new secondary metabolites have recently been characterized and named erythrolactones A2, B2, and C2. These are characterized by a central 4-hydroxy-unsaturated δ lactone ring bearing an alkyl saturated chain at carbon-2 and a butyl-benzenoid group at carbon-5 [65, 68]. These molecules were detected in the crude extract of whole cells together with their respective sulfate esters, erythrolactones A1, B1, and C1 (Figure 14). After the application of the cold-shock method on massive cell cultures of P. erythrina to induce the exclusive discharge of pigment granules, it was demonstrated that only non-sulfonated molecules A2, B2, and C2 were contained in the extrusomes of the ciliate [65]. The mixture of these three molecules has been proven to repel some predators, such as the ciliate C. hirtus, and to be toxic for a panel of ciliates and microinvertebrates [65]. Erythrolactones A2, B2, and C2 are the only toxins present in the extrusome discharge of P. erythrina, whereas their respective sulfate esters A1, B1, and C1 remain confined inside the cell environment [68]. 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. erythrina cell may be associated with their temporary inactivation, in order to prevent the phenomenon of self-toxicity that could occur before their definitive storing, as non-sulfonated and active compounds, in the cortical pigment granules.
External morphology of a living cell of Pseudokeronopsis erythrina. Scale bar = 100 μm.
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. virens are to date the most studied. 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. virens occurs when a predator, such as the ciliate D. margaritifer, contacts a C. virens cell with its toxicysts bearing proboscis (Figure 18A). D. margaritifer swims backward while dense material is visible under dark field microscopy, emerging from the site where the proboscis touched the C. virens (Figure 18B) which swims away [75]. Sometimes, together with the discharged material from C. virens, it is also possible to detect some hazy material consisting of needle-like structures which appear to be discharged toxicysts of D. margaritifer (Figure 19), suggesting a possible further protection against the toxic extrusomes of predators [75]. Interestingly, the chemical defense adopted by C. virens against D. margaritifer is also effective against some other protists and metazoans [44, 76].
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. Magnification ×70. Pictures from [75].
Hazy cloud consisting of needle-like structures discharged from the toxicysts of Dileptus margaritifer. Magnification ×720. Pictures from [75].
If the defensive function of cortical granules in C. virens is widely demonstrated, some evidences indicate that these extrusomes could be also successfully used for chemical offense. Differently from the Paramecium species which do not have trichocysts (exclusively for defense) localized in the oral apparatus, C. virens presents a wide number of cortical granules in the buccal cortex suggesting an additional offensive function for these extrusomes [71]. C. virens is able to catch and ingest prey of different sizes, from small flagellates such as Chlorogonium elongatum to large ciliates, such as B. japonicum or Spirostomum ambiguum [43, 77]. These prey are sucked up into the buccal cavity of C. virens, which is formed of a peristomial field and a buccal tube, and then ingested in a food vacuole, which arises at the end of the tube [43]. A cell of P. tetraurelia which is entirely taken into the buccal tube of C. virens is able to discharge the trichocysts and escape from the predator [43], different to what happens when an individual of the same species is totally caught in the pharynx of the microturbellarian S. sphagnetorum [44]. Perhaps, as in the case of contact with the toxicysts of the raptorial ciliate D. margaritifer, the trichocysts were discharged after contact with climacostol released from C. virens to kill the prey. A similar phenomenon also occurs with different preys which possess chemical extrusomes for defense such as the ciliate S. ambiguum. In this case, after a cell-cell contact, the S. ambiguum displays rapid cell contraction, and according to the authors, it is likely that this contraction is induced by the discharge of extrusomes by C. virens [77]. If this is the case, it is likely that the cortical granules of C. virens could be equally used as multifunctional extrusomes, both for chemical defense and offense.
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. pneumoniae and against a fungal pathogen, Candida albicans [79]. 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. teres. S. ambiguum (Figure 20) is a colorless freshwater species and one of the largest and elongated existing ciliates (800–2000 × 48–60 μm). 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]. S. ambiguum has numerous cortical granules which, under a phase contrast microscope, appear as dots placed in the region between ciliary lines that could be observed in a large transparent contractile vacuole placed at the posterior end of the cell (Figure 21A) [77]. The cold-shock method was applied to S. ambiguum to obtain the cortical granule-deficient cells, which showed a markedly reduced number of extrusomes (Figure 21B). Both untreated and cortical granule-deficient cells were exposed to the attack of C. virens, and when the buccal apparatus of the predator makes contact with an untreated cell of S. ambiguum, it showed a rapid contraction while the predator swam backwards (Figure 22A). Similarly to untreated cells, cortical granule-deficient cells of S. ambiguum also showed rapid contraction after attack by C. virens, but they were successfully captured and sucked up by the predator into its buccal cavity (Figure 22B) [77]. 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-methylbut-2-enyl)benzene-1,4-diol(mono-prenyl hydroquinone) (Figure 14). 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. teres, possesses a different colorless toxin used for defense, characterized as spiro[(2,5-dimethyl-5,6,7,8-tetrahydronaphthalene-1,4-dione)-8,6′-(pyrane2’,5′-dione)] and named spirostomin (Figure 14) [84]. 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. japonicum, B. stoltei and B. undulans share the same mixture of blepharismins even if produced in different proportions [56].
External morphology of living cells of Spirostomum ambiguum. Scale bar = 200 μm.
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. Magnification ×900. Pictures from [77].
Predator-prey interaction between Climacostomum virens and Spirostomum ambiguum. (A) 1: Cell of C. virens contacts a cell of S. ambiguum with its buccal apparatus. 2: S. ambiguum shows rapid contraction while the predator swims backwards. 3: The same cells as in 2, a second later, showing a retreated C. virens, while S. ambiguum swims away. (B) Predator-prey interaction between C. virens and extrusome-deficient cells of S. ambiguum obtained by cold-shock treatment. 1: C. virens cell contacts a S. ambiguum cell which instantly shows contraction. 2: C. virens engulfs the contracted S. ambiguum cell and continues to eat the S. ambiguum cell (3). Micrographs extracted from a film clip. Magnification ×50. Pictures from [77].
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. aurelia with that mediated by cortical granules in C. virens and S. ambiguum [44]. 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. In contrast, the chemical defense adopted by a toxic ciliate against metazoan predators can also be activated after the ingestion of the prey by the predator, but its effectiveness appears to be strictly linked to the cytotoxic potency of the compound stored in the protozoan cortical granules. It would also be interesting to compare these two mechanisms against unicellular predators.
In a general perspective, it is clear that the researches on predatory behavior and on the related defensive mechanisms in protists not only represent progress in knowledge about the ecological role played in nature by predator-prey interactions in aquatic microhabitats but will also provide new research opportunities for evolutionary biology and may also represent a relevant source of new natural products.
We are grateful to Dr. Gill Philip (University of Macerata) for the linguistic revision of the chapter. Financial support was provided by University of Macerata, Italy.
The authors have declared no conflict of interest.
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