When starting a new collaborative endeavor, it pays to establish upfront how strongly your partner commits to the common goal and what compensation can be expected in case the collaboration is violated. Diverse examples in biological and social contexts have demonstrated the pervasiveness of making prior agreements on posterior compensations, suggesting that this behavior could have been shaped by natural selection. Here, we analyze the evolutionary relevance of such a commitment strategy and relate it to the costly punishment strategy, where no prior agreements are made. We show that when the cost of arranging a commitment deal lies within certain limits, substantial levels of cooperation can be achieved. Moreover, these levels are higher than that achieved by simple costly punishment, especially when one insists on sharing the arrangement cost. Not only do we show that good agreements make good friends, agreements based on shared costs result in even better outcomes. Conventional wisdom suggests that cooperative interactions have a bigger chance of surviving when all participants are aware of the expectations and the possible consequences of their actions. All parties then clearly know to what they commit and can refuse such a commitment whenever the offer is made. A classical example of such an agreement is marriage13δ). Finally, when the co-player does not accept the deal, the game is not played and hence both obtain no payoff.Here we investigate analytically and numerically whether costly commitment strategies, in which players propose, initiate and honor a deal, are viable strategies for the evolution of cooperative behavior, using the symmetric, pairwise, and non-repeated Prisoner's Dilemma (PD) game to model a social dilemma. Next to the traditional cooperate (C) and defect (D) options, a player can propose its co-player to commit to cooperation before playing the PD game, willing to pay a personal cost imposes the commitment to the other players, i.e. defectors will always be punished even when they did not want to play with punishers. In addition, there is no notion of compensation incorporated in the model that remunerates the proposer when her accepted deal is violated, in contradistinction to our own model. Moreover, because the creation of the agreement occurs explicitly in the current work, players can behave conditionally , and that is not considered in the ongoing evolutionary models of peer and pool punishment. However, it is quite plausible to cooperate only when someone asks to commit and to otherwise defect in order to avoid exploitation, even in a one-shot interaction.Although there is a kind of punishment associated with the agreement, the notion of compensating a partner when not honoring a negotiated deal is not entirely equivalent to the general notion of punishment as has been studied in Evolutionary Game Theory67891011129129Nevertheless, in both punishment and of commitment strategies, a cost δ) on the punished161727One important conclusion derived from recent experiments is that CP may maintain cooperation only if the behavior is cost effective, meaning a low cost In summary, the main questions we ask here are whether proposing such commitments and honoring them is a viable strategy for the evolution of cooperative behavior and how such a commitment deal should be arranged in order to reach high levels of cooperation. That is, given the game at hand and the potential cost of arranging commitment, is it worth arranging it at all? And if yes, what kind of compensation should be associated with the commitment proposal? For commitment proposers to prevail, they need to justify the cost of arranging the commitment in order to win against the free-riders and the traditional defectors, as well as exhibiting a strong enough penalty for the fake committers. Additionally, we examine how this model of prior mutual commitment differs from CP.The fake committers (FAKE), who accept a commitment proposal yet defect when playing the game, assuming that they can exploit the COMP players without suffering a severe consequence.The commitment free-riders (FREE), who defect unless being proposed a commitment, which they then accept and cooperate afterwards in the PD game. In other words, these players are willing to cooperate when a commitment is proposed but are not prepared to pay the cost of setting it up.We consider here, next to the traditional pure cooperator (C) and defector (D) strategies, a commitment proposing (COMP) strategy, which proposes a commitment to others, is willing to pay the cost Every individual in the population will adhere to one of these five strategies whilst playing the PD game, where the game is traditionally defined by the following parametrized payoff matrix: R  for mutual cooperation . Unilateral cooperation provides the sucker's payoff S for the cooperative player and the temptation to defect T for the defecting one. The payoff matrix corresponds to the preferences associated with the Prisoner's Dilemma when the parameters satisfy the ordering, T > R > P > S30Once the interaction is established and both players have decided to play C or D (with or without commitment arrangements), both players receive the same reward Since pure cooperators (C) are always better off to engage in a commitment than otherwise— as cooperation is their default choice and a positive compensation is guaranteed when being exploited—they will always accept to commit when being asked to. On the other hand, pure defectors (D) will never accept commitment proposals since they would otherwise have to compensate the proposer, and as a consequence would be equivalent to the FAKE players. When two COMP players interact only one of them will need to pay the cost of setting up the commitment. Yet, as either one of them can take this action they pay this cost only half of the time. Hence, the payoff awarded to each COMP player is, on average, Together these five strategies define the following payoff matrix, capturing the average payoffs that each strategy will receive upon interaction with one of the other four strategies Note that given the current setting there are other possible strategies, such as those that propose commitment but do not cooperate or do not accept commitment proposed by others. These strategies are currently omitted for the sake of exposition, also for they get eliminated anyway since they are dominated by at least one of the strategies in the current model see .δ). One can determine this viability analytically by deducing under which conditions COMP is risk-dominant with respect to D, FAKE and FREE players (see Methods). Using the δ can be obtained concerning the risk-dominance of COMP against any of the three strategies D, FAKE and FREE: T = b, R = b − c, P = 0, S = −c and b > c, with b and c representing the benefit and cost of cooperation respectively. As a consequence, the previous conditions are simplified to: δ meet the conditions listed in Whether COMP is a viable strategy depends on the commitment parameters, i.e. the cost of arranging the deal These observations become even clearer when considering the fixation probabilities and stationary distributions (see Methods) of the different strategies, as visualized in see also , regardlb − c), will determine which arrangement cost, As game see .δ. Yet, more interestingly, this high frequency is not affected by changes in the compensation δ, once a certain threshold is reached. Accordingly, the arrangement cost is the essential parameter for the emergence and survival of mutual cooperation in the current extension of the PD game. This effect is absent in case of costly punishment (CP), as can be seen in the δ increases with the cost of punishment (see δ ≈ 14) is required for an equivalently small cost (δ), CP is more effective than COMP because, in the latter, commitment free-riders (FREE) become dominant  instead of 2(b − c)/3) is allowed for commitment proposers to still be risk-dominant. On the other hand, a smaller compensation is required to be successful against individuals playing the FAKE and FAKS strategies , or opt for not playing (FREE and FAKE), hence resolving the commitment free-riding issue.As was done for COMP individuals previously (see Constraints on the viability of commitment proposers), the conditions for which COMS strategists are risk-dominant against all defectors and free-riders  can be determined: In the literature of costly punishment, pool punishment is an effective solution towards the problem of second-order free riding, where cooperation can be subverted if those who contribute to the joint effort but do not punish defectors, are not punished themselves12generosity is traded for stability', assuming COMP is deemed more generous than COMS as it voluntarily pays the whole arrangement cost, expecting to attract more (non-fake) committers. We envisage that COMP may do better if reputation effects are considered, assigning higher reputation for COMP than COMS. Otherwise, it is better off proposing cost-sharing deals.Differently, in our commitment model, sharing costs does not reduce the efficiency. It is tantamount to ‘We show in this paper, in the context of the one-shot pairwise Prisoner's Dilemma, that even in the absence of repeated interactions, reputation effects, network reciprocity, as well as group and kin selection, asking to commit and spelling out of the consequences of not honoring the deal is an effective mechanism promoting the emergence of cooperation. If commitment can be arranged in such a way that its cost is justified with respect to the benefit of cooperation , by associating a sufficient compensation , commitment proposers pervade and dominate the population. On the one hand, such commitment proposers can get rid of those individuals that agree to cooperate yet act differently (fake committers) as well as avoid interacting, without any assurance of not being exploited, with the traditional defectors (D). On the other hand, they can maintain a sufficient advantage over those individuals that only cooperate when being asked to commit, because a commitment proposer will cooperate with players alike herself, while the latter defect among themselves. These results suggest our specialized capacity for commitment might have been shaped by natural selection132333436generosity is traded for stability. More precisely, when the option of sharing the cost is in place, cooperation is sustained for a much wider range of the parameters. Our model thus demonstrates that in societies with an adequate support for arranging shared costly commitments and resolving with compensation conflicts that follow from not honoring prior commitments, cooperation becomes widespread.When the cost of arranging commitments is increased and goes beyond the two conditions mentioned earlier, this advantage vanishes, and the commitment proposers become exploited by free-riders, thereby leading to the disappearance of cooperation. We have shown that this commitment free-riding issue can be dealt with by insisting explicitly on sharing the arrangement cost. Even when the free-riders still can refuse to share, δ, for instance proportional to the probability p of receiving the compensation, i.e. p × δ. Considering again the conditions shown in However, what happens when the society is not capable of resolving the conflict? In other words, what if a FAKE player can sometimes get away with cheating? In this case a COMP (or COMS) player would obtain an average compensation smaller than We have also highlighted the differences between the notions of costly commitment and costly punishment, where the latter has been abundantly described in the game theoretical literature68910111218Here we have analyzed commitments in the one-shot PD game. Clearly, as can be derived from some of the examples, commitments can be established for longer time periods and may be influenced by reputation effects3839404135good agreements make good friends provides a highly relevant guideline which, as we have shown here, is extendable to good cost-sharing agreements lead to even better friends.In short, our work demonstrates that the conventional wisdom or common knowledge stating that risk-dominant against B3045N when X,Yπ stands for the payoff an individual using strategy X obtained in an interaction with another individual using strategy Y (as given in the payoff matrix (1)).An important analytical criteria to determine the viability of a given strategy is whether it is risk-dominant with respect to other strategiesfitness or social success, and evolutionary dynamics is shaped by social learning3149A with fitness Af adopts the strategy of another individual B with fitness Bf with probability p given by the Fermi function, β represents the ‘imitation strength' or ‘intensity of selection', i.e., how strongly the individuals base their decision to imitate on fitness comparison. For β = 0, we obtain the limit of neutral drift – the imitation decision is random. For large β, imitation becomes increasingly deterministic.Both the analytical and numerical results obtained here use Evolutionary Game Theory methods for finite populations3147In the absence of mutations or exploration, the end states of evolution are inevitably monomorphic: once such a state is reached, it cannot be escaped through imitation. We thus further assume that, with a certain mutation probability, an individual switches randomly to a different strategy without imitating another individual. In the limit of small mutation rates, the dynamics will proceed with, at most, two strategies in the population, such that the behavioral dynamics can be conveniently described by a Markov Chain, where each state represents a monomorphic population, whereas the transition probabilities are given by the fixation probability of a single mutant4748N be the size of the population. Suppose there are at most two strategies in the population, say, k individuals using strategy A (0 ≤ k ≤ N) and (N − k) individuals using strategies B. Thus, the (average) payoff of the individual that uses A and B can be written as follows, respectively, k of individuals using strategy A by ± one in each time step can be written asN − 1) individuals using B is given by4751β = 0), B,Aρ equals the inverse of population size, 1/N.Let q} of different strategies, these fixation probabilities determine a transition matrix ij,j≠iT = jiρ/(q − 1) and M provides the stationary distribution described above4852Considering a set {1, …, T.A.H., L.M.P., F.C.S. and T.L. designed the research. The models were implemented by T.A.H. Results were analyzed and improved by T.A.H., L.M.P., F.C.S. and T.L. T.A.H., L.M.P., F.C.S. and T.L. wrote the paper together.Supplementary Information


In efforts to modernise the entire drug-development process, making it more efficient, less costly, and ultimately of real benefit to patients, The Federal Drug Administration (FDA) authorised the use of exploratory IND or early Phase I (Phase 0) studies. Quite different in structure from Phase I, II, and III studies, the Phase 0 construct understandably poses a set of ethical problems not seen in the other research phases and so far not adequately addressed by ethicists. In an effort to deal with this deficiency, this paper proposes an ethics critique, based not on the usual concept of benefit, but on the means–end relation, and placed within an ethic of science derived from the practice of science. To date, the ethics analysis of Phase 0 clinical trials has remained incomplete –3. FocusCritical Path Initiative issued by the Federal Drug Administration (FDA) in March 2004  new product-development toolkit – containing powerful new scientific methods, such as animal or computer-based predictive models, biomarkers for safety and effectiveness, and new clinical evaluation techniques – is urgently needed to improve predictability and efficiency along the critical path from laboratory concept to commercial product” .In concluding its critical path report, The FDA argued that progress along the path requires more than better medical ideas. It must also include producing reliable, safe and effective treatments for patients at affordable prices. “This is an essential step in achieving more timely, affordable, and predictable access to therapies based on the latest biomedical insights – that so far are having little impact on patient care . To thisGuidance for Industry, Investigators, and Reviewers: Exploratory IND Studies, issued on January 12, 2006  with that of another and grafts each on each; it cannot survive without justice and honour and respect between man and man”. These vTo a particularly relevant degree, the implications of Bronowski’s societal conception of science applies to the Phase 0 construct, in particular, its design. As Murgo and colleagues view it, the design calls for “a rational transition from pre-clinical to clinical development” . This, tIf we can presume collaboration, we do so because, as Bronowski observed, the nature of science is such that it values the search over the discovery. “In the society of scientists, each man by exploring for the truth has earned a dignity more profound than his doctrine” . But onlBronowski explains that Clifford’s point hinges on the phrase, “it may be true after all”, pointing out that while some might justify their actions with such credulity, the scientist cannot since the word true cannot have such a meaning in science. “The test of truth is the known factual evidence and no glib expediency . . . can justify the smallest self-deception in that” . In pursThe Phase 0 construct promises to be a welcome development in clinical research as long as we see it for what it is scientifically and what it should be ethically. However, different as its scientific constitution is, so also its ethics constitution. Scientifically, it has been developed as a means to a very specific end. And it is on that basis that an ethics critique of the construct must be developed. The concept of benefit, particularly direct benefit, which has been central to the standard ethics critique of the other research phases, is not relevant to Phase 0. But a critique based on the means-end relation as understood by Adler provides a reasonable alternative to understanding what it should be, given that relation. And placed within Bronowski’s formulation of an ethic of science derived from the practice of science, it affords a robust mechanism with which to address the ethical challenges posed by the Phase 0 construct since “the fine structure of science make(s) the grain of conscience” .


Paul Gauguin in his journal Noa Noa. “The only ‘wild game’ on the island are the pigs which have escaped into the forest, where they have multiplied and become entirely wild.”“There are neither carnivores nor reptiles in Tahiti,” wrote The artist’s first impression after the long voyage, “sixty-three days of feverish expectancy,” was “nothing very extraordinary; nothing, for instance, that could be compared with the magnificent bay of Rio de Janeiro.” A native Parisian, Gauguin had spent his childhood in South America, where his family had ties to Spanish nobility in Peru. His mother collected pre-Columbian pottery. These early life experiences he mythologized into a complex persona, often referring to himself as part savage. “You know that I have Indian blood, Inca blood in me, and it’s reflected in everything I do…. I try to confront rotten civilization with something more natural, based on savagery.”This desire to escape his own times and the vestiges of a civilization he considered corrupt and dehumanizing guided Gauguin’s work. He came to art late in life, first cautiously as a collector; then for pleasure, as friend of Edgar Degas, Paul Cézanne, and Camille Pissarro. He became entirely consumed by art when he worked for a time with Vincent van Gogh. “I am a great artist and I know it.” After the stock market crash in 1882, he left Paris to travel and seek the authentic life he believed would lead him to greatness. “You must remember,” he wrote to his wife, “that I have a dual nature, [that of] the Indian and [that of] the sensitive man.”He moved to the French coast. “I love Brittany; I find the wild and the primitive here. When my clogs resonate on this granite ground, I hear the muffled and powerful thud that I’m looking for in painting.” Then he settled in Martinique “to live as a savage,” but soon he left France altogether. “There in Tahiti,” he wrote to his wife, “in the silence of the tropical night, I shall be able to listen to the sweet murmuring music of my heart’s movements in loving harmony with the beings around me. Free at last with no money troubles and able to love, sing, and die.” He painted his best work during his first of two trips to Tahiti. He died of syphilis nearly penniless in the Marquesas.During his travels to the South Sea Islands, Gauguin found that many of his expectations for a life of wildness filled with savages and untamed nature existed only in myths and fables. “The Tahitian soil is becoming completely French.” Unable to find the world he dreamed about, he sought his own. This preferred world he created in his art. From the exotic islands he extracted magical colors he then placed side by side in unconventional combinations and clearly outlined. Despite his admiration for and friendship with the impressionists, he shunned their work in favor of the flat planes that anticipated modern art.Black Pigs, on this month’s cover, was painted the first year Gauguin was in Tahiti. Although he had not yet entirely abandoned perspective, spatial relationships are secondary to color and form. Traces of impressionist technique appear in the brushwork of the foliage and thatched roof of the hut. But the human and animal figures, including the namesake pigs, are simplified, almost archaic in their peacefulness, and entirely at ease with the surroundings.In island culture, the sacred pig was the bond between gods and humans, facilitating their intermarriages or mitigating their disputes. “Food for gods and men” and the backbone of household prosperity, pigs were mentioned in Gauguin’s writings again and again, his cordial interactions with island neighbors sprinkled with “daintily prepared little pig” or “little pigs roasted on hot stones.”Tahiti “Is the summit of a mountain submerged at the time of one of the ancient deluges,” the artist wrote. “Only the very point rose above the waters. A family fled thither and founded a new race—and then the corals climbed up and along it, surrounding the peak, and in the course of centuries builded a new land. It is still extending, but retains its original character of solitude and isolation, which is only accentuated by the immense expanse of the ocean.” Yet even this isolation that Gauguin sensed so acutely was an illusion. During a period of exotic introductions, local animals were modified along with the political, economic, spiritual, social, and physical landscape.Animal movement and trade around the globe, which already had altered the island fauna in Gauguin’s diminishing paradise, now have eliminated even the possibility of zoonotic isolation. As animals and the viruses, bacteria, and internal and external parasites that travel with them find themselves evolving independently from their forbearers in new ecologic niches with new pressures and pathogenic tools, genetic changes occur in them. These changes facilitate emergence of new pathogens that can then start the process over again as the pathogens continue to span the globe.Island people, Gauguin believed, “Had been richly endowed with an instinctive feeling for the harmony necessary between human creations and the animal and plant life which formed the setting and decoration of their existence.” This harmony, which included the savage element he sought and captured in garish tones, he viewed as a main ingredient of great art, along with the unity of humans with nature. This oneness in nature, with its inherent intimacy of all the macroscopic and microscopic players, requires concurrent attention to human health, animal health, and environmental health. For in this cauldron, as full participants and hosts of the wild microbes, which inhabit and kill us, we remain, like Gauguin, part savages.


For many centuries, scientists have debated the cause and best treatment of the disease now known as malaria. Two theories regarding malaria transmission – that of “bad air” and that of insect vectors – have been widely accepted at different times throughout history. Treatments and cures have varied accordingly over time. This paper traces the evolution of scientific consensus on malaria aetiology, transmission, and treatment from ancient times to the present day. There are certain diseases which have been historically shrouded in mystery, their causes attributed to magical or spiritual forces. Ovarian teratoma, for example, a tumour named after the Greek τέρας (monster), is a neoplasm composed of hair, teeth, skin and other mature tissues. Aristotle attributed teratomas to hair that had been swallowed by the patient and deposited in various body tissues. But other theorists claimed more ominous origins: teratomas were variously reported to be a consequence of sexual relations with the devil, an expression of a nightmare (incubus), evidence of engagement in witchcraft, or a punishment for wickedness.Malaria has long been a member of this fascinating group of diseases with veiled origins. For many centuries it was believed that certain diseases, such as malaria and cholera, were caused by miasma , a poisonous vapour or mist filled with particles from decomposed matter (miasmata). Prior to the introduction of the microscope, everything floating above ground that was invisible to the human eye, including dust particles and bacteria, was called “air” – so in a sense, perhaps this early aetiological speculation was not far from the truth.febris acuta), “tertian fevers”, “quartan fevers”, or “intermittent fevers.” Most terms originated from the writing of Hippocrates, who described the unhealthiness of the air in certain environments as it related to fatal diseases with quartan fevers:In 400 BCE, Hippocrates discussed the aetiology of selected diseases in his treatise “On Airs, Waters, and Places” . In anciThis disease is habitual to them both in summer and in winter, and in addition they are very subject to dropsies of a most fatal character; and in summer dysenteries, diarrhoeas, and protracted quartan fevers frequently seize them, and these diseases when prolonged dispose such constitutions to dropsies, and thus prove fatal.,23.22,23It is now known that this brown pigment is formed during the digestion of haemoglobin and produced by the malaria parasite through biocrystallization . The preIn the early years of microscopy, staining technology was in its infancy. The French army doctor, Laveran, had to use unfixed and unstained blood for his experiments, and could see in the blood of his patients a dark pigment that was, according to Virchow, the result of infection with malaria. Some years passed before Malachowski developed a stain which allowed for differential identification of blood parasites .Melanæmia is specially very pronounced in individuals who died from acute paludisme (pernicious attacks); the colour which it gives to certain organs, particularly to the spleen, the liver, and the grey substance of the brain, is almost always sufficient to show from microscopic examination if death is the result of paludisme”. It. It27]. Koch, whose prime quest was tuberculosis control, began malaria fieldwork in 1897. In his African studies he found that in some malaria-ridden villages, all children had malaria and splenomegaly, but as the children grew, the splenomegaly disappeared, and their blood no longer contained the parasites in demonstrable numbers. Eventually, the children became immune to malaria. He favoured the mosquito-borne theory based on concepts he developed during a visit to India in 1883. Koch describes the origins of his theory briefly in a letter he wrote to his Cambridge colleague GHF Nuttall in 1898  (Greek anofelís: good-for-nothing) as the sole vector of malaria in Italy . No. No32]. he blood .Malaria remains one of the most important causes of human morbidity and mortality worldwide, with a tremendous impact in the developing world. Theories on the cause and transmission of malaria have evolved over time, from Hippocrates’ theory of bad air in the Fifth Century BCE to our current understanding of Plasmodial organisms as the causative agent of the disease. Treatments for malaria have likewise changed over time, from Sammonicus’ magical amulets in the Third Century CE to today’s anti-malarial drugs. Current anti-malarial treatments are much more sophisticated than ancient therapies. However, as some of the drugs in the anti-malarial arsenal are losing effectiveness, perhaps over time there may be a resurgence of bygone therapies.The authors have declared that they have no competing interests.Both authors contributed to the research, drafting and editing of the paper. Both authors read and approved the final manuscript.


Principally: (1) Physically associated clonal groupings of bacteria can represent larger targets for phage adsorption than individual bacteria; and (2), due to a combination of proximity and similar phage susceptibility, individual bacteria should be especially vulnerable to phages infecting within the same clonal, bacterial grouping. Consistent with particle transport theory—the physics of movement within fluids—these considerations are suggestive that formation into arrangements, microcolonies, or biofilms could be either less profitable to bacteria when phage predation pressure is high or require more effective phage-resistance mechanisms than seen among bacteria not living within clonal clusters. I consider these ideas of bacterial ‘spatial vulnerability’ in part within a phage therapy context.The ability of bacteria to survive and propagate can be dramatically reduced upon exposure to lytic bacteriophages. Study of this impact, from a bacterium’s perspective, tends to focus on phage-bacterial interactions that are governed by mass action, such as can be observed within continuous flow or similarly planktonic ecosystems. Alternatively, bacterial molecular properties can be examined, such as specific phage‑resistance adaptations. In this study I address instead how limitations on bacterial movement, resulting in the formation of cellular arrangements, microcolonies, or biofilms, could  Development of alternative methods of biofilm removal therefore is desirable [Environments can be distinguished in terms of the degree of spatial structure that they exhibit, where spatial structure is a description of the extent to which diffusion, motility, and environmental mixing are constrained. Important spatially structured bacterial habitats include soils; sediments; surface tissues of plants, animals, and fungi; and bacterial biofilms in general. The latter are found both as suspended aggregates and on most submerged surfaces. It is an oft-repeated assertion that the majority of bacteria, or at least a large fraction, may be found within biofilms rather than as planktonic organisms ,2. Naturesirable .Bacteriophages, the viruses of bacteria, are a possible alternative to antibiotics, or disinfectants, as antibacterial agents. Such phage therapy or phage-mediated bacterial biocontrol ,7 has shHere I explore the costs to bacteria of ‘group living’ that can result from exposure to phages. This I term a ‘spatial vulnerability’ because bacteria that are physically attached together—as arrangements, microcolonies, “macrocolonies” , or othemust possess substantial phage-resistance mechanisms [Mechanisms that can reduce bacterial exposure to phages include existence within environments into which phage penetration is difficult or when bacteria exist at sufficiently low densities that they are unable to support phage amplification to “inundative” densities . The latchanisms ,31,32 inThe common theme is that living as physically associated and therefore spatially structured clonal groups, in and of itself, should not be expected to serve bacteria as a phage-resistance mechanism. Rather, I argue from first principles that group living can result in greater bacterial vulnerability to phages than may be experienced by bacteria that instead are physically separated from their clonal relations. With this perspective in mind, the utility of phages as an anti-bacterial as well as a specifically anti-biofilm strategy may be appreciated as an explicit ecological reversal of exactly those circumstances in which biofilms otherwise may flourish: Application of sufficient densities of phages, where phages otherwise are lacking, such that uncontrolled bacterial proliferation can be reversed.In this study the primary question being asked is what might be the ecological costs to bacteria, in light of phage predation, that are associated with bacterial growth as arrangements or microcolonies. To answer this question, I generally employ ecological models, arguments, and scenarios—that is, considerations of how bacteria may interact with their environments—and this is rather than primarily enlisting evolutionary approaches or perspectives. In addition, as the study represents a relatively novel exploration the ideas presented, I limit discussion to less complex scenarios, avoiding addressing for instance consideration of stochasticity, the growth in size of bacterial arrangements, or simulations of phage-bacterial ecological dynamics.not exist given a combination of bacteria that are “free” and substantial environmental mixing. In this section I consider the basics of phage adsorption, focusing particularly on issues of encounter rates between phages and bacteria rather than mechanisms of phage attachment or subsequent phage initiation of infection. For visualization of the spatial scale of environments in which these interactions take place, see Abedon [The interaction between phages and those bacteria that exist as individual, planktonic cells—here, collectively, “free” bacteria—is fairly straightforward. Beginning with phage attachment to a susceptible bacterium, phage-genome uptake occurs, initiating the infection proper. At some point mature virions must be released from the infected bacterium, beginning an extracellular search for new bacteria to infect . This see Abedon .The extracellular search at its most basic consists of a process of virion diffusion. Such diffusion, due to the comparatively small size of phages, occurs at a rate that is substantially greater than the diffusion of free-floating bacteria. As a result, phage extracellular movement towards an idealized bacterium can be described  (p. 104)Rates of virus diffusion along with the size of target bacteria are not the only factors affecting rates of encounter between these entities, as so too do their environmental densities. For instance, and as considered in some detail by Murray and Jackson , the speet al. [Ioannou et al.  provide et al. ,29.The higher the density at which phage virions are present within environments then the more likely that a given bacterium will encounter a phage, potentially resulting in bacterial conversion from phage uninfected to phage infected. Thus, tN is the density of bacteria that are not phage infected at the end of some interval (t), N0 the density of uninfected bacteria at the beginning of that interval, P is the density of free phages , and kPt represents an actual multiplicity of phage infection [actual as defined by Kasman et al. [k, is the probability that a single phage within a specified volume will encounter and then adsorb a single bacterium. This value is based in part on the rate of phage diffusion along with the size of bacterial targets , for this study I employ the simplifying assumption that phage densities do not vary over time. Operationally, this means that I am placing greater emphasis on consideration of bacterial vulnerability to phages than I am on the dynamics of phage generation and loss.where versus bacillus versus spirillum, can be viewed as presumptively adaptive [We can increase the complexity of phage-bacterial interactions by considering bacteria that are found predominantly as arrangements rather than as otherwise “free” cells . For example, bacteria can be arranged as doublets of cells (such as diplococci), strings of bacteria (streptococci or streptobacilli), or other, more complicated forms , and even, as indicated above, as microcolonies as well as biofilms. These arrangements are formed in the course of bacterial division and they differ in terms of the number of divisions that take place prior to cell separation as well as in terms of the planes of those divisions. Forming into arrangements presumably provides bacteria with selective advantages, as considered in adaptive ,41 or biadaptive .i.e., ten bacteria as a single target could be ten-times as likely to become phage adsorbed as a single bacterium). Again at an extreme, once an arrangement has become phage infected, then complete loss of all bacteria found in that arrangement could occur. In this section, I consider limitations on these extremes. I nevertheless retain the general conclusion that group living could increase bacterial vulnerability to phages.Existing as arrangements, or as microcolonies, may be costly in the face of phage-mediated predation. We can consider this proposed elevation in costs as a consequence of increases in the overall target size of arrangements relative to individual bacteria, which is relevant especially in combination with increased potential for phage propagation within arrangements. At an extreme, arrangement target size could increase directly as a multiple of the number of bacteria found within an arrangement , is get size ,35. If bget size  or, altenP, where n is the number of bacteria making up an individual arrangement. That is, the target size of an arrangement increases by a factor of n relative to free bacteria while at the same time decreases by a factor of /k. The increase due to n, however, likely is greater than the decrease described by /k, at least so long as arrangements are not sequestered within phage-excluding volumes such as (perhaps) defects in the glass walls of chemostats [The rate of phage adsorption to a bacterial arrangement can be described as emostats . Larger 1/3 = n), which is the increased diameter of a two-fold larger volume, and approximately two (n) times larger than the target size of individual cocci. These values in other words range from where shading is substantial (1.26 times) to where shading instead is minimal  withA stands for arrangement and tA is the number of arrangements that have not been phage adsorbed over an interval, t, given a constant phage density, P. So long as n > k holds, then t/NN0 > t/AA0. That is, fewer arrangements will remain fractionally unadsorbed (t/A0A) than would individual, free bacteria (t/N0 )N, holding bacterial size and adsorption susceptibility otherwise constant. Here nPt is equivalent to MOIactual for arrangements. Note though that it is my preference to instead use the term multiplicity of adsorption, i.e., MOA, rather than multiplicity of infection because while an arrangement can be wholly adsorbed by a phage, subsequent infection of the whole arrangement is a more complicated process versus the infection of individual phage-adsorbed bacteria.Here t/N N0 > t/AA0—is that MOA for arrangements can be up to n-fold higher than that for individual cells. A quantity that I will call MOAinput (M) can, after Kasman et al. [A0), as phage targets, is expected to be n-fold lower than that of free bacteria, i.e., 0 = N0/nA, assuming a constancy in both cell size and total species biomass [M for arrangements (AM) is expected to be n-fold higher than M for free bacteria (NM), since A = P/M(0/nN) whereas N = P/N0M. The fraction of targets expected to remain unabsorbed, in turn, is readily calculated as e-M, which is the frequency of the zero category—bacteria (t/NN0) or arrangements (t/AA0) experiencing no phage adsorption—given a Poisson distribution of phages adsorbing to targets. The larger M then the smaller the fraction of cells or arrangements remaining unadsorbed, and therefore t/NN0 > t/AA0 if A > MNM. More precisely, we can consider instead MOAactual, which are A = nMN=kPtPt versus M. With M defined in this manner, then the fraction of phage targets expected to remain unadsorbed is equal to  (= t/AA0) and e-kPt (= t/NN0), respectively, which are restatements of Equations (3) and (1), respectively. Note that  < e-kPt if as expected nPt > kPt, implying that t/AA0 < t/NN0.A complementary perspective on the above assertion—that is, that fewer arrangements will remain unadsorbed by phages relative to free bacteria,n et al. , be set  biomass ,43. Holdn, that is, as n contributes to arrangement diameter and therefore to target size, may be slowed to the extent that adsorption rates to the individual cells making up an arrangement, , also may decline as n increases. It may be harder, that is, for environmental phages on average to encounter individual bacteria that are found within larger arrangements (such as a large microcolony) versus individual bacteria that are found in smaller arrangements (such as a diplococcus). Arrangement vulnerability to adsorption, given this tendency, therefore might increase less rapidly as a function of the number of bacteria that they contain.These considerations come with the caveat that increases in the likelihood of arrangement adsorption that occur as a function of nPt should adequately describe the likelihood of arrangement encounter with a phage. Further, nPt defines the actual multiplicity, a.k.a., multiplicity of adsorption, of a bacterial arrangement with the value  specifying inefficiencies in this phage adsorption process in comparison to free bacteria. In addition, phage adsorption to arrangements is not identical to phage adsorption to free bacteria because only a fraction of the number of bacteria making up an arrangement become initially phage adsorbed  rather than all of the bacteria making up a free bacterium (1/1). Furthermore, with bacterial arrangements an initial (“primary”) phage adsorption could give rise to a variety of subsequent outcomes including infection of only the adsorbed bacterium, subsequent infection of a fraction of the bacteria found within the arrangement, or indeed subsequent infection of all of the bacteria making up an arrangement. The latter result, of course, is the more costly of outcomes to the affected bacteria, just as it is in terms of prey aggregation more generally [The expression enerally . The resWhen phage densities are higher, then the likelihood of phage adsorption of a given bacterium also should be higher, as described by Equation (1). Importantly, then, the density of phages immediately surrounding a lysing bacterium, that is, as made up predominantly of those phages released from that bacterium, will be the highest phage densities that can be readily attained within a given environment. The phages released in this burst will then diffuse outward, declining in density as they do. The result should be a higher rate of phage adsorption to any susceptible bacterium found within the immediate vicinity of a lysing bacterium, but lower rates at increasing distances .et al. [i.e., a constraining of bacterial location in arrangements to within the vicinity of agents infecting the same arrangement (p. 1): “Since many bacterial species grow naturally in chains, this intrachain transfer is likely a common mechanism for accelerating the spread of conjugative elements within microbial communities.”While free bacteria can randomly find themselves in the vicinity of a lysing bacterium, bacteria that are found in arrangements can be spatially constrained to that vicinity. The result is a higher likelihood of phage infection of other bacteria found within the same phage-infected arrangement than to other environmental bacteria . This aret al.  that tradensities of bacteria. This “mass” of bacteria within a bacterial arrangement would be more likely to exceed a “critical” level, such that phage propagation can be sustained, than may be achieved by an equivalent number of more locally dilute, free bacteria. This perspective is just as one can consider for nuclear fission, i.e., radioactive decay, and associated chain reactions [Analogously, greater bacterial densities found within bacterial arrangement can be viewed as possessing a higher “mass” relative to that associated with planktonic bacteria, where by “mass”—potentially confusingly—I am referring to immediately local eactions , which oeactions ,25. EachA target bacterium that is found in the immediate vicinity of a phage, in other words, displays a much higher local density from the perspective of that phage than may be case for bacteria that are randomly dispersed throughout an environment. A local concentration of bacterial “mass” thus can result in a high propensity for bacterial adsorption by phages that have been generated within the same “mass”. See Abedon , by cont. This represents the number of bacteria within an arrangement that will be lost, on average, as a consequence of phage adsorption of a single bacterium within that arrangement. This number, , and can range up to the total number of bacteria making up an arrangement (n). Another way of viewing , however, is that  > 1 implies a phage reproductive number within a bacterial arrangement that is greater than 0 such that some degree of phage propagation within an arrangement occurs along with consequent bacterial death. If insufficient phage release from infected bacteria and/or insufficient subsequent bacterial infection occurs within an arrangement, or subsequent infections don’t happen fast enough, then complete eradication of an arrangement by an infecting phage may not happen, such that  < n.Notwithstanding proposed tendencies for phages to more readily acquire bacteria that are within their immediate vicinity, it is as noted possible for inefficiencies to exist in the sequential phage infection of bacteria co-occupying the same arrangements. To incorporate such inefficiencies into models of arrangement vulnerability to phages, I employ the term, These ideas can be expressed as,) is the fraction of arrangements that become phage adsorbed over some interval, t, and  is the number of bacteria per arrangement that are lost to this adsorption . Were  = 1, then though arrangements are more likely to be adsorbed than free cells, nevertheless no more bacteria would be lost per arrangement adsorption. Indeed, to the extent that the initial bacterial infection is less likely, that is, given  < k along with  = 1, then overall existence as an arrangement could result in less vulnerability to phages rather than more. Such a situation would occur, for example, were phage infections abortive or perhaps could result instead were infections substantially reduced in burst size or extended in latent period such that phage propagation through an arrangement were substantially impaired. Alternatively, the equality  = n would imply complete arrangement loss following each phage adsorption of an arrangement. The parameter, , is thus a description of arrangement vulnerability to phages post-adsorption, ranging from minimal  to maximal ( = n ). A visual summary of the models represented by Equations (1) and (4) is presented in Where  does not reflect that if one adsorbing phage fails to clear an arrangement then perhaps more than one adsorbing phage will, with greater likelihood, succeed in doing so. One way to address this concern is simply by setting  = n, that is, an assumption of complete arrangement loss per primary phage adsorption.Equation (4) is less applicable given higher levels of phage adsorption, as can occur over longer periods. This is because, as noted, Equation (4) fails to take into account the impact, on the overall fraction of bacteria that are lost, of multiple phage adsorptions of individual arrangements. That is, for instance, we might have an expectation of greater bacterial loss with greater levels of arrangement adsorption by environmental phages under conditions where otherwise Alternatively, one can limit one’s considerations to circumstances in which phage multiplicities of adsorption to arrangements are relatively low (<<1). In such cases the equation can be simplified aswhich compares, for a free bacterium, with,tN (as defined by Equation (5)) is greater than tN (as defined by Equation (6)) when  > k/. That is, when this inequality holds then bacteria found within bacterial arrangements are more vulnerable to phages than are phage-susceptible bacteria that are “free”. In words: Bacteria found within arrangements are more susceptible to phage attack if losses due to existence within a phage‑adsorbed arrangement, , are greater than increases in individual bacterial vulnerability to “primary” adsorptions that come from not existing within an arrangement .Note that /k = 0.5 such that k/ = 2) but each primary adsorption results in the loss of ten bacteria ( = 10), then the resulting 10 > 2 would imply that arrangements are more vulnerable to phages than are free bacteria. Indeed, in this example bacteria found within arrangements would be five times more vulnerable. Note further that if arrangements can reduce  to 1, then arrangements will be expected to display lower vulnerability to phages than free bacteria so long as individual bacteria found within arrangements are less vulnerable to primary phage adsorptions than free bacteria . This is an observation that could very well explain the utility of abortive infection systems to bacteria, i.e., phage-resistance mechanisms in which both adsorbed bacteria and adsorbing phages die [ = 0, which is the case given successful bacterial display of, for example, antibacterial restriction-modification systems, then biologically the inequality no longer holds, i.e., 0 > k/. Logically, though, in this case arrangements should be no more or less vulnerable to phages than free bacteria since, in fact, neither would be vulnerable. See i.e., Equation (7), are not discussed until If a bacterium is half as likely to be subject to primary phage adsorption when found within an arrangement (ages die . Alterna) than for free bacteria (k) but nonetheless that arrangements can be more vulnerable to exploitation by phages. If we assume that arrangements nevertheless provide bacteria with selective benefits, then it should be possible to consider how large these benefits must be to offset costs stemming from this presumed increased susceptibility of arrangements to phage infection. Before moving on to that issue, however, I first address two underlying considerations, (1) how existence within clonal associations in fact might benefit bacteria and (2) experimental evidence that phages can propagate through bacterial arrangements and/or microcolonies.Above I argue that the likelihood of adsorption by any given phage can be lower for individual bacteria found within arrangements  are considered in terms of rates of cell division (μ) versus declines due to phage adsorption,These considerations of phage impact on the instantaneous population growth of bacteria existing within arrangements can be easily formulated as a differential equation in which changes in bacterial density , or simply kPN in Equation (8), describes those bacteria that have been lost from the unadsorbed bacterial pool, N, as a consequence of phage adsorption either of themselves (Equation (7) and Equation (8)) or of the arrangement in which those bacteria are located (Equation (7)). As noted above  is a description of the density of arrangements consisting of n bacteria that are found in the environment in question. Also as above, the parameter  describes the number of bacteria that will be lost to phage infection given phage adsorption of an arrangement. Lastly, nP is a description of the per-arrangement rate of bacterial adsorption by phages given an environmental phage density of P.The expression P and each of those adsorptions results in a loss of  bacteria. This compares with the rate of loss of individual bacteria as described by kP in Equation (8). The expression, μN with either subscript, by contrast, describes in both equations the gains in bacterial density that occur as a consequence of bacterial replication. Thus, If P > A µ, then the bacterial population will experience a net decline in number whereas P < Aµ indicates net gains and P = Aµ defines a steady state. For Equation (8) the equivalent expressions instead are respectively N kP > μ, N kP < μ, and NkP = μ, where P in the latter can be described as an inundation threshold or even phage minimum inhibitory concentration, that is, of bacteria [Aμ or Nμ.Per bacterium, Equation (7) thus describes adsorptions that occur at a rate of bacteria . In the P  from phages that instead are explicitly propagating through bacterial arrangements. What Equation (7) nevertheless indicates is that the phage impact on bacterial arrangements varies as a function of phage density (P) in combination with the susceptibility of individual bacteria making up an arrangement to phages . Specifically, the larger P then the more bacteria found within arrangements that are lost to phage infection. For example, twice as many bacteria will be lost per unit time for bacterial arrangements (P from Equation (7)) versus free bacteria (kP from Equation (8)) if P /k = 2. In Equation (7) the dynamics of microcolony formation are not considered and nor are various other complications such as multiple adsorption by “environmental” phages of individual bacterial arrangements, where by “environmental” I am distinguishing those phages defining P/k-fold more arrangement-associated bacteria will be lost versus free bacteria for any given phage density, P. Forming into an arrangement, and thereby incurring costs of additional vulnerability to phages, therefore should be worthwhile to bacteria only to the extent that Aμ, or some other measure of bacterial fitness, increases as a consequence of group living to a larger extent than bacterial fitness decreases as a result of incurring a greater spatial vulnerability to phages, i.e., as described by P /k.Alternatively, a total of P = 0) then there will be no phage-associated cost to arrangement, microcolony, or biofilm formation (in which case no advantage is required from group living to offset costs of phage adsorption) or (ii) if phages are present at effectively infinite densities (P = ∞), or simply sufficiently high densities, then potentially no amount of phage-independent benefit to group living could offset increases in phage-associated vulnerabilities. The latter situation is just as dire for free bacteria, however, i.e., for which  = 1 but nonetheless where it is possible for .tif (Equation (7) versus Equation (8)). At a minimum, however, Aμ = P is necessary for bacterial fitness, as measured here in terms of increases in bacterial growth rates, to offset costs due to phage adsorption, and this compares with N = kPμ for free bacteria; see Abedon and Thomas-Abedon [The extreme situations with regard to Equation (7) are as follows: (i) If no phages are present in an environment , to offset increased phage-associated costs that are borne by bacterial arrangements. This fitness improvement, however, need not be substantial unless phage densities (P) are also substantial. Thus, as  or  increase so too does the potential for phages to block the evolution of bacterial arrangements, but at the same time such increases do not serve as absolute blocks on this evolution. The alternative perspective is that given sufficiently high phage densities—but not too high, as indicated in the previous paragraph—then evolution could tend to favor reductions in  even if bacteria otherwise experience benefits from forming into arrangements, that is, reduced formation of arrangements could serve as a bacterial anti-phage strategy. In simple terms, a coccus might encounter a phage approximately half as often as a diplococcus. For circumstances in which versus free bacteria given higher versus lower arrangement vulnerability to phages.A generalization on these considerations is that bacteria that have formed into arrangements will have to avoid, on a per-bacterium basis, equivalent increases in vulnerability to phages in order to partake of whatever net advantages may be associated with forming into arrangements. This reduced vulnerability, furthermore, can result either from existing in environments in which phage densities are low  or, alternatively, from greater resistance by individual bacteria to phage attack when phage densities are high, that is, when phage-mediated losses dominate competitive dynamics ,31. Thisless able to attain “winner” densities within a given environment—as in “kill the winner” (et al. [et al. and that of kill the winner, however, is seen in terms of the degree of specialization of the predator species. For phages, specialization can be extreme such that bacteria phage-susceptibility types can “hide” by failing to support phage replication to inundative densities, that is, by not being “winners”. For the visual predators considered by Ioannou et al., by contrast, it is not that prey avoid predation by impacting predator densities but instead that they avoid being consumed by being less visible to predators that already exist at some more or less fixed density.Perhaps the simplest approach to reducing arrangement vulnerability to phages would be for those bacteria to exist at lower population densities, population densities, that is, which are insufficient to support phage population growth to levels at which those phages can substantially impact arrangement fitness. In other words, bacteria that are  winner” —may as a (et al. , as descBiofilms often can occupy only a small fraction of total environmental volumes, such as in aquatic environments. In such circumstances, biofilm bacteria as a consequence may be inherently unable to achieve winner-level densities. This low potential, furthermore, may be particularly the case to the extent that biofilms consist of mixed populations of bacteria rather than monocultures of specific phage susceptibility types, thereby implying even lower densities of individual bacterial phage susceptibility types than available surfaces might maximally hold. Alternatively, biofilms found within highly structured environments, such as undisturbed soils , may oveAn additional possibility may hold and that is that it is particularly those bacteria which, as a matter of luck, are resistant to all phages present within environments that may attain “winner” densities, either free or in association with arrangements. This latter idea is ecologically similar to the domination of animal-rich ecosystems by plants that are resistant to forage by those herbivores which happen to be present within an environment . That isSee Results and Discussion.populations to predators, with prey population density also playing a key ecological role.Group living bestows benefits—else why live in groups?—but also engenders costs. One cost comes from an increased vulnerability to exploitation that group living creates, such as a greater potential for infection of individual bacteria by bacteriophages. Here I have provided a simple model for quantifying those costs, one that points to the idea that bacterial arrangements along with microcolonies and biofilms may persist particularly under circumstances where high rates of lytic phage infection is unlikely. This conclusion is broadly consistent with Murray and Jackson’s  suggestidirectly serve as a means of avoiding phage-mediated predation. Observation of the existence of specific bacterial types at high environmental densities and particularly as arrangements, microcolonies, or biofilms, however, would suggest the existence of effective mechanisms by which their vulnerability to phages has been reduced, just as pathogen-resistance mechanisms have, of course, evolved in multicellular organisms such as animals [This perspective contrasts with notions that group living among bacteria might  animals . One rea animals .Alternatively, phages to which high-density bacterial populations are sensitive simply may not have reached those bacterial populations. That situation represents the default assumption that one might make with regard to small, relatively closed systems. The utility of phage therapy as an antibacterial strategy is that in many instances the infection to be treated can be described in the latter terms: small, relatively closed systems, such as localized or even systemic bacterial infections. Efficient bacterial eradication, including of bacterial biofilms, often can be achieved, therefore, simply by “opening” these systems sufficiently to phages, and the phage therapy literature, such as in terms of successful clinical treatment of infections in humans ,75, appe


Bosome book, a manuscript discovered in 1574 and believed to have been written in Ripley’s own hand. In 1583, Dee and his associate Edward Kelley left England for East Central Europe, taking with them a proportion of Dee’s vast library, including alchemical books—the contents of which would soon pique the interest of continental practitioners. Kelley used Ripley’s works, including the Bosome book, not only as sources of practical information, but as a means of furthering his own relationships with colleagues and patrons: transactions that in turn influenced Ripley’s posthumous continental reception. The resulting circulation of texts allows us to trace, with unusual precision, the spread of English alchemical ideas in the Holy Roman Empire from the late sixteenth century.This paper investigates John Dee’s relationship with two kinds of alchemist: the authorities whose works he read, and the contemporary practitioners with whom he exchanged texts and ideas. Both strands coincide in the reception of works attributed to the famous English alchemist, George Ripley (d. c. 1490). Dee’s keen interest in Ripley appears from the number of transcriptions he made of ‘Ripleian’ writings, including the  Less has been said, on the other hand, about Dee the reader of alchemical texts (and colleague of other readers), or about Dee the alchemical practitioner (and colleague of other practitioners). This paper focuses on Dee’s relationship with two kinds of alchemist: the authorities whose works he read, and the contemporary practitioners he knew, or may have known. Often the two groups come together, for one interest that Dee shared with many of his acquaintances was an active interest in recovering, reading, and putting into practice the works of past adepts.For a large part of his life John Dee was engaged in reading about and practising alchemy. His alchemical interests have been most closely studied in relation to his own John Dee’s library catalogue (1990).The earliest evidence for Dee’s interest in alchemy is his list of the alchemical books he read (but did not necessarily own) in 1556.Monas hieroglyphica . More specifically, scholars have pondered the extent to which Dee’s alchemical books were read, copied and discussed by others . In the second half of the sixteenth century, ‘Ripleian’ writings were well known in his native land, but less so in East Central Europe. This strand therefore stands out in relatively high relief against the rich backdrop of continental alchemy, enabling us more easily to pick out clues to its dissemination. The circulation of Ripleian texts coincides in time with the arrival of Dee and his associate Edward Kelley in Bohemia during the 1580s. Indeed, the spread of Ripley’s alchemy in mainland Europe is intimately related to the circulation of Dee’s books and Kelley’s burgeoning alchemical practice. The travels of the English party therefore leave us clues to the relationship between Dee and two alchemists: the living Kelley and long-dead Ripley.2From the mid-fourteenth century, a body of alchemical texts posthumously and pseudonymously attributed to the Majorcan philosopher Ramon Lull (1232–1316) became increasingly influential on the theory and practice of western alchemy. Detailed studies by 7ignis contra naturam) is a powerful and toxic corrosive, valuable for gold-making but deadly if ingested. The second, ‘natural fire’ (ignis naturae), embodies the vegetative power of nature, and may be used to heal both metals and the human body. Various works, describe the use of these fires in manufacturing the ‘mineral’ and ‘vegetable’ stones.As might be expected given the size of the corpus, pseudo-Lullian alchemical operations are many and varied. However, English commentaries on this tradition frequently invoke two powerful waters, sometimes described as ‘fires’, with contrary actions.fumus albus, which is collected in a receiver and condensed to form a liquid, which is then subjected to further procedures. Owing to the circumstances of its manufacture, the vegetable stone was regarded as combining both mineral and vegetable qualities. In this form, it was regarded as a safe and legitimate product for human consumption, and therefore provided one basis for the medicinal aurum potabile—an objective which cannot have been assisted, in practice, by the use of toxic lead compounds.One basic recipe for the vegetable stone, found in hundreds of permutations in alchemical literature, employs a metallic body called ‘sericon’ as its prime matter: usually taken to denote minium (red lead). The minium is dissolved in strong wine vinegar, and the resulting solution heated until a thick gum remains in the bottom of the glass.Compound of alchemy (1471) and Medulla alchimiae (1476).Medulla, for instance, Ripley gave instructions for the manufacture of the vegetable stone from sericon, and its subsequent combination with the mineral ‘fire against nature’  to produce a powerful transmutational elixir—the aqua composita, or compound water  for both medicine and transmutation.13Doctrines of both Lull (or ‘Raymond’) and Ripley would have been familiar to John Dee from the earliest days of his interest in alchemy, as appears from the list of alchemical books he read in 1556. These include ‘Quinta essentia Raymundi Lulli’, the abbreviated title of the Compound was composed in English verse, while the Medulla was translated into English in 1552 as the Marrow or Mary of alchemy. Several other Latin texts attributed to Ripley, including the Philorcium alchymistarum and Concordantia Guidonis et Raymundi, were translated around the same time, while two other well known treatises, the Pupilla alchimiae and Accurtations of Raymond, seem to have been originally composed in Middle English. All circulated widely in the second half of the sixteenth century.15The list also includes an unnamed text, labelled ‘Ripley, anglice.’Compound, or ‘Twelve gates.’ Dee later owned a mid-sixteenth-century copy of this work which he annotated on 21 December 1595 . It is likely that he was familiar with the Compound long before this—by the 1560s, it was already one of the best known alchemical works in England. Yet Dee’s interest was not confined to Ripley’s poetical masterpiece: he also copied out several Ripleian prose works in his own hand, all of which may be distinguished by their practical, ‘sericonian’ character. These include the Accurtations of Raymond, a theoretical treatise accompanied by a collection of recipes, which was transcribed (at least in part) by Dee, with copious annotations, in Wellcome Library MS 239 (pp. 1–44).Compound: ‘Riplay in his 12 Ga[tes] in the Chap. of Ferm[entation]’ .By the far the most popular of Ripley’s works was his Middle English poem, the mber pot in the vyneger, farder | to try what wold comme of it’ (fol. 3r–v). In extracting this slimy ‘mercury’, Dee was guided by an earlier authority:m warnes of. Whereby I perceyued that an [ounce] had byn enough | or 6 [drams] rather, to kepe the water in hand | and not to overcharge it, as Riplay in philortiuDee seems also to have drawn on Ripley when translating his textual studies into practical procedures. Bodleian Library MS Rawlinson D.241 is an alchemical notebook, written partly in Dee’s hand, that details experiments carried out between 22 June and 6 October 1581. This account of Dee’s daily practices and observations implies that Dee—no armchair alchemist—was willing to get his hands dirty. At one point, he describes how he obtained over 10 oz of ‘quick mercury’ from a sublimate, ‘by my diligence in pressing the soft stuff || betwene my fingers partly: and by washing it | destilled vineger.’ The result was, ‘a faynt fyne slick or slyme: which I yet | left in an stone ChaPhilorcium, in which Ripley advises his disciples to take care when adding liquid to their work:Draw a water from the same stone using an alembic, and with that water dissolve the stone by alembic, and with that water dissolve the stone infinite. But dissolve it by little and little, and you shall not incontinently suffocate it with water, for if it is correctly regulated you may with one pint of water make, if you wish, an infinite quantity of water.17Ripley was clearly regarded—at least by Dee—as having something more than theoretical application: his recipes and recommendations assigned practical value almost 90 years after his death.The reference is to a passage in the Opera omnia chemica (1649) in Kassel. When compiling the twelve texts that constituted this volume, the editor, Ludwig Combach, was concerned to secure reliable exemplary manuscripts—at least one of which was linked to Dee and Kelley, and their contacts within the Empire. These connections not only shed light on the alchemical activities of Dee and Kelley in Bohemia, but also suggest that these played a role in shaping Ripley’s own posthumous reception.Following the arrival of the scryer Edward Kelley in his household in 1582, Dee’s alchemical pursuits increasingly relied on spiritual as well as temporal authorities. Within two years of making the notes in MS Rawlinson D.241, Dee had relocated himself, his family, and many of his alchemical books to East Central Europe, where he and Kelley attempted to establish themselves first in Cracow and later in Prague, guided by the advice of their angelic interlocutors . Yet Dee3Dee, Kelley and their families left England on 21 September 1583, in the company of Albrecht Łaski, palatine of Sieradz, whose pretensions to the throne of Poland had received encouragement in the angelic conversations mediated by Kelley at Mortlake. Following an inauspicious start in Cracow and at the imperial capital of Prague, the Englishmen eventually settled in Třeboň (Wittingau) as clients of Vilém of Rožmberk (1535–1592), one of the greatest magnates in Bohemia, and like the Emperor, Rudolf II, a patron of alchemists.Until his death in poorly-documented circumstances, probably on 1 November 1597 , Kelley Compound.Kelley’s rise is mirrored by an increasing interest in the works of George Ripley in East Central Europe, which seem to have made their Bohemian debut at around the same time. For instance, nine Ripleian works were transcribed in Bohemia in 1605–06 by the alchemist Simon Thadeas Budek, including a Latin translation of the Compound and two other works (the Epistle to Edward IV and the Clavis aureae portae) to Kelley.Compound and Epistle were already circulating in France by the early 1570s, as the Liber duodecim portarum (‘The book of the twelve gates’) and the Epistola ad Regem Eduardum respectively.Liber was printed in Frankfurt in 1595, edited by the Paracelsian physician Bernard Georges Penot (c. 1522–1620), while a fuller version was published in Leiden in 1599 by Penot’s friend, the Protestant pamphleteer Nicholas Barnaud (c. 1539–1604?), in his Quadriga aurifera.Such connections were not lost on Ripley’s later editor, Combach, who attributed the Latin translation of both the Liber’s appearance in print. Several of the individuals involved in the later dissemination of Ripley’s works were staying in Prague at roughly the same time as Kelley, and in circumstances conducive to the sharing of alchemical ideas, or, for that matter, texts.Combach’s attributions might therefore be dismissed as an attempt to promote Ripley’s authority by linking it to the celebrated Kelley. Yet surviving manuscripts suggest that Kelley’s own fascination with Ripleian alchemy may have helped stimulate wider interest in the medieval English alchemist, subsequently contributing to the Testamentum, in which letters denote ingredients and chemical processes. The alphabet, published in Penot’s Apologia  as ‘Rubri & Albi vini a terra foliata extractio’ (‘The extraction of red and white wine from foliate earth’), yields a fairly straightforward sericonian recipe, beginning with the dissolution of the calcined ‘green lion’ in vinegar to create a gum.Penot and Barnaud actually met in Prague some time after 1586, while Barnaud was a guest of another alchemical enthusiast, the emperor’s physician, Thaddeus Hájek (1525–1600). During this time they collaborated on a ‘Practica alphabetica’, which—as Penot later reported—Barnaud ‘wanted to be arranged according to the time honoured model.’Liber to another Bohemian acquaintance: Sir Edward Dyer (1543–1607), an English courtier and poet sent to Prague on at least three occasions between 1588 and 1590, partly in repeated attempts to persuade Kelley either to return to England or to teach him alchemical secrets.De denario medico (1608), which refers to transmutations carried out by Kelley during Penot’s own sojourn in Prague .There would have been plenty of opportunities for both men to meet the English visitors. Barnaud, like Dee and Kelley, became a client of Vilém of Rožmberk, while his host, Hájek, had housed the Englishmen during their first visit to Prague. Barnaud would eventually dedicate his edition of the Compound was fanned by the presence of the English visitors in Prague. However, there is more tangible evidence that Kelley took an active role in promoting Ripley’s works amongst well placed colleagues. This includes the appearance of a second, alternative translation of the Liber, composed in elegiac verse by the German alchemist Nicolaus Mai, apparently at Kelley’s instigation.Since Penot’s and Barnaud’s exemplary manuscripts are no longer extant, we cannot know for certain whether their interest in Ripley’s Appellationsrat, prefect of the silver mines at Joachimsthal—a post previously vacated by another alchemist, Sebald Schwaertzer. From there he corresponded with another alchemical patron, Moritz, Landgraf of Hesse-Kassel. In one letter he apologises for being prevented from visiting Moritz, since the emperor requires his services as an envoy to the Counts of Mansfeld.28Mai, or Maius, was well connected in both political and alchemical circles.De denario to him, addressing him as a councillor to the Elector of Brandenburg . Mai contributed a eulogistic poem to the De signaturis internis rerum (1609) of the Paracelsian chymist Oswald Croll, and was the addressee of one of the letters collected in the Rerum chymicarum epistola (1595) of the Saxon alchemist and physician, Andreas Libavius.Compound into verse for presentation to the Emperor.31Mai also maintained links with other alchemical practitioners. Penot dedicated a tract in One man, since he grasps nothing, condemns the most learned writings of the wise ancients, calling them barbarous writings; another despises chymistry as false, and believes the books of Ripley vain dreams.32The Emperor, as patron and guardian of alchemists, of course knows better. Yet, Mai explains, he actually has two reasons for completing the translation:In his verse dedication, Mai complains that while his own age is not without learning, it struggles to regain the wisdom of earlier authorities:The first is that you understand the arts and are favourable to philosophers; you nurture artificers. The other is that Kelley, than whom no one is more excellent, ordered this work to be turned into Latin verse.33The Englishman’s influence is underscored by the inclusion of a distichon attributed to Kelley, inserted between Mai’s dedication and Ripley’s Prologue:Kellaeus lectoriCuicquid philosophum congesserat ordine Turba,patria Rypplaei carmine Lingva dedit:Haec eadem Maius, calamo facit esse Latino,Hinc notus Rypplai est, Notior ille magis. maius and magis (‘greater’), suggests that Kelley was able to trade on Ripley’s reputation as a means of forging or strengthening relationships with other adepts. Besides encouraging Mai to compose a more elegant verse translation of the Liber, Kelley also compiled a number of Ripleian works for presentation to a Silesian patron. These survive in a Latin manuscript, Książnica Cieszyńska SZ DD.vii.33: a compendium of Ripleian treatises transcribed in Prague between May and July 1592 by one ‘Jan Kapr.’34This artful verse, with its pun on the shared meaning of Medulla, he has added a note, indicating that the collection was compiled at Kelley’s behest:Edward Kelley wrote this book out of kindness and love for his most sincere friend, the noble lord Karl von Biberstein, 2 August in the year 1589: whom he wishes to have known as his adopted philosophical son, and to be esteemed above all other mortals.36Jan Kapr of Kaprštejn, also known as John Carpe or Johannes Carpio, was an administrator responsible for Rudolf’s vineyards.The recipient of this gift, the nobleman Karl von Biberstein, or Karl of Biberštejn (1528–1593), was a Silesian official and imperial councillor, who twice served as master of the Bohemian mint.Liber duodecim portarum , together with the Epistola ad Regem Eduardum (fols. 31v–37r), Liber de mercurio et lapide philosophorum , Clavis aureae portae (fols. 45r–62r), Medulla alchimiae (fols. 99r–119r), Pupilla alchimiae , and Philorcium alchimistarum (fols. 147r–165r). These are supplemented by other texts, including a treatise attributed to Ripley’s major authority, Raymond, and the Latin text of the Work of Dunstan, a theoretical and practical treatise pseudonymously ascribed to the tenth-century archbishop of Canterbury, but actually adapted from another work in the Ripley Corpus—the Accurtations of Raymond.38In adopting Biberstein as his ‘philosophical son’, Kelley assumed the role of his master and tutor, a relationship embodied in the transfer of alchemical knowledge. It is significant that the knowledge Kelley bestowed on his pupil centred on the work of his English predecessor, Ripley. SZ DD.vii.33 includes seven Ripleian works: the original Latin translation of the Besides its value as a storehouse of authoritative texts, the compendium thus drew attention to English expertise in the theory and practice of alchemy: a tradition to which Kelley was himself an heir. Ripley’s authority thus underwrote Kelley’s, while his assembled corpus offered a store of intellectual capital that could be disbursed as gifts and deployed as evidence of experience within a long established practical tradition. Yet the benefits of this diachronic relationship were not one-sided. Kelley’s interest in Ripley would have a lasting effect on the European reception of the medieval adept, as may be seen from the subsequent history of the Ripley corpus.Liber duodecim portarum, either the Emperor’s copy or Rožmberk’s, was borne away from Prague as booty by Swedish troops.o MS chem. 68, came to rest in the archives of the princely court of Hesse-Kassel, there to be noticed by Ludwig Combach, physician to the former landgrave, Moritz, and his successor Wilhelm. In the preface to the Opera, Combach expressed his regret at being unable to publish Mai’s version of Ripley’s poem, since the manuscript was not his own.41By 1649, Rudolf II and Moritz of Hesse-Kassel were long dead and their alchemical circles dispersed. The Thirty Years’ War had raged through Europe, and a presentation volume of Mai’s This manuscript, now 4° MS chem. 67, is a compendium of Ripleian texts, probably compiled in Prague some time after 1600, densely annotated and amended by several scribes. One of the annotating hands is that of Mai, while the compendium also includes one of Mai’s Latin verses, ‘Ænigma M. Nicolaii Maii’ (fol. 183r). Although Mai’s is not the principal hand, we would have to guess that the scribe, too, had some relationship with Kelley. The collection includes a recipe heard from Kelley’s own lips (‘Ex ore EK’); the ‘Deposition of Edward Kelley, Englishman’, dated Galway, 10 March 1593 (testifying to Kelley’s noble Irish lineage); and several extracts apparently taken from letters .42Opera, in his Tractatus aliquot , Dee’s name appears less frequently than that of Kelley in relation to alchemical receipts.47Within the circle of Kelley’s alchemical clients and correspondents, John Dee himself has so far played a surprisingly modest role. Although his Opuscule of Daniel Zeccaire for him out of French  is one of the works included in Ripley’s Opera . Unlike the widely travelled Liber duodecim portarum, it is possible to set a terminus post quem for this work’s appearance in East Central Europe, for the Viaticum includes several processes extracted from a manuscript that was only discovered in 1574, by the English alchemist Samuel Norton (1548–1621).The Key of alchemie, to Elizabeth I. Here he described his discovery of an old Latin commonplace book, containing Ripley’s personal notes and jottings—his daily ‘Bosome book’:hich havinge by profe found so many to be true, and little doubtinge of the accomplishment of the rest; I thought it but a point of dutie to reveall and vppen the Secrets heereof vnto your Highnes. Although it fortuned mee in manner vnloked for, to hitt vpon the secret bosome booke of Riple, wherby the true grounds are discovered, Of wOn 20 July 1577, Norton, son of the Somerset gentleman Sir George Norton, dedicated his alchemical treatise, the Bosome book into his own treatise, the Key.Norton says nothing of the provenance of this manuscript, which is apparently no longer extant. The Latin text of the original survives in only a single, later copy, now British Library MS Harley 2411. With the intention of presenting its contents to the Queen, however, Norton set about translating the collection of treatises, poems and practical recipes into English.Whole work of the stone philosophical  and the Practise by experience of the stone (pp. 19–25).Several copies of Norton’s English translation, dated February 1573 (i.e. 1574),Book were soon available in continental Europe as components of the Viaticum: a collection of short, practical tracts gleaned from various sources and gathered under sixteen subheadings. Of these, at least eight are items apparently extracted directly from the Book, as appears from comparison with the most complete surviving copy of the latter, MS Harley 2411. The Viaticum also shows evidence of revision, most obviously in ‘Oleum verò Solis fiet’ (‘Oil of the sun [i.e. gold] will truly be made’). Although framed as a single, practical process, this short text is actually a reworking of 31 of the Notable rules from Guido: a collection of 45 aphorisms supposedly gathered by Ripley from the works of Guido de Montanor, and a component of the Bosome book.Viaticum, ‘Elixir vitae’ and ‘Virtutes huius quintae essentiae hoc modo poterunt probari’ (‘The virtues of this quintessence may be proved in this way’) have been adapted from another Ripleian work, the Elixir vitae, that circulated widely in English in the later sixteenth century.Accurtations of Raymond. The Viaticum is therefore a compendium, prepared by a compiler who was clearly familiar with a variety of Ripleian works, including the original version of the recently discovered Bosome book.While Norton’s English translation seems to have circulated only in England, extracts from the original Latin Viaticum survive in English archives, the distribution of surviving copies suggests that the work may actually have been compiled in Bohemia. The earliest manuscript copies date from around the turn of the century, in the compilations of Nicolaus Mai (MS chem. 67) and Simon Budek (ÖNB Codex 11133). The presence of these excerpts in Prague within a few decades of Norton’s discovery merits some investigation, since their appearance of course postdates the arrival of Dee and Kelley in Třeboň.Although a few later manuscripts of the When our menstrual ‘mercury’ ascends from the sericon by the violence of the fire, a certain part of it is found cleaving to the side of the flask after the complete distillation and cooling of the glass, like salt and of crystalline appearance. And that crystalline earth is a fixed material, and apt to receive any form whatsoever. Let it therefore be gathered and kept. And the form of this earth is like mercury sublimed, and therefore shines brightly … This secret I learned through practice: G[eorge] R[ipley], as God is my witness.55Beneath, the scribe has sketched a small picture of a flask and receiver, depicting the crystalline residue as a ring around the inner circumference of the flask. A note adds, ‘So in a circle aboue the matter was the cleare matter lyke [mercury]’ . The picture is dated 1588, while additional information is supplied by a marginal note:A clue is provided by the copy in MS Harley 2411. This early seventeenth-century manuscript includes a recipe entitled ‘Magna philosophorum corrosiva’ (‘The great corrosive of the philosophers’). This begins with the distillation of vitriol with ‘salt of sericon’, resulting in a curious residue:I saw the same on 8 February 1588 (new style) in Trebon in Bohemia. From 2 lb. of sericon dissolved in distilled vinegar, and by means of spirit of wine cleansed of much sediment, came 4 oz. of red wine or oil. J:D. E:K.56These notes apparently describe a sericonian experiment carried out by Dee and Kelley in Třeboň, together with its results: the red oil and crystalline residue, complete with sketch. Since MS Harley 2411 dates from after 1588, it is likely that the scribe copied both recipes and annotations from a copy of the Bosome book in the possession of Dee or Kelley.Mr. E.K. at 9 of the clock after none sent for me to his laboratory over the gate: to se[e] how hee | distilled sericon, according as in tyme past & of late he hard [sic] of me out of Riplay. , as long as it is fixed.58This short passage draws on two axioms of Guido de Montanor.fumus albus), the stinking water (aza foetida), and the green lion (leo viridis). However, Kelley has modified his source by substituting ‘water of life’ for the ‘white fume.’ The second axiom, concerning the earth to be used, is another saying of Guido, in this case an exact quote.Accurtations of Raymond and the Notable rules from Guido. Kelley’s usage is slightly closer to the latter, suggesting that the model for his alchemical composition, as for his laboratory practice, was a component of Ripley’s Bosome book.Kelley’s personal interest in the Book with him in Třeboň, this must be considered a likely source for the extracts that began to appear in Bohemian manuscripts soon after, in the form of the Viaticum: a distillation of noteworthy processes from an authoritative source, in which we can discern echoes of Dee and Kelley’s own alchemical practice. This new compilation went on to acquire an afterlife of its own in manuscript and print. The reworked process ‘Oleum verò Solis fiet’, based on Guido’s axioms, may also owe its existence to Dee or Kelley, since MS Harley 2411 includes a copy towards the end of the Book, with the note ‘Revised’ . Grouped with other items taken from the Book and elsewhere in the Ripley Corpus, this process yielded the Viaticum: a ‘new’ Ripleian work with a predominantly continental circulation and an unambiguously practical flavour.If Dee had a copy of the Book’s dissemination. These included Edward Cradock, the Lady Margaret Professor of Divinity at the University of Oxford, who translated one item from the Book, the Somnium, in June 1582, a year before Dee’s departure abroad.Book was later commissioned by another friend of Dee, Gawin Smith, a prominent engineer in the service of both Elizabeth I and James I.iesties Engines’, had visited Dee for several days in Bremen in October 1589, and petitioned the queen on his behalf in July 1590  that accompanied him to Bohemia.Irrespective of whether or not Norton’s discovery was an authentic manuscript of George Ripley, these connections suggest that the newly discovered works of England’s master alchemist provoked keen interest among Elizabethan and Rudolfine Book, Dee’s copy has not survived, although its vestiges remain: in the diary record of Kelley’s practical experiments; in the copy in MS Harley 2411; in Kelley’s letter; and in Bohemian copies of the Viaticum. Such traces also hint at a vanished network of readers and practitioners, who circulated and transcribed new and authoritative works, translated them into or out of Latin, and even prepared them for presentation to princely patrons. One outcome of such correspondences was the publication of the otherwise obscure Viaticum: a work linked to a great English authority, translated by Norton for presentation to an English queen, harboured by Dee, and tested and revised by Edward Kelley.Like the original 5In March 1589, disheartened by his lack of fortune and under pressure by Rožmberk to leave Třeboň, Dee departed from Bohemia with his family, leaving Kelley in possession of the field. Over the previous few years, Dee’s preoccupation with the angelic conversations and their millenarian implications seems to have come increasingly into conflict with Kelley’s practical interest in transmutation—an interest more congenial to his patrons, Rožmberk and Rudolf II, than the specious pledges of the showstone. From 1587, Kelley’s attempts to extricate himself from his role as Dee’s scryer were paralleled by his own increasing success as an alchemist .Although Dee was unable to muster the same level of support for his own projects, he continued to make some contributions to his colleague’s practice. In an entry dated 28–29 October 1587, a few days after Kelley’s return from a visit to Prague, he noted that ‘Jo. Carp. did begyn to make furnaces over the gate &c.: & he vsed of | my rownd bricks’ . This poem was already available in manuscript: in Copenhagen, Royal Library GKS 242 (fol. 37v), one of a group of documents connected with the Kelley circle in Prague . Rabbards’ change of title to include ‘G.S. Gent.’ may perhaps suggest a link with Dee’s friend, ‘Mr Gawyn Smithe gentleman’: the royal projector and reader of the Bosome book.While Rabbards doubtless had his own skills in mind, another such practitioner was surely Edward Kelley, whose absent presence is recorded by the inclusion of a poem by ‘Sr. E. K. concerning the Philosophers Stone, written to his especiall good friend, G.S. Gent’ (ibid., sig. n Prague . This coCompound—by Rabbards, Penot, Barnaud and Combach—were not set in isolation, but lay enmeshed within webs of communication, authority and patronage: a pan-European network in which John Dee, practising alchemist and one of the great bibliophiles of Renaissance Europe, was an enthusiastic and influential participant. This network now survives only in fragmentary form: in friendly dedications, in marginal notes, and in the appearance of particular works in unexpected places. Such clues guide us to the routes by which Ripley’s poem attained a level of success that Dee, indifferently successful petitioner to a host of European monarchs, might well have envied: written for an English king, printed for an English queen, and translated for a Holy Roman Emperor.The host of coincidences surrounding the publication of Ripley’s works draws attention to the vigorous, scribal transmission of early modern alchemical texts.