Microbial rights?

Our ability to disrupt habitats and manipulate living organisms requires a discussion of the ethics of microbiology, even if we argue that microbes themselves have no rights.Synthetic biology and the increasing complexity of molecular biology have brought us to the stage at which we can synthesize new microorganisms. This has generated pressing questions about whether these new organisms have any place in our system of ethics and how we should treat them.The idea that microbes might have some moral claims on us beyond their practical uses or instrumental value is not a new question. Microbiologist Bernard Dixon (1976) presciently asked whether it was ethical to take the smallpox virus to extinction at the height of the attempts of the World Health Organization in the 1970s to eradicate it. There is no unambiguous answer. Today, we might still ask this question, but we might extend it to ask whether the destruction or extinction of a synthetic microbe that was made by humans is also ethically questionable or is such an entity—in that it is designed—more like a machine, which we have no compunction in terminating? Would two lethal pathogens, one of them synthetic and one of them natural, but otherwise identical, command the same moral claims?In a colloquial way, we might ask whether microbes have rights. In previous papers (Cockell, 2004) I have discussed the ‘rights'' of microbes and further explored some issues about the ethics we apply to them (Cockell, 2008). Julian Davies, in a recent opinion article in EMBO reports (Davies, 2010) described my assertion that they should have constitutional rights as ‘ridiculous''. Although I did suggest that environmental law could be changed to recognize the protection of microbial ecosystems—which would imply statutory rights or protection—nowhere have I claimed that microbes should have ‘constitutional'' rights. Nevertheless, this misattribution provides a useful demonstration of the confusion that exists about exactly how we should treat microbes.Few people are in any doubt that microbes should be conserved for their direct uses to humans, for example, in food and drug production, and their indirect uses such as the crucial role they have in the health of ecosystems. Indeed, these motivations can be used to prioritize microbial conservation and protection efforts (Cockell & Jones, 2009). The crucial question is whether microbes have ‘intrinsic value'' beyond their practical uses. If the answer is ‘no'', then we should have no guilt about deliberately driving microbes to extinction for our benefit. However, there are people who feel uneasy with this conclusion, a feeling that calls forth more complex ethical questions.The question is whether microbes have some sort of ‘interests'' that make demands on our treatment of them that go beyond a mere utilitarian calculation. These arguments themselves question what we define as ‘interests'' and whether interests make demands on us. A microbe has no future plans or thought processes; the sorts of interests that are accepted as being of sufficient scope to place demands on our treatment of other human beings, for instance. However, microbes do have biological interests. A halophilic microbe might eventually die if it is dropped into freshwater. Does our knowledge of what is in the biological interests of a microbe mean that we must show it any consideration beyond practical uses? The answer is not obviously negative (Taylor, 1981), but even if we decide that it is, this does not let us off the hook quite yet.There are other intrinsic value arguments that are more obscure, particularly those around the notion of ‘respect''; the idea that we should show empathy towards the trajectory, however deterministic, of other life forms. These unquantifiable and controversial arguments might, nevertheless, partly explain any unease that we have in watching a group of people smash up and destroy some exquisite microbial mats, just because they were bored.Clearly, human instrumental needs do trump microbes at some level. If they did not, we could not use bleach in our houses, an absurd end-point raised in a 1970s science fiction story that explored the futuristic ramifications of full microbial rights, in which household bleaches and deodorants are banned (Patrouch, 1977).However, we should not be so quick to ridicule ideas about microbial ethics and rights. Although it might be true that phages kill a large percentage of the bacterial population of the world every few days, as Julian Davies points out, human society has achieved an unprecedented capacity for destruction and creation. Our ability to poison and disrupt habitats has been unquantified, with respect to the loss of microbial species. Both synthetic biology and bioterrorism raise the spectre of creating new organisms, including pathogens, which we might need to control or deliberately pursue to extinction. Dixon''s dilemma about the smallpox virus, raised more than 30 years ago, has become an urgent point of discussion in the ethics of molecular biology and microbiology.     

The social structure of microbial community involved in colonization resistance

It is well established that host-associated microbial communities can interfere with the colonization and establishment of microbes of foreign origins, a phenomenon often referred to as bacterial interference or colonization resistance. However, due to the complexity of the indigenous microbiota, it has been extremely difficult to elucidate the community colonization resistance mechanisms and identify the bacterial species involved. In a recent study, we have established an in vitro mice oral microbial community (O-mix) and demonstrated its colonization resistance against an Escherichia coli strain of mice gut origin. In this study, we further analyzed the community structure of the O-mix by using a dilution/regrowth approach and identified the bacterial species involved in colonization resistance against E. coli. Our results revealed that, within the O-mix there were three different types of bacterial species forming unique social structure. They act as ‘Sensor'', ‘Mediator'' and ‘Killer'', respectively, and have coordinated roles in initiating the antagonistic action and preventing the integration of E. coli. The functional role of each identified bacterial species was further confirmed by E. coli-specific responsiveness of the synthetic communities composed of different combination of the identified players. The study reveals for the first time the sophisticated structural and functional organization of a colonization resistance pathway within a microbial community. Furthermore, our results emphasize the importance of ‘Facilitation'' or positive interactions in the development of community-level functions, such as colonization resistance.     

The secret garden's gardeners

The role of the microbial fauna in our gut for health and well-being is undisputed. Now, scientists are discovering that gut viruses also play a crucial role in modulating our risk for a wide range of diseases.Research has shown that the microbiota—the population of micro-organisms inhabiting the gut—has a profound influence on health in both humans and animals. However, most studies have largely ignored the viral population of the gut—the virome—although it is much larger, both in number of organisms and in genetic diversity. This is because the virome was thought to be less important for health and immunity, as it mainly comprises bacteriophages that only affect bacteria. However, researchers are beginning to realize that the viruses present might well be important in human health, as they manipulate the microbiota, swapping genetic virulence factors among bacteria, and through interaction with the host immune system.There are two distinct categories of virus in the gut: phages, which infect bacteria, and viruses that target host cells. Although these two categories are apparently independent of each other, there is a relationship between them, as indicated by growing evidence that the microbiota as a whole, including phages, has a crucial role in protecting against bacterial and viral infections [1].The phage and bacteria populations of the gut have an intricate relationship, which raises the potential therapeutic use of phages to treat a variety of conditions caused by bacteria in the gut, especially those involving chronic inflammation. The first step, however, is to explore and analyse the phage populations in the gut in terms of diversity and number, along with their interactions with their bacterial targets. This has proven to be a major challenge, given the enormous difficulties in identifying, isolating and amplifying genetic material from the phage population.Nevertheless, researchers from the Weizmann Institute of Science and Tel Aviv University in Israel have made substantial progress by indirectly identifying phages through clustered regularly interspaced short palindromic repeats (CRISPRs) in their bacterial hosts [2]. CRISPRs function as a prokaryotic adaptive immune system against genetic invaders such as phages by recognizing foreign DNA and then silencing its expression in a manner analogous to RNA interference (RNAi) in eukaryotes. Short segments of the foreign DNA, known as spacers, are incorporated into the bacterial genome to provide the memory of past exposures to enable recognition of phage DNA.The phage and bacteria populations of the gut have an intricate relationship, which raises the potential therapeutic use of phages to treat a variety of conditions…The Israeli study reconstructed the CRISPR bacterial immune system in the human gut microbiomes of 124 European individuals, and from that identified 991 phages targeted in at least one of the individuals. Of these phages, 78% were present in at least two individuals and some turned out to be the same ones that had already been identified in Japanese and American people. This global distribution of particular phages was a surprise, given that in other ecological niches, notably seawater, where phages are highly abundant, there is great genetic diversity among the populations, even over short distances.The Israeli team further succeeded in deducing the bacterial hosts of 130 of the phages, which allowed them to study the associated phage–bacteria interactions. It turned out that a subset of the phages had developed closer associations with their host bacteria as lysogenized prophages after fusing their DNA with the bacterial chromo-some or as plasmids. Rotem Sorek, a specialist in microbial warfare at the Weizmann Institute of Science and co-author of the Israel study, commented that this behaviour allows bacteria to take advantage of the phage by incorporating and transmitting genes that provide vital functions and occasionally aid pathogenesis. “There are clear instances of phages ‘helping'' pathogenic bacteria to attack humans,” Sorek said. “The toxins of the Cholera, Diphtheria and Shigella (disenteria) are all carried by phages that are integrated into the bacterial genome.”Horizontal gene transfer among bacteria has long been known to increase the adaptability of several potentially virulent bacterial species, but it is only recently that the mechanisms involving prophages have begun to be elucidated. A significant advance was made in a Japanese study from the University of Miyazaki, inspired by the observation that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes involved in virulence, and that these often seem to contain genetic defects [3]. The team analysed a virulent strain of Escherichia coli, known as O157, which contains 18 prophages that encode various genes involved in the production of virulence factors, including two potent cytotoxins: Shiga toxins 1 and 2. Most of the prophages they identified contained multiple genetic defects, yet they seemed to be capable of transporting virulence elements between not only members of the same strain but also different E. coli strains.The conclusion from their study was that defective prophages in close proximity within E. coli cells were still capable of recombining to yield a new phage that was released from the cell and could infect other cells nearby in the gut. It seems that these defective prophages were not just evolutionary leftovers, but were important components of the bacterial genome, conferring additional adaptive flexibility through horizontal gene transfer. Many other bacteria contain multiple prophages with genetic defects, so it is probable that this mechanism is not confined to E. coli.Other studies have focused on the composition of phage virus populations outside bacteria in the gut, as part of initiatives to compare and contrast the virome and bacteriome in response to individual genetic variation and environmental factors such as diet. One might imagine that phage and bacteria populations should be closely correlated, but it turns out that there are significant differences in the level of variation between individuals, as well as over time within the same individual. A study at the Washington University School of Medicine, USA, on monozygotic twins, found that in contrast to bacteriomes, viromes tended to be unique to individuals and less varied over time in response to changes in diet or other factors. By contrast, the bacterial population changed much more with diet and was also quite similar between twins.“There are clear instances of phages ‘helping'' pathogenic bacteria to attack humans”…Given that there is a direct relationship between the bacteria of the gut and the immune system of the individual—which is not the case for phage viruses—these findings make a degree of sense. Furthermore, as noted by Jeffrey Gordon, co-author of the Washington University study, there is not a one-to-one relationship between bacteria and the phages they host: “It''s been shown in other environments that you can have several different viruses capable of infecting the same bacterial host, while the specificity of each virus is usually quite narrow, typically extending only to a few strains within a species-level phylotype,” he explained. “This leads to a greater genetic diversity in the virome. Furthermore, a viral genome is enriched for genes involved in genome replication and virion assembly. Thus, the functional composition of the virome and the microbiome are quite different.”The situation is different for non-phage viruses in the gut that have a direct relationship with the human or animal host. The main research interest here is the three-way relationship between the virus, the bacteriome and the host''s immune system. Research on this front has already led to a new understanding of the role played by the entire microbiome in immunity. Often, the microbiome provides protection against viruses, but in some cases it can encourage their propagation. This is relevant in the context of human immunodeficiency virus (HIV), for example, given that the virus infects immune cells such as helper T cells, macrophages and dendritic cells, the activity and production of which in turn are related to the microbiota. An important question is whether the course of HIV and its possible development into symptomatic acquired immunodeficiency syndrome (AIDS) might be encouraged by the microbiota, if it stimulates production of such immune cells. Whilst this has yet to be established for AIDS, there is evidence that it is the case in monkeys carrying the related simian immunodeficiency virus (SIV), which infects at least 45 species of African primates. Unlike HIV in humans, SIV is usually non-pathogenic, as many primates evolved to coexist with the virus; but it does cause AIDS in rhesus macaque monkeys.Another Washington University School of Medicine study, this time looking at the link between viruses, the bacteriome and host immunity, began with the insight that animals developing AIDS experience immune hyperactivation, including higher levels of inflammatory chemokines, cytokines and activated T cells. This observation suggested that excessive inflammation is an important factor in progression to AIDS, presumably because it increases the number of cells vulnerable to HIV and SIV infection. The US team investigated whether there were any corresponding changes in the virome, finding that whilst it remains unchanged in uninfected animals, including rhesus macaque monkeys that had not succumbed to AIDS, its diversity increases significantly in infected macaques with full-blown AIDS [4].Often, the microbiome provides protection against viruses, but in some cases it can encourage their propagationThe team is following up by probing the relationship between enteric viruses and AIDS and how the immune system is stimulated. The animals suffer from progressive damage to their intestinal walls, which could increase the absorption of viruses that in turn stimulate the immune system, promoting replication of SIV and possibly encouraging more opportunistic viral infection, thereby creating a vicious circle. “This is one of several possible scenarios that we are actively investigating,” commented Scott Handley, lead author of the study. “What is uncertain is what is causing the damage to the intestinal wall. It could be the viruses which expand in the enteric virome during SIV infection, or some other factor, which could be immune-mediated or some other microbe or microbial product in the enteric microbiome.”Handley''s team has identified some of the viruses involved, including both common ones and some previously undiscovered. “Many of the viruses we identified have been associated with [gastrointestinal] disease in one form or another,” Handley said. “It is true that many of the viruses we identified are common; however, we identified at least 32 novel subtypes of these viruses never seen before.” He added that it remains to be established whether SIV infection encourages opportunistic infection by these viruses or not: “We don''t really have a good handle on what viruses would be considered ‘commensal'' viruses in any animal, including research primates. So whether they are already there when infection with SIV occurs, or are just more susceptible to opportunistic infection, is unclear.”Handley further commented that this work could lead to new therapeutic targets for treating HIV infection and preventing AIDS, and he is investigating whether the same expansion of the enteric virome occurs in humans infected with HIV. “In addition, we are interested to see if vaccination can reverse the enteric virome expansion,” he explained. “We would also like to better understand if the viruses we have identified were already circulating in these primates, or are succumbing to opportunistic infections.”Handley argued that his team''s work and that of others provides a compelling case for devoting more resources to studying the role of viruses in the gut, which would require further advances in laboratory and analytical techniques. “One challenge with studying the viral members of a microbiome is that there are no well-defined marker sequences,” he said. “Therefore, we are largely dependent on random shotgun sequencing approaches, which are less efficient and more expensive. Not only do you have to gather much more data, the computational analysis required is much more complex than the well-established techniques developed for studying the bacterial microbiome. While we know there is a great deal of interest in studying the virome, current techniques and technology tend to limit the number of labs that can participate in these efforts. We are very interested in developing new tools and techniques to help alleviate this issue.”An important question is whether the course of HIV […] might be encouraged by the microbiota, if it stimulates production of such immune cellsWork undertaken so far has already shown that probiotic or prebiotic treatments that provide or encourage beneficial gut bacteria can benefit patients infected with HIV. Improvements in intestinal health could reduce the leakage of all antigens, including viral ones, through the intestinal wall. More generally, a better understanding of how phages, viruses and bacteria in the gut interact could lead to new therapies that manipulate the microbiome to restore intestinal health in sufferers of a variety of conditions, including those involving chronic inflammation.     

A ‘metaphising’ dung beetle

Humans and beetles both have a species-specific Umwelt circumscribed by their sensory equipment. However, Ladislav Kováč argues that humans, unlike beetles, have invented scientific instruments that are able to reach beyond the conceptual borders of our Umwelt.You may have seen the film Microcosmos, produced in 1996 by the French biologists Claude Nuridsany and Marie Perrenou. It does not star humans, but much smaller creatures, mostly insects. The filmmakers'' magnifying camera transposes the viewer into the world of these organisms. For me, Microcosmos is not an ordinary naturalist documentary; it is an exercise in metaphysics.One sequence in the film shows a dung beetle—with the ‘philosophical'' generic name Sisyphus—rolling a ball of horse manure twice its size that becomes stuck on a twig. As the creature struggles to free the dung, it gives the impression that it is both worried and obstinate. As we humans know, the ball represents a most valuable treasure for the beetle: it will lay its eggs into the manure that will later feed its offspring. The behaviour of the beetle is biologically meaningful; it serves its Darwinian fitness.Yet, the dung beetle knows nothing of the function of manure, nor of the horse that dropped the excrement, nor of the human who owned the horse. Sisyphus lives in a world that is circumscribed by its somatic sensors—a species-specific world that the German biologist and philosopher Jakob von Uexküll would have called the dung beetle''s ‘Umwelt''. The horse, too, has its own Umwelt, as does the human. Yet, the world of the horse, just like the world of the man, does not exist for the beetle.If a ‘scholar'' among dung beetles attempted to visualize the world ‘out there'', what would be the dung-beetles'' metaphysics—their image of a part of the world about which they have no data furnished by their sensors? What would be their religions, their truths, or the Truth—revealed, and thus indisputable?Beetles are most successful animals; one animal in every four is a beetle, leading the biologist J.B.S. Haldane to quip that the Creator must have “had an inordinate fondness for beetles”. Are we humans so different from dung beetles? By birth we are similar: inter faeces et urinas nascimur—we are born between faeces and urine—as Aurelius Augustine remarked 1,600 years ago. Humans also have a species-specific Umwelt that has been shaped by biological evolution. A richer one than is the Umwelt of beetles, as we have more sensors than have they. Relative to the body size, we also possess a much larger brain and with it the capacity to make versatile movements with our hands and to finely manipulate with our fingers.This manual dexterity has enabled humans to fabricate artefacts that are, in a sense, extensions and refinements of the human hand. The simplest one, a coarse-chipped stone, represents the evolutionary origins of artefacts. Step-by-step, by a ratchet-like process, artefacts have become ever more complicated: as an example, a Boeing 777 is assembled from more than three million parts. At each step, humans have just added a tiny improvement to the previously achieved state. Over time, the evolution of artefacts has become less dependent on human intention and may soon result in artefacts with the capacity for self-improvement and self-reproduction. In fact, it is by artefacts that humans transcend their biology; artefacts make humans different from beetles. Here is the essence of the difference: humans roll their artefactual balls, no less worried and obstinate than beetles, but, in contrast to the latter, humans often do it even if the action is biologically meaningless, at the expense of their Darwinian fitness. Humans are biologically less rational than are beetles.Artefacts have immensely enriched the human Umwelt. From among them, scientific instruments should be singled out, as they function as novel, extrasomatic sensors of the human species. They have substantially fine-grained human knowledge of the Umwelt. But they are also reaching out—both to a distance and at a rate that is exponentially increasing—behind the boundary of the human Umwelt, behind its conceptual confines that we call Kant''s barriers. Into the world that has long been a subject of human ‘dung-beetle-like'' metaphysics. Nevertheless, our theories about this world could now be substantiated by data coming from the extrasomatic sensors. These instruments, fumbling in the unknown, supply reliable and reproducible data such that their messages must be true. They supersede our arbitrary guesses and fancies, but their truth seems to be out of our conceptual grasp. Conceptually, our mind confines us to our species-specific Umwelt.We continue to share the common fate of our fellow dung beetles: There is undeniably a world outside the confinements of our species-specific Umwelt, but if the world of humans is too complex for the neural ganglia of beetles, the world beyond Kant''s barriers may similarly exceed the capacity of the human brain. The physicist Richard Feynman (1965) stated, perhaps resignedly, “I can safely say that nobody today understands quantum mechanics.” Frank Gannon (2007) likewise commented that biological research, similarly to research in quantum mechanics, might be approaching a state “too complex to comprehend”. New models of the human brain itself may turn out to be “true and effective—and beyond comprehension” (Kováč, 2009).The advances of science notwithstanding, the knowledge of the universe that we have gained on the planet Earth might yet be in its infancy. However, in contrast to the limited capacity of humans, the continuing evolution of artefacts may mean that they face no limits in their explorative potential. They might soon dispense with our conceptual assistance exploring the realms that will remain closed to the human mind forever.     

By their genes ye shall know them: genomic signatures of predatory bacteria

Predatory bacteria are taxonomically disparate, exhibit diverse predatory strategies and are widely distributed in varied environments. To date, their predatory phenotypes cannot be discerned in genome sequence data thereby limiting our understanding of bacterial predation, and of its impact in nature. Here, we define the ‘predatome,'' that is, sets of protein families that reflect the phenotypes of predatory bacteria. The proteomes of all sequenced 11 predatory bacteria, including two de novo sequenced genomes, and 19 non-predatory bacteria from across the phylogenetic and ecological landscapes were compared. Protein families discriminating between the two groups were identified and quantified, demonstrating that differences in the proteomes of predatory and non-predatory bacteria are large and significant. This analysis allows predictions to be made, as we show by confirming from genome data an over-looked bacterial predator. The predatome exhibits deficiencies in riboflavin and amino acids biosynthesis, suggesting that predators obtain them from their prey. In contrast, these genomes are highly enriched in adhesins, proteases and particular metabolic proteins, used for binding to, processing and consuming prey, respectively. Strikingly, predators and non-predators differ in isoprenoid biosynthesis: predators use the mevalonate pathway, whereas non-predators, like almost all bacteria, use the DOXP pathway. By defining predatory signatures in bacterial genomes, the predatory potential they encode can be uncovered, filling an essential gap for measuring bacterial predation in nature. Moreover, we suggest that full-genome proteomic comparisons are applicable to other ecological interactions between microbes, and provide a convenient and rational tool for the functional classification of bacteria.     

Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus)

Bacterial gut symbiont communities are critical for the health of many insect species. However, little is known about how microbial communities vary among host species or how they respond to anthropogenic disturbances. Bacterial communities that differ in richness or composition may vary in their ability to provide nutrients or defenses. We used deep sequencing to investigate gut microbiota of three species in the genus Bombus (bumble bees). Bombus are among the most economically and ecologically important non-managed pollinators. Some species have experienced dramatic declines, probably due to pathogens and land-use change. We examined variation within and across bee species and between semi-natural and conventional agricultural habitats. We categorized as ‘core bacteria'' any operational taxonomic units (OTUs) with closest hits to sequences previously found exclusively or primarily in the guts of honey bees and bumble bees (genera Apis and Bombus). Microbial community composition differed among bee species. Richness, defined as number of bacterial OTUs, was highest for B. bimaculatus and B. impatiens. For B. bimaculatus, this was due to high richness of non-core bacteria. We found little effect of habitat on microbial communities. Richness of non-core bacteria was negatively associated with bacterial abundance in individual bees, possibly due to deeper sampling of non-core bacteria in bees with low populations of core bacteria. Infection by the gut parasite Crithidia was negatively associated with abundance of the core bacterium Gilliamella and positively associated with richness of non-core bacteria. Our results indicate that Bombus species have distinctive gut communities, and community-level variation is associated with pathogen infection.     

On the lack of evidence that non-human animals possess anything remotely resembling a 'theory of mind'

After decades of effort by some of our brightest human and non-human minds, there is still little consensus on whether or not non-human animals understand anything about the unobservable mental states of other animals or even what it would mean for a non-verbal animal to understand the concept of a ‘mental state’. In the present paper, we confront four related and contentious questions head-on: (i) What exactly would it mean for a non-verbal organism to have an ‘understanding’ or a ‘representation’ of another animal''s mental state? (ii) What should (and should not) count as compelling empirical evidence that a non-verbal cognitive agent has a system for understanding or forming representations about mental states in a functionally adaptive manner? (iii) Why have the kind of experimental protocols that are currently in vogue failed to produce compelling evidence that non-human animals possess anything even remotely resembling a theory of mind? (iv) What kind of experiments could, at least in principle, provide compelling evidence for such a system in a non-verbal organism?     

Cooperation beyond the dyad: on simple models and a complex society

Players in Axelrod and Hamilton''s model of cooperation were not only in a Prisoner''s Dilemma, but by definition, they were also trapped in a dyad. But animals are rarely so restricted and even the option to interact with third parties allows individuals to escape from the Prisoner''s Dilemma into a much more interesting and varied world of cooperation, from the apparently rare ‘parcelling’ to the widespread phenomenon of market effects. Our understanding of by-product mutualism, pseudo-reciprocity and the snowdrift game is also enriched by thinking ‘beyond the dyad’. The concepts of by-product mutualism and pseudo-reciprocity force us to think again about our basic definitions of cooperative behaviour (behaviour by a single individual) and cooperation (the outcome of an interaction between two or more individuals). Reciprocity is surprisingly rare outside of humans, even among large-brained ‘intelligent’ birds and mammals. Are humans unique in having extensive cooperative interactions among non-kin and an integrated cognitive system for mediating reciprocity? Perhaps, but our best chance for finding a similar phenomenon may be in delphinids, which also live in large societies with extensive cooperative interactions among non-relatives. A system of nested male alliances in bottlenose dolphins illustrates the potential and difficulties of finding a complex system of cooperation close to our own.     

Convergence,constraint and the potential for mutualism between ants and gut microbes

Ants are a hugely diverse family of eusocial insects that dominate terrestrial ecosystems all over the planet. Did mutualistic gut microbes help ants to achieve their diversity and ecological dominance? Initial studies suggested the potential for widespread convergence in ant gut bacterial communities based on dietary niche, but it now seems possible that dedicated bacterial symbionts are restricted to a minority of ant lineages (Russell et al., 2017). Nevertheless, as most ants are omnivores, the evidence so far has suggested a broad, positive correlation between the evolution of dietary specialization and ant investment in nutrient‐provisioning gut bacteria. In this issue of Molecular Ecology, Sapountzis et al. (2019) and Rubin et al. (2019) examine the evolution of gut bacterial communities in two iconic ant taxa—the attine fungus farmers and the Pseudomyrmex plant bodyguards, respectively—in a comparative context. By comparing gut bacteria between ant species of differing dietary specialization within each taxon, these studies demonstrate a hint of convergence in the midst of widespread apparent constraints. These results raise numerous interesting questions about the nature of these apparent constraints and whether they are causes or consequences of varying investment by ants to mutualism with their gut microbes.     

Complexity: The ultimate frontier?

Complexity remains a slippery concept for explaining living systems. Although the first cells and their descendants must have been much more complex than previously thought, this raises the question of whether there is an upper limit to the complexity of life.Murmur the word ‘complexity'' and it is a bit like tasting a fine wine: ‘forward oaty nose, thrilling undertones of blackberry, whiff of giraffe urine …''. Everybody knows what you are talking about, but it is all so very elusive. Oats? Giraffe pee? The history of life shouts ‘Look! Once there was bacteria, now there is New York'': thermogenic plants, Bombardier beetles, ballistic fungal spores. The biological world is not only fascinating, but dazzlingly complex, but how do we capture it? Take two pinches of Shannon, a dash of self-organization, sprinkle Kolmogorov liberally, stir with a fractal spoon, and serve immediately. In the computer such a recipe might work, but in the forests and oceans, concepts of complexity slip through our fingers.Perhaps we need to take a step back; if we can define some of the boundaries, then maybe our mathematical colleagues can step into the cage and pin the beast with their equations. Let''s begin by applying Conway Morris'' Fourth Law of Biology: whenever the word ‘surprising'' is used, be prepared to smell a rat. Such terminology is employed when we look at ancestral forms. Far from being slobberingly simple, such ancestors are ‘surprisingly'' complex. A striking example involves the earliest eukaryotes. In terms of gene complements crucial for subsequent multicellularity and bodyplan construction, such as SNAREs (Kloepper et al, 2007) and homeodomain TALE/non-TALEs (Derelle et al, 2007), the archaic eukaryote must have been, well, unexpectedly complex. Much the same can be inferred from other molecular machines, such as the kinesins (Wickstead et al, 2010). That such may be the norm, even the rule, is apparent from the nature of the first vertebrates (Heimberg et al, 2010). As Alysha Heimberg and co-workers remark, the first worm-like vertebrate “was a more complex organism than conventionally accepted”. To be sure, there are further elaborations, not least by the engine of gene duplication. But ancestral complexity is a non-trivial problem. In part, the solution must lie in co-options (and maybe horizontal gene transfer), but the suspicion remains that self-organization has a crucial role as each biological threshold is breached.But what happens when evolution runs in reverse, towards supposedly simpler forms? In fact, are they any less complex? In the world of bacteria, for example, think of either pathogens or those inhabiting the bacteriomes of sap-feeding insects. Convergence is the rule both in terms of their multiple origins and the striking reductions in genome size as innumerable operations, such as amino-acid synthesis, are passed to the host. These associations are not only extraordinarily intimate, indeed dangerously so with little charmers like rickettsialids, but also incredibly sophisticated. Can we make the argument that the degree of integration between bacteria and host defines another boundary in the world of complexity? That it does is suggested by another striking symbiosis. This involves the dicyemid metazoans and chromodinid ciliates, whose habitation on the surface of cephalopod kidneys not only represents an extraordinary convergence, but also, as I speculate, enhances kidney function to a near-vertebrate capacity.Perhaps there are not only boundary lines to complexity, but also a ceiling. Can we make the argument that evolution is running out of things to do? Sarah Adamowicz and colleagues (2008), for example, argue that in terms of tagmosis in crustaceans—whereby the segmental series of more or less identical limbs are transformed into a linear array of highly specialized units (such as in the lobster)—not only is this a trend that has evolved several times, but as a group the crustaceans are also approaching the limits of possible tagmosis. Fascinatingly, they discuss whether this evolutionary journey is incomplete, or whether other factors will prevent crustaceans reaching the final limit of complexity.Perhaps the latter, if Conway Morris'' Seventh Law of Biology holds: that all systems evolve to the most complex of possible forms. Nowhere is this more hauntingly evident than in the evolution of nervous systems. First, such configurations are prodigiously expensive to run. As Simon Laughlin et al (1998) point out, to transport a single bit of information across a synapse requires a staggering 10⁴ ATP molecules. The retina of the blow-fly is consuming a jaw-dropping 8% of total metabolic energy, even at rest. Nervous systems, therefore, are masters of economy; not surprisingly, strategies that would be the envy of any Green Officer have evolved to attempt to circumvent the energetic penalties. Yet there are other limits to the complexity of any nervous system. Michel Hofman (2001), for example, has shown that because of the contrasting allometries of grey and white matter in anthropoid brains, there is an upper limit to their size. At about three times our current size we might be mistaken for extra-terrestrials, but even this limit might be forever beyond reach, on account of the challenges of connecting such a massive piece of neurological machinery.Here lies an irony. Does this ceiling constrain our very powers of thought? Are there neurological limits to what we can understand; are there things ‘out there'' that are literally beyond our comprehension? Or are you willing to subscribe to Conway Morris'' Ninth Law of Biology, that paradoxically states: there is no limit to our understanding or knowledge? And if you do, then what does that suggest about the nature of the Universe in which we are embedded?     

Cellular promiscuity: explaining cellular fidelity in vivo against unrestrained pluripotency in vitro

The differentiation of pluripotent stem cells into various progeny is perplexing. In vivo, nature imposes strict fate constraints. In vitro, PSCs differentiate into almost any phenotype. Might the concept of ‘cellular promiscuity'' explain these surprising behaviours?John Gurdon''s [1] and Shinya Yamanaka''s [2] Nobel Prize involves discoveries that vex fundamental concepts about the stability of cellular identity [3,4], ageing as a rectified path and the differences between germ cells and somatic cells. The differentiation of pluripotent stem cells (PSCs) into progeny, including spermatids [5] and oocytes [6], is perplexing. In vivo, nature imposes strict fate constraints. Yet in vitro, reprogrammed PSCs liberated from the body government freely differentiate into any phenotype—except placenta—violating even somatic cell against germ cell segregations. Albeit that it is anthropomorphic, might the concept of ‘cellular promiscuity'' explain these surprising behaviours?Fidelity to one''s differentiated state is nearly universal in vivo—even cancers retain some allegiance. Appreciating the mechanisms in vitro that liberate reprogrammed cells from the numerous constraints governing development in vivo might provide new insights. Similarly to highway guiderails, a range of constraints preclude progeny cells within embryos and organisms from travelling too far away from the trajectory set by their ancestors. Restrictions are imposed externally—basement membranes and intercellular adhesions; internally—chromatin, cytoskeleton, endomembranes and mitochondria; and temporally by ageing.‘Cellular promiscuity'' was glimpsed previously during cloning; it was seen when somatic cells successfully ‘fertilized'' enucleated oocytes in amphibians [1] and later with ‘Dolly'' [7]. Embryonic stem cells (ESCs) corroborate this. The inner cell mass of the blastocyst cells develops faithfully, but liberation from the trophoectoderm generates pluripotent ESCs in vitro, which are freed from fate and polarity restrictions. These freedom-seeking ESCs still abide by three-dimensional rules as they conform to chimaera body patterning when injected into blastocysts. Yet if transplanted elsewhere, this results in chaotic teratomas or helter-skelter in vitro differentiation—that is, pluripotency.August Weismann''s germ plasm theory, 130 years ago, recognized that gametes produce somatic cells, never the reverse. Primordial germ cell migrations into fetal gonads, and parent-of-origin imprints, explain how germ cells are sequestered, retaining genomic and epigenomic purity. Left uncontaminated, these future gametes are held in pristine form to parent the next generation. However, the cracks separating germ and somatic lineages in vitro are widening [5,6]. Perhaps, they are restrained within gonads not for their purity but to prevent wild, uncontrolled misbehaviours resulting in germ cell tumours.The ‘cellular promiscuity'' concept regarding PSCs in vitro might explain why cells of nearly any desired lineage can be detected using monospecific markers. Are assays so sensitive that rare cells can be detected in heterogeneous cultures? Certainly population heterogeneity is considered for transplantable cells—dopaminergic neurons and islet cells—compared with applications needing few cells—sperm and oocytes. This dilemma of maintaining cellular identity in vitro after reprogramming is significant. If not addressed, the value of unrestrained induced PSCs (iPSCs) as reliable models for ‘diseases in a dish'', let alone for subsequent therapeutic transplantations, might be diminished. X-chromosome re-inactivation variants in differentiating human PSCs, epigenetic imprint errors and copy number variations are all indicators of in vitro infidelity. PSCs, which are held to be undifferentiated cells, are artefacts after all, as they undergo their programmed development in vivo.If correct, the hypothesis accounts for concerns raised about the inherent genomic and epigenomic unreliability of iPSCs; they are likely to be unfaithful to their in vivo differentiation trajectories due to both the freedom from in vivo developmental programmes, as well as poorly characterized modifications in culture conditions. ‘Memory'' of the PSC''s identity in vivo might need to be improved by using approaches that might not fully erase imprints. Regulatory authorities, including the Food & Drug Administration, require evidence that cultured PSCs do retain their original cellular identity. Notwithstanding fidelity lapses at the organismal level, the recognition that our cells have intrinsic freedom-loving tendencies in vitro might generate better approaches for only partly releasing somatic cells into probation, rather than full emancipation.     

Evidence for polymicrobic flora translocating in peripheral blood of HIV-infected patients with poor immune response to antiretroviral therapy

In advanced HIV infection, the homeostatic balance between gastrointestinal indigenous bacteria and gut immunity fails and microbes are able to overcome the intestinal barrier and gain the systemic circulation. Because microbial translocation is not fully controlled by antiviral therapy and is associated with inefficient CD4+ reconstitution, we investigated the profile of translocating bacteria in peripheral blood of 44 HIV-infected patients starting therapy with advanced CD4+ T-lymphopenia and displaying poor CD4+ recovery on virologically suppressive HAART. According to CD4+ reconstitution at 12-months HAART, patients were considered Partial Immunological Responders, PIRs (CD4+≥250/µl, n = 29) and Immunological non Responders, INRs (CD4+<200/µl, n = 15)). We show that PIRs and INRs present similarly elevated plasma levels of lipopolysaccharide (LPS) and its ligand sCD14 that were not lowered by virologically suppressive therapy. Bacterial 16S rRNA gene amplification and sequencing resulted in a highly polymicrobic peripheral blood microbiota both prior and after 12-month HAART. Several differences in bacterial composition were shown between patients'' groups, mainly the lack of probiotic Lactobacillaceae both prior and after therapy in INRs. Failure to control microbial translocation on HAART is associated with a polymicrobic flora circulating in peripheral blood that is not substantially modified by therapy.     

Positive epistasis between co-infecting plasmids promotes plasmid survival in bacterial populations

Plasmids have a key role in the horizontal transfer of genes among bacteria. Although plasmids are catalysts for bacterial evolution, it is challenging to understand how they can persist in bacterial populations over the long term because of the burden they impose on their hosts (the ‘plasmid paradox''). This paradox is especially perplexing in the case of ‘small'' plasmids, which are unable to self-transfer by conjugation. Here, for the first time, we investigate how interactions between co-infecting plasmids influence plasmid persistence. Using an experimental model system based on interactions between a diverse assemblage of ‘large'' plasmids and a single small plasmid, pNI105, in the pathogenic bacterium Pseudomonas aeruginosa, we demonstrate that positive epistasis minimizes the cost associated with carrying multiple plasmids over the short term and increases the stability of the small plasmid over a longer time scale. In support of these experimental data, bioinformatic analysis showed that associations between small and large plasmids are more common than would be expected owing to chance alone across a range of families of bacteria; more generally, we find that co-infection with multiple plasmids is more common than would be expected owing to chance across a wide range of bacterial phyla. Collectively, these results suggest that positive epistasis promotes plasmid stability in bacterial populations. These findings pave the way for future mechanistic studies aimed at elucidating the molecular mechanisms of plasmid–plasmid interaction, and evolutionary studies aimed at understanding how the coevolution of plasmids drives the spread of plasmid-encoded traits.     

The interplay of cognition and cooperation

Cooperation often involves behaviours that reduce immediate payoffs for actors. Delayed benefits have often been argued to pose problems for the evolution of cooperation because learning such contingencies may be difficult as partners may cheat in return. Therefore, the ability to achieve stable cooperation has often been linked to a species'' cognitive abilities, which is in turn linked to the evolution of increasingly complex central nervous systems. However, in their famous 1981 paper, Axelrod and Hamilton stated that in principle even bacteria could play a tit-for-tat strategy in an iterated Prisoner''s Dilemma. While to our knowledge this has not been documented, interspecific mutualisms are present in bacteria, plants and fungi. Moreover, many species which have evolved large brains in complex social environments lack convincing evidence in favour of reciprocity. What conditions must be fulfilled so that organisms with little to no brainpower, including plants and single-celled organisms, can, on average, gain benefits from interactions with partner species? On the other hand, what conditions favour the evolution of large brains and flexible behaviour, which includes the use of misinformation and so on? These questions are critical, as they begin to address why cognitive complexity would emerge when ‘simple’ cooperation is clearly sufficient in some cases. This paper spans the literature from bacteria to humans in our search for the key variables that link cooperation and deception to cognition.     

Motivated research

While Europe is locked in the debate about basic versus applied research, Louis Pasteur solved the problem more than 100 years ago. Antoine Danchin comments on Pasteur''s notion of ‘motivated research'' and how it leads both to new discoveries and to new applications.Three years ago, a senior politician attended his country''s Annual Congress for the Advancement of Science to give the introductory lecture. He asked the attending scientists to make science and research more attractive to young students and the general public, and asked his countrymen to support scientists to address the urgent challenges of global climate change, energy needs and dwindling water resources. It was neither a European nor a US politician, but the Indian Prime Minister Manmohan Singh who made this speech about the relationship between research and its practical applications. This is such an important topic that one might think it deserves appropriate attention in Europe, yet we fail to address it properly. Instead, we just discuss how science should serve society or contribute to the ‘knowledge-based economy'', or how ‘basic'' or ‘fundamental'' research is opposed to ‘applied'' or ‘industrial'' research and how funding for ‘big science'' comes at the expense of ‘little academic'' research.This dichotomy between the research to generate knowledge and the application of that knowledge to benefit humankind seems to be a recent development. In fact, more than 100 years ago Louis Pasteur avoided this debate altogether: one of his major, yet forgotten, contributions to science was the insight that research and its applications are not opposed, but orthogonal to each other (Stokes, 1997). If Niels Bohr ‘invented'' basic academic research—which was nevertheless the basis for many technological inventions and industrial applications—Pasteur developed what we might call ‘motivated'' research.How is research motivated and by what? By definition, scientists are citizens and members of the general public and, like the public, they are motivated by two forces: on the one hand, in Rudyard Kipling''s words, “man''s insatiable curiosity”; on the other hand, a desire for maintaining and improving their well-being. These are not contradictory to one another; curiosity nourishes dreams of a brighter future and leads to discoveries that contribute to well-being.Pasteur understood that it is essential to take account of society''s demands and desires; that science must be motivated by what people want. Still, there are severe misgivings about the nature of research. These stem from the mistaken but popular assumption that the scientists'' main task is to find solutions to current problems or to fulfil our desires. Problems and desires, however, are not enough, because finding solutions also requires creativity and discovery, which, by their very nature, are unpredictable. Often we do not even know what we need or desire and it is only through curiosity and more knowledge that we find new ways to improve our well-being. Motivation by itself is, therefore, not enough to lead to discovery. Motivation simply helps us choose between many different goals and an infinite number of paths to gain novel knowledge. Subsequently, each path, once chosen, must be explored using the scientific method, which is the only way to new discoveries.Motivation helps us to ask relevant questions. For example, why do wine and beer go sour without any apparent reason? Pasteur set out to design experiments that showed that fermentation is caused by microorganisms. A few years later, silkworms were suddenly dying of a terrible disease in the silk factories of southern France. The French government called on Pasteur for help, who eventually found that a parasite had infected silkworm eggs and proposed solutions to eradicate the disease. The original question therefore led to germ theory and bacteriology, helped to develop solutions to infectious diseases, and eventually created the whole field of microbiology.Motivation leads to conceptual and experimental research, which generates discoveries and new technologies. Discoveries, in turn, are the basic resource for the creation of general knowledge and the development of new products, services and other goods that fulfil public demands and generate jobs. The study of the ‘diseases'' of beer and wine also led to the development of fermentation processes that are still in use today. The same motivation that drove Pasteur in the nineteenth century now enables us to tackle current problems, such as pollution, by studying microbial communities that make compost or thrive in garbage dumps. Motivated research therefore reconciles our curiosity with the creation of knowledge and enables us to address pressing needs for humanity.Because it is strongly inspired by—even rooted in—society''s demands and desires, motivated research also raises accompanying ethical, legal, social and safety issues that should be compelling for all research. As mentioned above, scientists are members of the public who share the same concerns and demands as their fellow citizens and therefore participate with a general, public intelligence that, too often, is absent from academic research. This absence of ‘common sense'' or societal expectations generates the misunderstandings concerning research in biology and the development of biotechnology. These misconceptions—whether about the purported risks of genetically modified organisms or the exaggerated expectations for cancer therapies—can create real suffering in society and inefficient allocation of limited resources. It is therefore advisable for researchers to listen more to the public at large in order to find the motivation for their work.     

微生物组学研究的“淘金时代”

人体及动物肠道中生存着数量庞大的共生微生物;这些微生物无时无刻不参与着宿主的生命活动。揭示这些共生微生物在宿主体内的变化规律、与宿主之间的依存和博弈关系等,将使人类更加全面的认知高等生物体的生命本质。本专刊从肠道微生物与疾病、肠道微生物群落结构、肠道微生物与宿主互作、肠道微生物资源和肠道微生物研究方法 5个层面展示了我国科研工作者在肠道微生物研究领域的新进展及新观点。     

Domestic Dogs Use Contextual Information and Tone of Voice when following a Human Pointing Gesture

Domestic dogs are skillful at using the human pointing gesture. In this study we investigated whether dogs take contextual information into account when following pointing gestures, specifically, whether they follow human pointing gestures more readily in the context in which food has been found previously. Also varied was the human''s tone of voice as either imperative or informative. Dogs were more sustained in their searching behavior in the ‘context’ condition as opposed to the ‘no context’ condition, suggesting that they do not simply follow a pointing gesture blindly but use previously acquired contextual information to inform their interpretation of that pointing gesture.Dogs also showed more sustained searching behavior when there was pointing than when there was not, suggesting that they expect to find a referent when they see a human point. Finally, dogs searched more in high-pitched informative trials as opposed to the low-pitched imperative trials, whereas in the latter dogs seemed more inclined to respond by sitting. These findings suggest that a dog''s response to a pointing gesture is flexible and depends on the context as well as the human''s tone of voice.     

Not too much and not too little

We tend to think in black and white terms of good versus bad alleles and their meaning for disease. However, in doing so, we ignore the potential importance of heterozygous alleles.The structure and function of any protein is determined by its amino acid sequence. Thus, the substitution of one amino acid for another can alter the activity of a protein or its function. Mutations—or rather, polymorphism, once they become fixed in the population—can be deleterious, such that the altered protein is no longer able to fulfil its role with potentially devastating effects on the cell. Rarely, they can improve protein function and cell performance. In either case, any changes in the amino acid sequence, whether they affect only one amino acid or larger parts of the protein, are encoded by polymorphisms in the nucleotide sequence of that protein''s gene. For any given polymorphism, diploid organisms with two sets of chromosomes can therefore exist in either a heterozygous state or one of two homozygous states. When the polymorphism is rare, most individuals are homozygous for the ‘wild-type'' state, some individuals are heterozygous and a few are homozygous for the rare polymorphic variant. Conversely, if the polymorphism occurs in 50% of the alleles, the heterozygous state is common.At first glance, the deleterious homozygous state seems to be something that organisms try to avoid: close relatives usually do not breed, probably to prevent the homozygous accumulation of deleterious alleles. Thus, human cultural norms, founded in our biology, actively select for heterozygosity as many civilizations and societies regard incest as a social taboo. The fields of animal husbandry and conservation biology are littered with information about the significant positive correlation between genetic diversity, evolutionary advantage and fitness [1]. In sexually reproducing organisms, heterozygosity is generally regarded as ‘better'' in terms of adaptability and evolutionary advantage.Why then do we seldom, if ever, regard allelic heterozygosity as an advantage when it comes to genes linked with health and disease? Perhaps it is because we tend to distinguish between the ‘good'' allele, the ‘bad'' allele and the ‘ugly'' heterozygote—since it is burdened with one ‘bad'' allele. Maybe this attitude is a remnant of the outdated ‘one gene, one disease'' model, or of the early studies on inheritable diseases that focused on monogenic or autosomal-dominant genetic disorders. Even modern genetics almost always assigns ‘risk'' to an allele that is associated with a health condition or disadvantaged phenotype; clearly, then, the one homozygous state must have an advantage—sometimes referred to as wild-type—but the heterozygote is often ignored altogether.Maybe we also shun heterozygosity because it is hard to prove, beyond a few examples, that it might offer advantage. A 2010 paper published in Cell claimed that heterozygosity of the lth4A locus conveys protection against tuberculosis [2]. There is a mechanistic basis for the claim: lth4A encodes leukotriene A4 hydrolase, which is the final catalyst to synthesize leukotriene B4, an efficient pro-inflammatory eicosanoid. However, an extensive case–control study could not confirm the association between heterozygosity and protection against tuberculosis [3]. Therefore, many in the field dismiss the prior claim to protection conferred by the heterozygous state.Yet, we know that most biochemical and physiological processes are highly complex systems that involve multiple, interlinked steps with extensive control and feedback mechanisms. Heterozygosity might be one strategy by which an organism maintains flexibility, as it provides more than one allele to fall back on, should conditions change. We may therefore hypothesize that heterozygosity can be either a risk or an advantage, depending on the penetrance or dominance of the alleles. Indeed, there are a few cases in which heterozygosity confers some advantage. For example, individuals who are homozygous for the CCR5 deletion polymorphism (D32/D32) are protected against HIV1 infection, whereas CCR5/D32 heterozygotes have a slower progression to acquired immunodeficiency syndrome (AIDS). In sickle-cell anaemia, heterozygotes have a protective advantage against malaria, whereas the homozygotes either lack protection or suffer health consequences. Thus, although heterozygosity might not create a general fitness advantage, it is advantageous under certain specific conditions, namely the presence of the malaria parasite.In most aspects of life, there are few absolutes and many shades of grey. The ‘normal'' range of parameters in medicine is a clear example of this: optimal functioning of the relevant physiological processes depends on levels that are ‘just right''. As molecular and genetic research tackles the causes and risk factors of complex diseases, we may perhaps find more examples of how heterozygosity at the genetic level conveys health advantages in humans. As the above example regarding tuberculosis indicates, it is difficult to demonstrate any advantage of the heterozygous state. We simply need to be receptive to such possibilities, and improve and reconcile our understanding of allelic diversity and heterozygosity. Researchers working on human disease could benefit from the insights of evolutionary biologists and breeders, who are more appreciative of the heterozygous state.     

How many scientists does it take to change a paradigm?: New ideas to explain scientific observations are everywhere—we just need to learn how to see them

The scientific process requires a critical attitude towards existing hypotheses and obvious explanations. Teaching this mindset to students is both important and challenging.People who read about scientific discoveries might get the misleading impression that scientific research produces a few rare breakthroughs—once or twice per century—and a large body of ‘merely incremental'' studies. In reality, however, breakthrough discoveries are reported on a weekly basis, and one can cite many fields just in biology—brain imaging, non-coding RNAs and stem cell biology, to name a few—that have undergone paradigm shifts within the past decade.The truly surprising thing about discovery is not just that it happens at a regular pace, but that most significant discoveries occurred only after the scientific community had already accepted another explanation. It is not merely the accrual of new data that leads to a breakthrough, but a willingness to acknowledge that a problem that is already ‘solved'' might require an entirely different explanation. In the case of breakthroughs or paradigm shifts, this new explanation might seem far-fetched or nonsensical and not even worthy of serious consideration. It is as if new ideas are sitting right in front of everyone, but in their blind spots so that only those who use their peripheral vision can see them.Scientists do not all share any single method or way of working. Yet they tend to share certain prevalent attitudes: they accept ‘facts'' and ‘obvious'' explanations only provisionally, at arm''s length, as it were; they not only imagine alternatives, but—almost as a reflex—ask themselves what alternative explanations are possible.When teaching students, it is a challenge to convey this critical attitude towards seemingly obvious explanations. In the spring semester of 2009, I offered a seminar entitled The Process of Scientific Discovery to Honours undergraduate students at the University of Illinois-Chicago in the USA. I originally planned to cover aspects of discovery such as the impact of funding agencies, the importance of mentoring and hypothesis-driven as opposed to data-driven research. As the semester progressed, however, my sessions moved towards ‘teaching moments'' drawn from everyday life, which forced the students to look at familiar things in unfamiliar ways. These served as metaphors for certain aspects of the process by which scientists discover new paradigms.For the first seven weeks of the spring semester, the class read Everyday Practice of Science by Frederick Grinnell [1]. During the discussion of the first chapter, one of the students noted that Grinnell referred to a scientist generically as ‘she'' rather than ‘he'' or the neutral ‘he or she''. This use is unusual and made her vaguely uneasy: she wondered whether the author was making a sexist point. Before considering her hypothesis, I asked the class to make a list of assumptions that they took for granted when reading the chapter, together with the possible explanations for the use of ‘she'' in the first chapter, no matter how far-fetched or unlikely they might seem.For example, one might assume that Frederick Grinnell or ‘Fred'' is from a culture similar to our own. How would we interpret his behaviour and outlook if we knew that Fred came from an exotic foreign land? Another assumption is that Fred is male; how would we view the remark if we discover that Frederick is short for Fredericka? We have equally assumed that Fred, as with most humans, wants us to like him. Instead, perhaps he is being intentionally provocative in order to get our attention or move us out of our comfort zone. Perhaps he planted ‘she'' as a deliberate example for us to discuss, as he does later in the second chapter, in which he deliberately hides a strange item in plain sight within one of the illustrations in order to make a point about observing anomalies. Perhaps the book was written not by Fred but by a ghost writer? Perhaps the ‘she'' was a typo?The truly surprising thing about discovery is […] that most significant discoveries occurred only after the scientific community had already accepted another explanationLooking for patterns throughout the book, and in Fred''s other writing, might persuade us to discard some of the possible explanations: does ‘she'' appear just once? Does Fred use other unusual or provocative turns of phrase? Does Fred discuss gender bias or sexism explicitly? Has anyone written or complained about him? Of course, one could ask Fred directly what he meant, although without knowing him personally, it would be difficult to know how to interpret his answer or whether to take his remarks at face value. Notwithstanding the answer, the exercise is an important lesson about considering and weighing all possible explanations.Arguably, the most prominent term used in science studies is the notion of a ‘paradigm''. I use this term with reluctance, as it is extraordinarily ambiguous. For example, it could simply refer to a specific type of experimental design: a randomized, placebo-controlled clinical trial could be considered a paradigm. In the context of science studies, however, it most often refers to the idea of large-scale leaps in scientific world views, as promoted by Thomas Kuhn in The Structure of Scientific Revolutions [2]. Kuhn''s notion of a paradigm can lead one to believe—erroneously in my opinion—that paradigm shifts are the opposite of practical, everyday scientific problem-solving.A paradigm is recognized by the set of assumptions that an observer might not realize he or she is making…Instead, I propose here a definition of ‘paradigm'' that emphasizes not the nature of the problem, the type of discovery or the scope of its implications, but rather the psychology of the scientist. A scientist viewing a problem or phenomenon resides within a paradigm when he or she does not notice, and cannot imagine, that an alternative way of looking at things needs to be considered seriously. Importantly, a paradigm is not a viewpoint, model, interpretation, hypothesis or conclusion. A paradigm is not the object that is viewed but the lenses through which it is viewed. A paradigm is recognized by the set of assumptions that an observer might not realize he or she is making, but which imply many automatic expectations and simultaneously prevent the observer from seeing the issue in any other fashion.For example, the teacher–student paradigm feels natural and obvious, yet it is merely set up by habit and tradition. It implies lectures, assignments, grades, ways of addressing the professor and so on, all of which could be done differently, if we had merely thought to consider alternatives. What feels most natural in a paradigm is often the most arbitrary. When we have a birthday, we expect to have a cake with candles, yet there is no natural relationship at all between birthdays, cakes and candles. In fact, when something is arbitrary or conventional yet feels entirely natural, that is an important clue that a paradigm is present.It is certainly natural for people to colour their observations according to their expectations: “To a man with a hammer, everything looks like a nail,” as Mark Twain put it. However, this is a pitfall that scientists (and doctors) must try hard to avoid. When I was a first-year medical student at Albert Einstein College of Medicine in New York City, we took a class on how to approach patients. As part of this course, we attended a session in which a psychiatrist interviewed a ‘normal, healthy old person'' in order to understand better the lives and perspectives of the elderly.A man came in, and the psychiatrist began to ask him some benign questions. After about 10 minutes, however, the man began to pause before answering; then his answers became terse; then he said he did not feel well, excused himself and abruptly left the room. The psychiatrist continued to lecture to the students for another half-hour, analysing and interpreting the halting responses in terms of the emotional conflicts that the man was experiencing. ‘Repression'', ‘emotional blocks'', and ‘reaction formation'' were some of the terms bandied about.However, unbeknown to the class, the man had collapsed just on the other side of the classroom door. Two cardiologists happened to be walking by and instantly realized the man was having an acute heart attack. They instituted CPR on the spot, but the man died within a few minutes.The psychiatrist had been told that the man was healthy, and thus interpreted everything that he saw in psychological terms. It never entered his mind that the man might have been dying in front of his eyes. The cardiologists saw a man having a heart attack, and it never entered their minds that the man might have had psychological issues.The movie The Sixth Sense [3] resonated particularly well with my students and served as a platform for discussing attitudes that are helpful for scientific investigation, such as “keep an open mind”, “reality is much stranger than you can imagine” and “our conclusions are always provisional at best”. Best of all, The Sixth Sense demonstrates the tension that exists between different scientific paradigms in a clear and beautiful way. When Haley Joel Osment says, “I see dead people,” does he actually see ghosts? Or is he hallucinating?…when scientists reach a conclusion, it is merely a place to pause and rest for a moment, not a final destinationIt is important to emphasize that these are not merely different viewpoints, or different ways of defining terms. If we argued about which mountain is higher, Everest or K2, we might disagree about which kind of evidence is more reliable, but we would fundamentally agree on the notion of measurement. By contrast, in The Sixth Sense, the same evidence used by one paradigm to support its assertion is used with equal strength by the other paradigm as evidence in its favour. In the movie, Bruce Willis plays a psychologist who assumes that Osment must be a troubled youth. However, the fact that he says he sees ghosts is also evidence in favour of the existence of ghosts, if you do not reject out of hand the possibility of their existence. These two explanations are incommensurate. One cannot simply weigh all of the evidence because each side rejects the type of evidence that the other side accepts, and regards the alternative explanation not merely as wrong but as ridiculous or nonsensical. It is in this sense that a paradigm represents a failure of imagination—each side cannot imagine that the other explanation could possibly be true, or at least, plausible enough to warrant serious consideration.The failure of imagination means that each side fails to notice or to seek ‘objective'' evidence that would favour one explanation over the other. For example, during the episodes when Osment saw ghosts, the thermostat in the room fell precipitously and he could see his own breath. This certainly would seem to constitute objective evidence to favour the ghost explanation, and the fact that his mother had noticed that the heating in her apartment was erratic suggests that the temperature change was not simply another imagined symptom. But the mother assumed that the problem was in the heating system and did not even conceive that this might be linked to ghosts—so the ‘objective'' evidence certainly was not compelling or even suggestive on its own.Osment did succeed eventually in convincing his mother that he saw ghosts, and he did it in the same way that any scientist would convince his colleagues: namely, he produced evidence that made perfect sense in the context of one, and only one, explanation. First, he told his mother a secret that he said her dead mother had told him. This secret was about an incident that had occurred before he was born, and presumably she had never spoken of it, so there was no obvious way that he could have learned about it. Next, he told her that the grandmother had heard her say “every day” when standing near her grave. Again, the mother had presumably visited the grave alone and had not told anyone about the visit or about what was said. So, the mother was eventually convinced that Osment must have spoken with the dead grandmother after all. No other explanation seemed to fit all the facts.Is this the end of the story? We, the audience, realize that it is possible that Osment had merely guessed about the incidents, heard them second-hand from another relative or (as with professional psychics) might have retold his anecdotes whilst looking for validation from his mother. The evidence seems compelling only because these alternatives seem even less likely. It is in this same sense that when scientists reach a conclusion, it is merely a place to pause and rest for a moment, not a final destination.Near the end of the course, I gave a pop-quiz asking each student to give a ‘yes'' or ‘no'' answer, plus a short one-sentence explanation, to the following question: Donald Trump seems to be a wealthy businessman. He dresses like one, he has a TV show in which he acts like one, he gives seminars on wealth building and so on. Everything we know about him says that he is wealthy as a direct result of his business activities. On the basis of this evidence, are we justified in concluding that he is, in fact, a wealthy businessman?About half the class said that yes, if all the evidence points in one direction, that suffices. About half the class said ‘no'', the stated evidence is circumstantial and we do not know, for example, what his bank balance is or whether he has more debt than equity. All the evidence we know about points in one direction, but we might not know all the facts.Even when looked at carefully, not every anomaly is attractive enough or ‘ripe'' enough to be pursued when first noticedHow do we know whether or not we know all the facts? Again, it is a matter of imagination. Let us review a few possible alternatives. Maybe his wealth comes from inheritance rather than business acumen; or from silent partners; or from drug running. Maybe he is dangerously over-extended and living on borrowed money; maybe his wealth is more apparent than real. Maybe Trump Casinos made up the role of Donald Trump as its symbol, the way McDonald''s made up the role of Ronald McDonald?Several students complained that this was a ridiculous question. Yet I had posed this just after Bernard Madoff''s arrest was blanketing the news. Madoff was known as a billionaire investor genius for decades and had even served as the head of the Securities and Exchange Commission. As it turned out, his money was obtained by a massive Ponzi scheme. Why was Madoff able to succeed for so long? Because it was inconceivable that such a famous public figure could be a common con man and the people around him could not imagine the possibility that his livelihood needed to be scrutinized.To this point, I have emphasized the benefits of paying attention to anomalous, strange or unwelcome observations. Yet paradoxically, scientists often make progress by (provisionally) putting aside anomalous or apparently negative findings that seem to invalidate or distract from their hypothesis. When Rita Levi-Montalcini was assaying the neurite-promoting effects of tumour tissue, she had predicted that this was a property of tumours and was devastated to find that normal tissue had the same effects. Only by ‘ignoring'' this apparent failure could she move forward to characterize nerve growth factor and eventually understand its biology [4].Another classic example is Huntington disease—a genetic disorder in which an inherited alteration in the gene that encodes a protein, huntingtin, leads to toxicity within certain types of neuron and causes a progressive movement disorder associated with cognitive decline and psychiatric symptoms. Clinicians observed that the offspring of Huntington disease patients sometimes showed symptoms at an earlier age than their parents, and this phenomenon, called ‘genetic anticipation'', could affect successive generations at earlier and earlier ages of onset. This observation was met with scepticism and sometimes ridicule, as everything that was known about genetics at the time indicated that genes do not change across generations. Ascertainment bias was suggested as a much more probable explanation; in other words, once a patient is diagnosed with Huntington disease, their doctors will look at their offspring much more closely and will thus tend to identify the onset of symptoms at an earlier age. Eventually, once the detailed genetics of the disease were understood at the molecular level, it was shown that the structure of the altered huntingtin gene does change. Genetic anticipation is now an accepted phenomenon.…in fact, schools teach a lot about how to test hypotheses but little about how to find good hypotheses in the first placeWhat does this teach us about discovery? Even when looked at carefully, not every anomaly is attractive enough or ‘ripe'' enough to be pursued when first noticed. The biologists who identified the structure of the abnormal huntingtin gene did eventually explain genetic anticipation, although they set aside the puzzling clinical observations and proceeded pragmatically according to their (wrong) initial best-guess as to the genetics. The important thing is to move forward.Finally, let us consider the case of Grigori Perelman, an outstanding mathematician who solved the Poincaré Conjecture a few years ago. He did not tell anyone he was working on the problem, lest their ‘helpful advice'' discourage him; he posted his historic proof online, bypassing peer-reviewed journals altogether; he turned down both the Fields Medal and a million dollar prize; and he has refused professorial posts at prestigious universities. Having made a deliberate decision to eschew the external incentives associated with science as a career, his choices have been written off as examples of eccentric anti-social behaviour. I suggest, however, that he might have simply recognized that the usual rules for success and the usual reward structure of the scientific community can create roadblocks, which had to be avoided if he was to solve a supposedly unsolvable problem.If we cannot imagine new paradigms, then how can they ever be perceived, much less tested? It should be clear by now that the ‘process of scientific discovery'' can proceed by many different paths. However, here is one cognitive exercise that can be applied to almost any situation. (i) Notice a phenomenon, even if (especially if) it is familiar and regarded as a solved problem; regard it as if it is new and strange. In particular, look hard for anomalous and strange aspects of the phenomenon that are ignored by scientists in the field. (ii) Look for the hidden assumptions that guide scientists'' thinking about the phenomenon, and ask what kinds of explanation would be possible if the assumptions were false (or reversed). (iii) Make a list of possible alternative explanations, no matter how unlikely they seem to be. (iv) Ask if one of these explanations has particular appeal (for example, if it is the most elegant theoretically; if it can generalize to new domains; and if it would have great practical impact). (v) Ask what kind of evidence would allow one to favour that hypothesis over the others, and carry out experiments to test the hypothesis.The process just outlined is not something that is taught in graduate school; in fact, schools teach a lot about how to test hypotheses but little about how to find good hypotheses in the first place. Consequently, this cognitive exercise is not often carried out within the brain of an individual scientist. Yet this creative tension happens naturally when investigators from two different fields, who have different assumptions, methods and ways of working, meet to discuss a particular problem. This is one reason why new paradigms so often emerge in the cross-fertilization of different disciplines.There are of course other, more systematic ways of searching for hypotheses by bringing together seemingly unrelated evidence. The Arrowsmith two-node search strategy [5], for instance, is based on distinct searches of the biomedical literature to retrieve articles on two different areas of science that have not been studied in relation to each other, but that the investigator suspects might be related in some fashion. The software identifies common words or phrases, which might point to meaningful links between them. This is but one example of ‘literature-based discovery'' as a heuristic technique [6], and in turn, is part of the larger data-driven approach of ‘text mining'' or ‘data mining'', which looks for unusual, new or unexpected patterns within large amounts of observational data. Regardless of whether one follows hypothesis-driven or data-driven models of investigation, let us teach our students to repeat the mantra: ‘odd is good''!? Open in a separate windowNeil R Smalheiser