Response to “Vast (but avoidable) underestimation of global biodiversity”

This Formal Comment provides clarifications on the authors’ recent estimates of global bacterial diversity and the current status of the field, and responds to a Formal Comment from John Wiens regarding their prior work.

We welcome Wiens’ efforts to estimate global animal-associated bacterial richness and thank him for highlighting points of confusion and potential caveats in our previous work on the topic [1]. We find Wiens’ ideas worthy of consideration, as most of them represent a step in the right direction, and we encourage lively scientific discourse for the advancement of knowledge. Time will ultimately reveal which estimates, and underlying assumptions, came closest to the true bacterial richness; we are excited and confident that this will happen in the near future thanks to rapidly increasing sequencing capabilities. Here, we provide some clarifications on our work, its relation to Wiens’ estimates, and the current status of the field.First, Wiens states that we excluded animal-associated bacterial species in our global estimates. However, thousands of animal-associated samples were included in our analysis, and this was clearly stated in our main text (second paragraph on page 3).Second, Wiens’ commentary focuses on “S1 Text” of our paper [1], which was rather peripheral, and, hence, in the Supporting information. S1 Text [1] critically evaluated the rationale underlying previous estimates of global bacterial operational taxonomic unit (OTU) richness by Larsen and colleagues [2], but the results of S1 Text [1] did not in any way flow into the analyses presented in our main article. Indeed, our estimates of global bacterial (and archaeal) richness, discussed in our main article, are based on 7 alternative well-established estimation methods founded on concrete statistical models, each developed specifically for richness estimates from multiple survey data. We applied these methods to >34,000 samples from >490 studies including from, but not restricted to, animal microbiomes, to arrive at our global estimates, independently of the discussion in S1 Text [1].Third, Wiens’ commentary can yield the impression that we proposed that there are only 40,100 animal-associated bacterial OTUs and that Cephalotes in particular only have 40 associated bacterial OTUs. However, these numbers, mentioned in our S1 Text [1], were not meant to be taken as proposed point estimates for animal-associated OTU richness, and we believe that this was clear from our text. Instead, these numbers were meant as examples to demonstrate how strongly the estimates of animal-associated bacterial richness by Larsen and colleagues [2] would decrease simply by (a) using better justified mathematical formulas, i.e., with the same input data as used by Larsen and colleagues [2] but founded on an actual statistical model; (b) accounting for even minor overlaps in the OTUs associated with different animal genera; and/or (c) using alternative animal diversity estimates published by others [3], rather than those proposed by Larsen and colleagues [2]. Specifically, regarding (b), Larsen and colleagues [2] (pages 233 and 259) performed pairwise host species comparisons within various insect genera (for example, within the Cephalotes) to estimate on average how many bacterial OTUs were unique to each host species, then multiplied that estimate with their estimated number of animal species to determine the global animal-associated bacterial richness. However, since their pairwise host species comparisons were restricted to congeneric species, their estimated number of unique OTUs per host species does not account for potential overlaps between different host genera. Indeed, even if an OTU is only found “in one” Cephalotes species, it might not be truly unique to that host species if it is also present in members of other host genera. To clarify, we did not claim that all animal genera can share bacterial OTUs, but instead considered the implications of some average microbiome overlap (some animal genera might share no bacteria, and other genera might share a lot). The average microbiome overlap of 0.1% (when clustering bacterial 16S sequences into OTUs at 97% similarity) between animal genera used in our illustrative example in S1 Text [1] is of course speculative, but it is not unreasonable (see our next point). A zero overlap (implicitly assumed by Larsen and colleagues [2]) is almost certainly wrong. One goal of our S1 Text [1] was to point out the dramatic effects of such overlaps on animal-associated bacterial richness estimates using “basic” mathematical arguments.Fourth, Wiens’ commentary could yield the impression that existing data are able to tell us with sufficient certainty when a bacterial OTU is “unique” to a specific animal taxon. However, so far, the microbiomes of only a minuscule fraction of animal species have been surveyed. One can thus certainly not exclude the possibility that many bacterial OTUs currently thought to be “unique” to a certain animal taxon are eventually also found in other (potentially distantly related) animal taxa, for example, due to similar host diets and or environmental conditions [4–7]. As a case in point, many bacteria in herbivorous fish guts were found to be closely related to bacteria in mammals [8], and Song and colleagues [6] report that bat microbiomes closely resemble those of birds. The gut microbiome of caterpillars consists mostly of dietary and environmental bacteria and is not species specific [4]. Even in animal taxa with characteristic microbiota, there is a documented overlap across host species and genera. For example, there are a small number of bacteria consistently and specifically associated with bees, but these are found across bee genera at the level of the 99.5% similar 16S rRNA OTUs [5]. To further illustrate that an average microbiome overlap between animal taxa at least as large as the one considered in our S1 Text (0.1%) [1] is not unreasonable, we analyzed 16S rRNA sequences from the Earth Microbiome Project [6,9] and measured the overlap of microbiota originating from individuals of different animal taxa. We found that, on average, 2 individuals from different host classes (e.g., 1 mammalian and 1 avian sample) share 1.26% of their OTUs (16S clustered at 100% similarity), and 2 individuals from different host genera belonging to the same class (e.g., 2 mammalian samples) share 2.84% of their OTUs (methods in S1 Text of this response). A coarser OTU threshold (e.g., 97% similarity, considered in our original paper [1]) would further increase these average overlaps. While less is known about insect microbiomes, there is currently little reason to expect a drastically different picture there, and, as explained in our S1 Text [1], even a small average microbiome overlap of 0.1% between host genera would strongly limit total bacterial richness estimates. The fact that the accumulation curve of detected bacterial OTUs over sampled insect species does not yet strongly level off says little about where the accumulation curve would asymptotically converge; rigorous statistical methods, such as the ones used for our global estimates [1], would be needed to estimate this asymptote.Lastly, we stress that while the present conversation (including previous estimates by Louca and colleagues [1], Larsen and colleagues [2], Locey and colleagues [10], Wiens’ commentary, and this response) focuses on 16S rRNA OTUs, it may well be that at finer phylogenetic resolutions, e.g., at bacterial strain level, host specificity and bacterial richness are substantially higher. In particular, future whole-genome sequencing surveys may well reveal the existence of far more genomic clusters and ecotypes than 16S-based OTUs.     

Sleep loss leads to the withdrawal of human helping across individuals,groups, and large-scale societies

Humans help each other. This fundamental feature of homo sapiens has been one of the most powerful forces sculpting the advent of modern civilizations. But what determines whether humans choose to help one another? Across 3 replicating studies, here, we demonstrate that sleep loss represents one previously unrecognized factor dictating whether humans choose to help each other, observed at 3 different scales (within individuals, across individuals, and across societies). First, at an individual level, 1 night of sleep loss triggers the withdrawal of help from one individual to another. Moreover, fMRI findings revealed that the withdrawal of human helping is associated with deactivation of key nodes within the social cognition brain network that facilitates prosociality. Second, at a group level, ecological night-to-night reductions in sleep across several nights predict corresponding next-day reductions in the choice to help others during day-to-day interactions. Third, at a large-scale national level, we demonstrate that 1 h of lost sleep opportunity, inflicted by the transition to Daylight Saving Time, reduces real-world altruistic helping through the act of donation giving, established through the analysis of over 3 million charitable donations. Therefore, inadequate sleep represents a significant influential force determining whether humans choose to help one another, observable across micro- and macroscopic levels of civilized interaction. The implications of this effect may be non-trivial when considering the essentiality of human helping in the maintenance of cooperative, civil society, combined with the reported decline in sufficient sleep in many first-world nations.

Helping behavior between humans has been one of the most influential forces sculpting modern civilizations, but what factors influence this propensity to help? This study demonstrates that a lack of sleep dictates whether humans choose to help each other at three different scales: within individuals, across individuals, and across societies.

“Service to others is the rent you pay for your room here on earth.”― Muhammad Ali

Humans help each other. Helping is a prominent feature of homo sapiens [1], and represents a fundamental force sculpting the advent and preservation of modern civilizations [2,3].The ubiquity of helping is evident across the full spectrum of societal strata. From global government-to-government aid packages (e.g., the international aid following the 2004 Indian Ocean tsunami [4]), to country-wide pledge drives (e.g., the 2010 Haiti disaster) [5], and to individuals altruistically gifting money or donating their own blood to strangers, the expression of helping is abundant and pervasive [6]. So much so that this fundamental act has scaled into a lucent and sizable “helping economy” [7], with charitable giving in the United States amounting to $450 billion in 2019; a value representing 5.5% of the gross domestic product. In the United Kingdom, 10 billion pounds were donated to charity in 2017 and 2018. Indeed, more than 50% of individuals across the US, Europe, and Asia will have reported donating to charity or helping a stranger within the past month (The World Giving index).Human helping is therefore globally abundant, common across diverse societies, sizable in scope, substantive in financial magnitude, consequential in ramification, and frequent in occurrence.The motivated drive for humans to help each other has been linked to a range of underlying factors, from evolutionary forces (e.g., kin selection and reciprocal altruism that bias helping toward close others [2]), cultural norms and expectations (e.g., individualistic versus collectivistic cultures [8,9]), to socioeconomic factors (e.g., helping is less common in larger cities relative to rural areas [10,11]), as well as personality traits (e.g., individual empathy) [12,13].Ultimately, however, the decisional act to help others involves the human brain. Prosocial helping of varied kinds consistently engages a set of brain regions known as the social cognition network. Comprised of the medial prefrontal cortex (mPFC), mid and superior temporal sulcus, temporal-parietal junction (TPJ), and the precuneus [14,15], this network is activated when considering the mental states, needs, and perspectives of others [16–19], and the active choice to help them [20–23]. In contrast, lesions within key regions of this network result in “acquired sociopathy” [24], associated with a loss of both empathy and the withdrawal of compassionate helping [25–27].Yet the possibility that sleep loss represents another significant factor determining whether or not humans help each other, linked to underlying impairments within the social cognition brain network, remains unknown. Several lines of evidence motivate this prediction. First, insufficient sleep impairs emotional processing, including deficits in emotion recognition and expression, while conversely increasing basic emotional reactivity, further linked to antisocial behavior [28,29] (such as increased interpersonal conflict [30] and reduced trust in others [31,32]). Second, sleep loss reliably decreases activity in, and disrupts functional connectivity between, numerous regions within the social cognition brain network [33], including the mPFC [34], TPJ, and precuneus [35].Building on this overarching hypothesis, here, we test the prediction that a lack of sleep impairs human helping at a neural, individual, group, and global societal level. More specifically, we tested whether: (i) within individuals, a night of experimental sleep loss decreases the fundamental desire to help others, the underlying neural mechanism of which is linked to impaired activity within the social cognition brain network when considering other individuals (Study 1), (ii) in a micro-longitudinal study, night-to-night fluctuations in sleep result in a corresponding next-day deficit in the desire to act altruistically and helping others (Study 2), and (iii) at a large-scale national level, the loss of 1 h of sleep opportunity, using the manipulation of daylight saving time (DST), impairs the real-world behavioral act of altruistic human helping at a large-scale, societal level (Study 3).     

Gauging the Purported Costs of Public Data Archiving for Long-Term Population Studies

It was recently proposed that long-term population studies be exempted from the expectation that authors publicly archive the primary data underlying published articles. Such studies are valuable to many areas of ecological and evolutionary biological research, and multiple risks to their viability were anticipated as a result of public data archiving (PDA), ultimately all stemming from independent reuse of archived data. However, empirical assessment was missing, making it difficult to determine whether such fears are realistic. I addressed this by surveying data packages from long-term population studies archived in the Dryad Digital Repository. I found no evidence that PDA results in reuse of data by independent parties, suggesting the purported costs of PDA for long-term population studies have been overstated.Data are the foundation of the scientific method, yet individual scientists are evaluated via novel analyses of data, generating a potential conflict of interest between a research field and its individual participants that is manifested in the debate over access to the primary data underpinning published studies [1–5]. This is a chronic issue but has become more acute with the growing expectation that researchers publish the primary data underlying research reports (i.e., public data archiving [PDA]). Studies show that articles publishing their primary data are more reliable and accrue more citations [6,7], but a recent opinion piece by Mills et al. [2] highlighted the particular concerns felt by some principal investigators (PIs) of long-term population studies regarding PDA, arguing that unique aspects of such studies render them unsuitable for PDA. The "potential costs to science" identified by Mills et al. [2] as arising from PDA are as follows:

• Publication of flawed research resulting from a "lack of understanding" by independent researchers conducting analyses of archived data
• Time demands placed on the PIs of long-term population studies arising from the need to correct such errors via, e.g., published rebuttals
• Reduced opportunities for researchers to obtain the skills needed for field-based data collection because equivalent long-term population studies will be rendered redundant
• Reduced number of collaborations
• Inefficiencies resulting from repeated assessment of a hypothesis using a single dataset

Each "potential cost" is ultimately predicated on the supposition that reuse of archived long-term population data is common, yet the extent to which this is true was not evaluated. To assess the prevalence of independent reuse of archived data—and thereby examine whether the negative consequences of PDA presented by Mills et al. [2] may be realised—I surveyed datasets from long-term population studies archived in the Dryad Digital Repository (hereafter, Dryad). Dryad is an online service that hosts data from a broad range of scientific disciplines, but its content is dominated by submissions associated with ecological and evolutionary biological research [8]. I examined all the Dryad packages associated with studies from four journals featuring ecological or evolutionary research: The American Naturalist, Evolution, Journal of Evolutionary Biology, and Proceedings of the Royal Society B: Biological Sciences (the latter referred to hereafter as Proceedings B). These four journals together represent 23.3% of Dryad''s contributed packages (as of early February 2016). Mills et al. [2] refer to short- versus long-term studies but do not provide a definition of this dichotomy. However, the shortest study represented by their survey lasted for 5 years, so I used this as the minimum time span for inclusion in my survey. This cut-off seems reasonable, as it will generally exclude studies resulting from single projects, such that included datasets likely relate to studies resulting from a sustained commitment on the part of researchers—although one included package contains data gathered via “citizen science” [9], and two others contain data derived from archived human population records [10,11]. However, as these datasets cover extended time spans and were used to address ecological questions [12–14], they were retained in my survey sample. Following Mills et al. [2], my focus was on population studies conducted in natural (or seminatural) settings, so captive populations were excluded. Because I was assessing the reuse of archived data, I excluded packages published by Dryad after 2013: authors can typically opt to impose a 1-year embargo, and articles based on archived data will themselves take some time to be written and published.Of the 1,264 archived data packages linked to one of the four journals and published on the Dryad website before 2014, 72 were identified as meeting the selection criteria. This sample represents a diverse range of taxa (Fig 1) and is comparable to the 73 studies surveyed by Mills et al. [2], although my methodology permits individual populations to be represented more than once, since the survey was conducted at the level of published articles (S1 Table). Of these 72 data packages, five had long-term embargoes remaining active (three packages with 5-year embargoes [15–17]; two packages with 10-year embargoes [18,19]). For two of these [17,19], the time span of the study could not be estimated because this information is not provided in the associated articles [20,21]. For a third package [22], the archived data indicated 10 years were represented (dummy coding was used to disguise factor level identities, including for year), yet the text of the associated paper suggests data collection covered a considerably greater time span [23]. However, since the study period is not stated in the text, I followed the archived data [22] in assuming data collection spanned a 10-year period. The distribution of study time spans is shown in Fig 2.Open in a separate windowFig 1Taxonomic representation of the 72 data packages included in the survey.The number of packages for each taxon is given in parentheses (note: one data package included data describing both insects and plants [9], while other data packages represented multiple species within a single taxonomic category).Open in a separate windowFig 2The study periods of the 70 data packages included in the survey for which this could be calculated.For each year from 2000 to 2004, these four journals contributed no more than a single data package to Dryad between them. However, around the time that the Joint Data Archiving Policy (JDAP; [24]) was adopted by three of these, we see a surge in PDA by ecologists and evolutionary biologists (Fig 3), such that in 2015 these four journals were collectively represented by 709 data packages. Of course, Mills et al. [2] argue against mandatory archiving of primary data for long-term studies in particular. For this subset of articles published in these four journals, the same pattern is observed: prior to adoption of the JDAP, only two data packages associated with long-term studies had been archived in Dryad, but following the implementation of the JDAP as a condition of publication in The American Naturalist, Evolution, and Journal of Evolutionary Biology, there is a rapid increase in the number of data packages being archived, despite the continuing availability of alternative venues should authors wish to avoid the purported costs of PDA as Mills et al. [2] contend. As the editorial policy of Proceedings B has shifted towards an increasingly strong emphasis on PDA (it is now mandatory), there has similarly been an increase in the representation of articles from this journal in Dryad, both overall (Fig 3) and for long-term studies in particular (Fig 4). These observations suggest that authors rarely chose to publicly archive their data prior to the adoption of PDA policies by journals and that uptake of PDA spread rapidly once it became a prerequisite for publication. In this respect, researchers using long-term population studies are no different to those in other scientific fields, despite the assertion by Mills et al. [2] that they are a special case owing to the complexity of their data. In reality, researchers in many other scientific disciplines also seek to identify relationships within complex systems. Within neuroscience, for example, near-identical objections to PDA were raised at the turn of the century [25], while archiving of genetic and protein sequences by molecular biologists has yielded huge advances but was similarly resisted until revised journal policies stimulated a change in culture [1,26].Open in a separate windowFig 3Total number of data packages archived in the Dryad Digital Repository each year for four leading journals within ecology and evolutionary biology.Arrow indicates when the Joint Data Archiving Policy (JDAP) was adopted by Evolution, Journal of Evolutionary Biology, and The American Naturalist. Note that because data packages are assigned a publication date by Dryad prior to journal publication (even if an embargo is imposed), some data packages will have been published in the year preceding the journal publication of their associated article.Open in a separate windowFig 4Publication dates of the 72 data packages from long-term study populations that were included in the survey.A primary concern raised by opponents of PDA is that sharing their data will see them “scooped” by independent researchers [6,8,27–30]. To quantify this risk for researchers maintaining long-term population studies, I used the Web of Science (wok.mimas.ac.uk) to search for citations of each data package (as of November 2015). For the 67 Dryad packages that were publicly accessible, none were cited by any article other than that from which it was derived. However, archived data could conceivably have been reused without the data package being cited, so I examined all journal articles that cited the study report associated with each data package (median citation count: 9; range: 0–58). Although derived metrics from the main articles were occasionally included in quantitative reviews [31,32] or formal meta-analyses [33], I again found no examples of the archived data being reused by independent researchers. As a third approach, I emailed the corresponding author(s) listed for each article, to ask if they were themselves aware of any examples. The replies I received (n = 35) confirmed that there were no known cases of long-term population data being independently reused in published articles. The apparent concern of some senior researchers that PDA will see them "collect data for 30 years just to be scooped" [30] thus lacks empirical support. It should also be noted that providing primary data upon request precedes PDA as a condition of acceptance for most major scientific journals [8]. PDA merely serves to ensure that authors meet this established commitment, a step made necessary by the failure rate that is otherwise observed, even after the recent revolution in communications technology [34–36]. As my survey shows, in practice the risk of being scooped is a monster under the bed: empirical assessment fails to justify the level of concern expressed. While long-term population studies are unquestionably a highly valuable resource for ecologists [2,37–39] and will likely continue to face funding challenges [37–39], there is no empirical support for the contention of Mills et al. [2] that PDA threatens their viability, although this situation may deserve reassessment in the future if the adoption of PDA increases within ecology and evolutionary biology. Nonetheless, in the absence of assessments over longer time frames (an inevitable result of the historical reluctance to adopt PDA), my survey results raise doubts over the validity of arguments favouring extended embargoes for archived data [29,40], and particularly the suggestion that multidecadal embargoes should be facilitated for long-term studies [2,41].Authors frequently assert that unique aspects of their long-term study render it especially well suited to addressing particular issues. Such claims contradict the suggestion that studies will become redundant if PDA becomes the norm [2] while simultaneously highlighting the necessity of making primary data available for meaningful evaluation of results. For research articles relying on data collected over several decades, independent replication is clearly impractical, such that reproducibility (the ability for a third party to replicate the results exactly [42]) is rendered all the more crucial. Besides permitting independent validation of the original results, PDA allows assessment of the hypotheses using alternative analytical methods (large datasets facilitate multiple analytical routes to test a single biological hypothesis, which likely contributes to poor reproducibility [43]) and reassessment if flaws in the original methodology later emerge [44]. Although I was not attempting to use archived data to replicate published results, and thus did not assess the contents of each package in detail, at least six packages [10,45–49] failed to provide the primary data underlying their associated articles, including a quantitative genetic study [50] for which only pedigree information was archived [47]. This limits exploration of alternative statistical approaches to the focal biological hypothesis and impedes future applications of the data that may be unforeseeable by the original investigators (a classic example being Bumpus'' [51] dataset describing house sparrow survival [52]), but it seems to be a reality of PDA within ecology and evolution at present [53].The "solutions" proffered by Mills et al. [2] are, in reality, alternatives to PDA that would serve to maintain the status quo with respect to data accessibility for published studies (i.e., subject to consent from the PI). This is a situation that is widely recognised to be failing with respect to the availability of studies'' primary data [34–36,54]. Indeed, for 19% (13 of 67 nonembargoed studies) of the articles represented in my survey, the correspondence email addresses were no longer active, highlighting how rapidly access to long-term primary data can be passively lost. It is unsurprising, then, that 95% of scientists in evolution and ecology are reportedly in favour of PDA [1]. Yet, having highlighted the value and irreplaceability of data describing long-term population studies, Mills et al. [2] reject PDA in favour of allowing PIs to maintain postpublication control of primary data, going so far as to discuss the possibility of data being copyrighted. Such an attitude risks inviting public ire, since asserting private ownership ignores the public funding that likely enabled data collection, and is at odds with a Royal Society report urging scientists to "shift away from a research culture where data is viewed as a private preserve" [55]. I contend that primary data would better be considered as an intrinsic component of a published article, alongside the report appearing in the pages of a journal that presents the data''s interpretation. In this way, an article would move closer to being a self-contained product of research that is fully accessible and assessable. For issues that can only be addressed using data covering an extended time span [2,37–39], excusing long-term studies from the expectation of publishing primary data would potentially render the PIs as unaccountable gatekeepers of scientific consensus. PDA encourages an alternative to this and facilitates a change in the treatment of published studies, from the system of preservation (in which a study''s contribution is fixed) that has been the historical convention, towards a conservation approach (in which support for hypotheses can be reassessed and updated) [56]. Given the fundamentally dynamic nature of science, harnessing the storage potential enabled by the Information Age to ensure a study''s contribution can be further developed or refined in the future seems logical and would benefit both the individual authors (through enhanced citations and reputation) and the wider scientific community.The comparison Mills et al. [2] draw between PIs and pharmaceutical companies in terms of how their data are treated is inappropriate: whereas the latter bear the financial cost of developing a drug, a field study''s costs are typically covered by the public purse, such that the personal risks of a failed project are largely limited to opportunity costs. It is inconsistent to highlight funding challenges [2,37] while simultaneously acting to inhibit maximum value for money being derived from funded studies. Several of the studies represented in the survey by Mills et al. [2] comfortably exceed a 50-year time span, highlighting the possibility that current PIs are inheritors rather than initiators of long-term studies. In such a situation, arguments favouring the rights of the PI to maintain control of postpublication access to primary data are weakened still further, given that the data may be the result of someone else''s efforts. Indeed, given the undoubted value of long-term studies for ecological and evolutionary research [2,37,39], many of Mills et al.''s [2] survey respondents will presumably hope to see these studies continue after their own retirement. Rather than owners of datasets, then, perhaps PIs of long-term studies might better be considered as custodians, such that—to adapt the slogan of a Swiss watchmaker—“you never really own a long-term population study; you merely look after it for the next generation.”     

Which New Health Technologies Do We Need to Achieve an End to HIV/AIDS?

In the last 15 years, antiretroviral therapy (ART) has been the most globally impactful life-saving development of medical research. Antiretrovirals (ARVs) are used with great success for both the treatment and prevention of HIV infection. Despite these remarkable advances, this epidemic grows relentlessly worldwide. Over 2.1 million new infections occur each year, two-thirds in women and 240,000 in children. The widespread elimination of HIV will require the development of new, more potent prevention tools. Such efforts are imperative on a global scale. However, it must also be recognised that true containment of the epidemic requires the development and widespread implementation of a scientific advancement that has eluded us to date—a highly effective vaccine. Striving for such medical advances is what is required to achieve the end of AIDS.In the last 15 years, antiretroviral therapy (ART) has been the most globally impactful life-saving development of medical research. Antiretrovirals (ARVs) are used with great success for both the treatment and prevention of HIV infection. In the United States, the widespread implementation of combination ARVs led to the virtual eradication of mother-to-child transmission of HIV from 1,650 cases in 1991 to 110 cases in 2011, and a turnaround in AIDS deaths from an almost 100% five-year mortality rate to a five-year survival rate of 91% in HIV-infected adults [1]. Currently, the estimated average lifespan of an HIV-infected adult in the developed world is well over 40 years post-diagnosis. Survival rates in the developing world, although lower, are improving: in sub-Saharan Africa, AIDS deaths fell by 39% between 2005 and 2013, and the biggest decline, 51%, was seen in South Africa [2].Furthermore, the association between ART, viremia, and transmission has led to the concept of “test and treat,” with the hope of reducing community viral load by testing early and initiating treatment as soon as a diagnosis of HIV is made [3]. Indeed, selected regions of the world have begun to actualize the public health value of ARVs, from gains in life expectancy to impact on onward transmission, with a potential 1% decline in new infections for every 10% increase in treatment coverage [2]. In September 2015, WHO released new guidelines removing all limitations on eligibility for ART among people living with HIV and recommending pre-exposure prophylaxis (PrEP) to population groups at significant HIV risk, paving the way for a global onslaught on HIV [4].Despite these remarkable advances, this epidemic grows relentlessly worldwide. Over 2.1 million new infections occur each year, two-thirds in women and 240,000 in children [2]. In heavily affected countries, HIV infection rates have only stabilized at best: the annualized acquisition rates in persons in their first decade of sexual activity average 3%–5% yearly in southern Africa [5–7]. These figures are hardly compatible with the international health community’s stated goal of an “AIDS-free generation” [8,9]. In highly resourced settings, microepidemics of HIV still occur, particularly among gays, bisexuals, and men who have sex with men (MSM) [10]. HIV epidemics are expanding in two geographic regions in 2015—the Middle East/North Africa and Eastern Europe/Central Asia—largely due to challenges in implementing evidence-based HIV policies and programmes [2]. Even for the past decade in the US, almost 50,000 new cases recorded annually, two-thirds among MSM, has been a stable figure for years and shows no evidence of declining [1].While treatment scale-up, medical male circumcision [11], and the implementation of strategies to prevent mother-to-child transmission [12] have received global traction, systemic or topical ARV-based biomedical advances to prevent sexual acquisition of HIV have, as yet, made limited impressions on a population basis, despite their reported efficacy. Factors such as their adherence requirements, cost, potential for drug resistance, and long-term feasibility have restricted the appetite for implementation, even though these approaches may reduce HIV incidence in select populations.Already, several trials have shown that daily oral administration of the ARV tenofovir disoproxil fumarate (TDF), taken singly or in combination with emtricitabine, as PrEP by HIV-uninfected individuals, reduces HIV acquisition among serodiscordant couples (where one partner is HIV-positive and the other is HIV-negative) [13], MSM [14], at-risk men and women [15], and people who inject drugs [16,17] by between 44% and 75%. Long-acting injectable antiretroviral agents such as rilpivirine and cabotegravir, administered every two and three months, respectively, are also being developed for PrEP. All of these PrEP approaches are dependent on repeated HIV testing and adherence to drug regimens, which may challenge effectiveness in some populations and contexts.The widespread elimination of HIV will require the development of new, more potent prevention tools. Because HIV acquisition occurs subclinically, the elimination of HIV on a population basis will require a highly effective vaccine. Alternatively, if vaccine development is delayed, supplementary strategies may include long-acting pre-exposure antiretroviral cocktails and/or the administration of neutralizing antibodies through long-lasting parenteral preparations or the development of a “genetic immunization” delivery system, as well as scaling up delivery of highly effective regimens to eliminate mother-to-child HIV transmission (Fig 1).Open in a separate windowFig 1Medical interventions required to end the epidemic of HIV.Image credit: Glenda Gray.     

Dredging in the Spratly Islands: Gaining Land but Losing Reefs

Coral reefs on remote islands and atolls are less exposed to direct human stressors but are becoming increasingly vulnerable because of their development for geopolitical and military purposes. Here we document dredging and filling activities by countries in the South China Sea, where building new islands and channels on atolls is leading to considerable losses of, and perhaps irreversible damages to, unique coral reef ecosystems. Preventing similar damage across other reefs in the region necessitates the urgent development of cooperative management of disputed territories in the South China Sea. We suggest using the Antarctic Treaty as a positive precedent for such international cooperation.Coral reefs constitute one of the most diverse, socioeconomically important, and threatened ecosystems in the world [1–3]. Coral reefs harbor thousands of species [4] and provide food and livelihoods for millions of people while safeguarding coastal populations from extreme weather disturbances [2,3]. Unfortunately, the world’s coral reefs are rapidly degrading [1–3], with ~19% of the total coral reef area effectively lost [3] and 60% to 75% under direct human pressures [3,5,6]. Climate change aside, this decline has been attributed to threats emerging from widespread human expansion in coastal areas, which has facilitated exploitation of local resources, assisted colonization by invasive species, and led to the loss and degradation of habitats directly and indirectly through fishing and runoff from agriculture and sewage systems [1–3,5–7]. In efforts to protect the world’s coral reefs, remote islands and atolls are often seen as reefs of “hope,” as their isolation and uninhabitability provide de facto protection against direct human stressors, and may help impacted reefs through replenishment [5,6]. Such isolated reefs may, however, still be vulnerable because of their geopolitical and military importance (e.g., allowing expansion of exclusive economic zones and providing strategic bases for military operations). Here we document patterns of reclamation (here defined as creating new land by filling submerged areas) of atolls in the South China Sea, which have resulted in considerable loss of coral reefs. We show that conditions are ripe for reclamation of more atolls, highlighting the need for international cooperation in the protection of these atolls before more unique and ecologically important biological assets are damaged, potentially irreversibly so.Studies of past reclamations and reef dredging activities have shown that these operations are highly deleterious to coral reefs [8,9]. First, reef dredging affects large parts of the surrounding reef, not just the dredged areas themselves. For example, 440 ha of reef was completely destroyed by dredging on Johnston Island (United States) in the 1960s, but over 2,800 ha of nearby reefs were also affected [10]. Similarly, at Hay Point (Australia) in 2006 there was a loss of coral cover up to 6 km away from dredging operations [11]. Second, recovery from the direct and indirect effects of dredging is slow at best and nonexistent at worst. In 1939, 29% of the reefs in Kaneohe Bay (United States) were removed by dredging, and none of the patch reefs that were dredged had completely recovered 30 years later [12]. In Castle Harbour (Bermuda), reclamation to build an airfield in the early 1940s led to limited coral recolonization and large quantities of resuspended sediments even 32 years after reclamation [13]; several fish species are claimed extinct as a result of this dredging [14,15]. Such examples and others led Hatcher et al. [8] to conclude that dredging and land clearing, as well as the associated sedimentation, are possibly the most permanent of anthropogenic impacts on coral reefs.The impacts of dredging for the Spratly Islands are of particular concern because the geographical position of these atolls favors connectivity via stepping stones for reefs over the region [16–19] and because their high biodiversity works as insurance for many species. In an extensive review of the sparse and limited data available for the region, Hughes et al. [20] showed that reefs on offshore atolls in the South China Sea were overall in better condition than near-shore reefs. For instance, by 2004 they reported average coral covers of 64% for the Spratly Islands and 68% for the Paracel Islands. By comparison, coral reefs across the Indo-Pacific region in 2004 had average coral covers below 25% [21]. Reefs on isolated atolls can still be prone to extensive bleaching and mortality due to global climate change [22] and, in the particular case of atolls in the South China Sea, the use of explosives and cyanine [20]. However, the potential for recovery of isolated reefs to such stressors is remarkable. Hughes et al. [20] documented, for instance, how coral cover in several offshore reefs in the region declined from above 80% in the early 1990s to below 6% by 1998 to 2001 (due to a mixture of El Niño and damaging fishing methods that make use of cyanine and explosives) but then recovered to 30% on most reefs and up to 78% in some reefs by 2004–2008. Another important attribute of atolls in the South China Sea is the great diversity of species. Over 6,500 marine species are recorded for these atolls [23], including some 571 reef coral species [24] (more than half of the world’s known species of reef-building corals). The relatively better health and high diversity of coral reefs in atolls over the South China Sea highlights the uniqueness of such reefs and the important roles they may play for reefs throughout the entire region. Furthermore, these atolls are safe harbor for some of the last viable populations of highly threatened species (e.g., Bumphead Parrotfish [Bolbometopon muricatum] and several species of sawfishes [Pristis, Anoxypristis]), highlighting how dredging in the South China Sea may threaten not only species with extinction but also the commitment by countries in the region to biodiversity conservation goals such as the Convention of Biological Diversity Aichi Targets and the United Nations Sustainable Development Goals.Recently available remote sensing data (i.e., Landsat 8 Operational Land Imager and Thermal Infrared Sensors Terrain Corrected images) allow quantification of the sharp contrast between the gain of land and the loss of coral reefs resulting from reclamation in the Spratly Islands (Fig 1). For seven atolls recently reclaimed by China in the Spratly Islands (names provided in Fig 1D, S1 Data for details); the area of reclamation is the size of visible areas in Landsat band 6, as prior to reclamation most of the atolls were submerged, with the exception of small areas occupied by a handful of buildings on piers (note that the amount of land area was near zero at the start of the reclamation; Fig 1C, S1 Data). The seven reclaimed atolls have effectively lost ~11.6 km² (26.9%) of their reef area for a gain of ~10.7 km² of land (i.e., >75 times increase in land area) from February 2014 to May 2015 (Fig 1C). The area of land gained was smaller than the area of reef lost because reefs were lost not only through land reclamation but also through the deepening of reef lagoons to allow boat access (Fig 1B). Similar quantification of reclamation by other countries in the South China Sea (Fig 1Reclamation leads to gains of land in return for losses of coral reefs: A case example of China’s recent reclamation in the Spratly Islands.Table 1List of reclaimed atolls in the Spratly Islands and the Paracel Islands.The impacts of reclamation on coral reefs are likely more severe than simple changes in area, as reclamation is being achieved by means of suction dredging (i.e., cutting and sucking materials from the seafloor and pumping them over land). With this method, reefs are ecologically degraded and denuded of their structural complexity. Dredging and pumping also disturbs the seafloor and can cause runoff from reclaimed land, which generates large clouds of suspended sediment [11] that can lead to coral mortality by overwhelming the corals’ capacity to remove sediments and leave corals susceptible to lesions and diseases [7,9,25]. The highly abrasive coralline sands in flowing water can scour away living tissue on a myriad of species and bury many organisms beyond their recovery limits [26]. Such sedimentation also prevents new coral larvae from settling in and around the dredged areas, which is one of the main reasons why dredged areas show no signs of recovery even decades after the initial dredging operations [9,12,13]. Furthermore, degradation of wave-breaking reef crests, which make reclamation in these areas feasible, will result in a further reduction of coral reefs’ ability to (1) self-repair and protect against wave abrasion [27,28] (especially in a region characterized by typhoons) and (2) keep up with rising sea levels over the next several decades [29]. This suggests that the new islands would require periodic dredging and filling, that these reefs may face chronic distress and long-term ecological damage, and that reclamation may prove economically expensive and impractical.The potential for land reclamation on other atolls in the Spratly Islands is high, which necessitates the urgent development of cooperative management of disputed territories in the South China Sea. First, the Spratly Islands are rich in atolls with similar characteristics to those already reclaimed (Fig 1D); second, there are calls for rapid development of disputed territories to gain access to resources and increase sovereignty and military strength [30]; and third, all countries with claims in the Spratly Islands have performed reclamation in this archipelago (20]. One such possibility is the generation of a multinational marine protected area [16,17]. Such a marine protected area could safeguard an area of high biodiversity and importance to genetic connectivity in the Pacific, in addition to promoting peace in the region (extended justification provided by McManus [16,17]). A positive precedent for the creation of this protected area is that of Antarctica, which was also subject to numerous overlapping claims and where a recently renewed treaty froze national claims, preventing large-scale ecological damage while providing environmental protection and areas for scientific study. Development of such a legal framework for the management of the Spratly Islands could prevent conflict, promote functional ecosystems, and potentially result in larger gains (through spillover, e.g. [31]) for all countries involved.     

Bacterial Ventures into Multicellularity: Collectivism through Individuality

Multicellular eukaryotes can perform functions that exceed the possibilities of an individual cell. These functions emerge through interactions between differentiated cells that are precisely arranged in space. Bacteria also form multicellular collectives that consist of differentiated but genetically identical cells. How does the functionality of these collectives depend on the spatial arrangement of the differentiated bacteria? In a previous issue of PLOS Biology, van Gestel and colleagues reported an elegant example of how the spatial arrangement of differentiated cells gives rise to collective behavior in Bacillus subtilus colonies, further demonstrating the similarity of bacterial collectives to higher multicellular organisms.Introductory textbooks tend to depict bacteria as rather primitive and simple life forms: the billions of cells in a population are all supposedly performing the exact same processes, independent of each other. According to this perspective, the properties of the population are thus nothing more than the sum of the properties of the individual cells. A brief look at the recent literature shows that life at the micro scale is much more complex and far more interesting. Even though cells in a population share the same genetic material and are exposed to similar environmental signals, they are individuals: they can greatly differ from each other in their properties and behaviors [1,2].One source of such phenotypic variation is that individual cells experience different microenvironments and regulate their genes in response. However, and intriguingly, phenotypic differences can also arise in the absence of environmental variation [3]. The stochastic nature of biochemical reactions makes variation between individuals unavoidable: reaction rates in cells will fluctuate because of the typical small number of the molecules involved, leading to slight differences in the molecular composition between individual cells [4]. While cells cannot prevent fluctuations from occurring, the effect of these extracellular and intracellular perturbations on a cell’s phenotype can be controlled by changing the biochemical properties of molecules or the architecture of gene regulatory networks [4,5]. The degree of phenotypic variation could thus evolve in response to natural selection. This raises the question of whether the high degree of phenotypic variation observed in some traits could offer benefits to the bacteria [5].One potential benefit of phenotypic variation is bet hedging. Bet hedging refers to a situation in which a fraction of the cells express alternative programs, which typically reduce growth in the current conditions but at the same time allow for increased growth or survival when the environment abruptly changes [6–8]. Another potential benefit can arise through the division of labor: phenotypic variation can lead to the formation of interacting subpopulations that specialize in complementary tasks [9]. As a result, the population as a whole can perform existing functions more efficiently or attain new functionality [10]. Division of labor enables groups of bacteria to engage in two tasks that are incompatible with each other but that are both required to attain a certain biological function.One of the most famous examples of division of labor in bacteria is the specialization of multicellular cyanobacteria into photosynthesizing and nitrogen-fixing subpopulations [11]. Here, the driving force behind the division of labor is the biochemical incompatibility between photosynthesis and nitrogen fixation, as the oxygen produced during photosynthesis permanently damages the enzymes involved in nitrogen fixation [12]. Other examples include the division of labor between two subpopulations of Salmonella Typhimurium (Tm) during infections [9] and the formation of multicellular fruiting bodies in Myxococcus xanthus [13]. Division of labor is not restricted to interactions between only two subpopulations; for example, the soil-dwelling bacteria Bacillus subtilis can differentiate into at least five different cell types [14]. Multiple types can simultaneously be present in Bacillus biofilms and colonies, each contributing different essential tasks [14,15].An important question is whether a successful division of labor requires the different subpopulations to coordinate their behavior and spatial arrangement. For some systems, it turns out that spatial coordination is not required. For example, the division of labor in clonal groups of Salmonella Tm does not require that the two cell types are spatially arranged in a particular way [9]. In other systems, spatial coordination between the different cell types seems to be beneficial. For example, differentiation into nitrogen-fixing and photosynthetic cells in multicellular cyanobacteria is spatially regulated in a way that facilitates sharing of nitrogen and carbon [16]. In general, when cell differentiation is combined with coordination of behavior between cells, this can allow for the development of complex, group-level behaviors that cannot easily be deduced from the behavior of individual cells [17–20]. In these cases, a population can no longer be treated as an assembly of independent individuals but must be seen as a union that together shows collective behavior.The study by van Gestel et al. [21] in a previous issue of PLOS Biology offers an exciting perspective on how collective behavior can arise from processes operating at the level of single cells. Van Gestel and colleagues [21] analyzed how groups of B. subtilis cells migrate across solid surfaces in a process known as sliding motility. The authors found that migration requires both individuality—the expression of different phenotypes in clonal populations—and spatial coordination between cells. Migration depends critically on the presence of two cell types: surfactin-producing cells, which excrete a surfactant that reduce surface tension, and matrix-producing cells, which excrete extracellular polysaccharides and proteins that form a connective extracellular matrix (Fig 1B) [14]. These two cell types are not randomly distributed across the bacterial group but are rather spatially organized (Fig 1C). The matrix-producing cells form bundles of interconnected and highly aligned cells, which the authors refer to as “van Gogh” bundles. The surfactant producing cells are not present in the van Gogh bundle but are essential for the formation of the bundles [21].Open in a separate windowFig 1Collective behavior through the spatial organization of differentiated cells.(A) Initially cells form a homogenous population. (B) Differentiation: cells start to differentiate into surfactin- (orange) and matrix- (blue) producing cells. The two cell types perform two complementary and essential tasks, resulting in a division of labor. (C) Spatial organization: the matrix-producing cells form van Gogh bundles, consisting of highly aligned and interconnected cells. Surfactin-producing cells are excluded from the bundles and have no particular spatial arrangement. (D) Collective behavior: growth of cells in the van Gogh bundles leads to buckling of these bundles, resulting in colony expansion. The buckling and resulting expansion depend critically on the presence of the two cell types and on their spatial arrangement.The ability to migrate is a collective behavior that can be linked to the biophysical properties of the multicellular van Gogh bundles. The growth of cells in these bundles causes them to buckle, which in turn drives colony migration (Fig 1D) [21]. This is a clear example of an emergent (group-level) phenotype: the buckling of the van Gogh bundles and the resulting colony motility cannot easily be deduced from properties of individual cells. Rather, to understand colony migration we have to understand the interactions between the two cells type as well as their spatial organization. Building on a rich body of work on the regulation of gene expression and cellular differentiation in Bacillus [14], van Gestel et al. [21] are able to show how these molecular mechanisms lead to the formation of specialized cell types that, through coordinated spatial arrangement, provide the group the ability to move (Fig 1). The study thus uniquely bridges the gap between molecular mechanisms and collective behavior in bacterial multicellularity.This study raises a number of intriguing questions. A first question pertains to the molecular mechanisms underlying the spatial coordination of the two cell types. Can the spatial organization be explained based on known mechanisms of the regulation of gene expression in this organism or does the formation of these patterns depend on hitherto uncharacterized gene regulation based on spatial gradients or cell–cell interaction? A second question is about the selective forces that lead to the evolution of collective migration of this organism. The authors raise the interesting hypothesis that van Gogh bundles evolved to allow for migration. Although this explanation is very plausible, it also raises the question of how selection acting on a property at the level of the group can lead to adaptation at the individual cell level. Possible mechanisms for such selective processes have been described within the framework of multilevel selection theory. However, there are still many questions regarding how, and to what extent, multilevel selection operates in the natural world [22–24]. The system described by van Gestel and colleagues [21] offers exciting opportunities to address these questions using a highly studied and experimentally amenable model organism.Bacterial collectives (e.g., colonies or biofilms) have been likened to multicellular organisms partly because of the presence of cell differentiation and the importance of an extracellular matrix [25,26]. Higher multicellular organisms share these properties; however, they are more than simple lumps of interconnected, differentiated cells. Rather, the functioning of multicellular organisms critically depends on the precise spatial organization of these cells [27]. Even though spatial organization has been suggested before in B. subtilis biofilms [28], there was a gap in our understanding of how spatial organization of unicellular cells can lead to group-level function. The van Gogh bundles in the article by van Gestel et al. [21] provide direct evidence on how differentiated cells can spatially organize themselves to give rise to group-level behavior. This shows once more that bacteria are not primitive “bags of chemicals” but rather are more like us “multicellulars” than we might have expected.     

Whisking,Sniffing, and the Hippocampal θ-Rhythm: A Tale of Two Oscillators

The hippocampus has unique access to neuronal activity across all of the neocortex. Yet an unanswered question is how the transfer of information between these structures is gated. One hypothesis involves temporal-locking of activity in the neocortex with that in the hippocampus. New data from the Matthew E. Diamond laboratory shows that the rhythmic neuronal activity that accompanies vibrissa-based sensation, in rats, transiently locks to ongoing hippocampal θ-rhythmic activity during the sensory-gathering epoch of a discrimination task. This result complements past studies on the locking of sniffing and the θ-rhythm as well as the relation of sniffing and whisking. An overarching possibility is that the preBötzinger inspiration oscillator, which paces whisking, can selectively lock with the θ-rhythm to traffic sensorimotor information between the rat’s neocortex and hippocampus.The hippocampus lies along the margins of the cortical mantle and has unique access to neuronal activity across all of the neocortex. From a functional perspective, the hippocampus forms the apex of neuronal processing in mammals and is a key element in the short-term working memory, where neuronal signals persist for tens of seconds, that is independent of the frontal cortex (reviewed in [1,2]). Sensory information from multiple modalities is highly transformed as it passes from primary and higher-order sensory areas to the hippocampus. Several anatomically defined regions that lie within the temporal lobe take part in this transformation, all of which involve circuits with extensive recurrent feedback connections (reviewed in [3]) (Fig 1). This circuit motif is reminiscent of the pattern of connectivity within models of associative neuronal networks, whose dynamics lead to the clustering of neuronal inputs to form a reduced set of abstract representations [4] (reviewed in [5]). The first way station in the temporal lobe contains the postrhinal and perirhinal cortices, followed by the medial and lateral entorhinal cortices. Of note, olfactory input—which, unlike other senses, has no spatial component to its representation—has direct input to the lateral entorhinal cortex [6]. The third structure is the hippocampus, which contains multiple substructures (Fig 1).Open in a separate windowFig 1Schematic view of the circuitry of the temporal lobe and its connections to other brain areas of relevance.Figure abstracted from published results [7–15]. Composite illustration by Julia Kuhl.The specific nature of signal transformation and neuronal computations within the hippocampus is largely an open issue that defines the agenda of a great many laboratories. Equally vexing is the nature of signal transformation as the output leaves the hippocampus and propagates back to regions in the neocortex (Fig 1)—including the medial prefrontal cortex, a site of sensory integration and decision-making—in order to influence perception and motor action. The current experimental data suggest that only some signals within the sensory stream propagate into and out of the hippocampus. What regulates communication with the hippocampus or, more generally, with structures within the temporal lobe? The results from studies in rats and mice suggest that the most parsimonious hypothesis, at least for rodents, involves the rhythmic nature of neuronal activity at the so-called θ-rhythm [16], a 5–10 Hz oscillation (reviewed in [17]). The origin of the rhythm is not readily localized to a single locus [10], but certainly involves input from the medial septum [17] (a member of the forebrain cholinergic system) as well as from the supramammillary nucleus [10,18] (a member of the hypothalamus). The medial septum projects broadly to targets in the hippocampus and entorhinal cortex (Fig 1) [10]. Many motor actions, such as the orofacial actions of sniffing, whisking, and licking, occur within the frequency range of the θ-rhythm [19,20]. Thus, sensory input that is modulated by rhythmic self-motion can, in principle, phase-lock with hippocampal activity at the θ-rhythm to ensure the coherent trafficking of information between the relevant neocortical regions and temporal lobe structures [21–23].We now shift to the nature of orofacial sensory inputs, specifically whisking and sniffing, which are believed to dominate the world view of rodents [19]. Recent work identified a premotor nucleus in the ventral medulla, named the vibrissa region of the intermediate reticular zone, whose oscillatory output is necessary and sufficient to drive rhythmic whisking [24]. While whisking can occur independently of breathing, sniffing and whisking are synchronized in the curious and aroused animal [24,25], as the preBötzinger complex in the medulla [26]—the oscillator for inspiration—paces whisking at nominally 5–10 Hz through collateral projections [27]. Thus, for the purposes of reviewing evidence for the locking of orofacial sensory inputs to the hippocampal θ-rhythm, we confine our analysis to aroused animals that function with effectively a single sniff/whisk oscillator [28].What is the evidence for the locking of somatosensory signaling by the vibrissae to the hippocampal θ-rhythm? The first suggestion of phase locking between whisking and the θ-rhythm was based on a small sample size [29,30], which allowed for the possibility of spurious correlations. Phase locking was subsequently reexamined, using a relatively large dataset of 2 s whisking epochs, across many animals, as animals whisked in air [31]. The authors concluded that while whisking and the θ-rhythm share the same spectral band, their phases drift incoherently. Yet the possibility remained that phase locking could occur during special intervals, such as when a rat learns to discriminate an object with its vibrissae or when it performs a memory-based task. This set the stage for a further reexamination of this issue across different epochs in a rewarded task. Work from Diamond''s laboratory that is published in the current issue of PLOS Biology addresses just this point in a well-crafted experiment that involves rats trained to perform a discrimination task.Grion, Akrami, Zuo, Stella, and Diamond [32] trained rats to discriminate between two different textures with their vibrissae. The animals were rewarded if they turned to a water port on the side that was paired with a particular texture. Concurrent with this task, the investigators also recorded the local field potential in the hippocampus (from which they extracted the θ-rhythm), the position of the vibrissae (from which they extracted the evolution of phase in the whisk cycle), and the spiking of units in the vibrissa primary sensory cortex. Their first new finding is a substantial increase in the amplitude of the hippocampal field potential at the θ-rhythm frequency—approximately 10 Hz for the data of Fig 2A—during the two, approximately 0.5 s epochs when the animal approaches the textures and whisks against it. There is significant phase locking between whisking and the hippocampal θ-rhythm during both of these epochs (Fig 2B), as compared to a null hypothesis of whisking while the animal whisked in air outside the discrimination zone. Unfortunately, the coherence between whisking and the hippocampal θ-rhythm could not be ascertained during the decision, i.e., turn and reward epochs. Nonetheless, these data show that the coherence between whisking and the hippocampal θ-rhythm is closely aligned to epochs of active information gathering.Open in a separate windowFig 2Summary of findings on the θ-rhythm in a rat during a texture discrimination task, derived from reference [32]. (A) Spectrogram showing the change in spectral power of the local field potential in the hippocampal area CA1 before, during, and after a whisking-based discrimination task. (B) Summary index of the increase in coherence between the band-limited hippocampal θ-rhythm and whisking signals during approach of the rat to the stimulus and subsequent touch. The index reports 〈sin(ϕH−ϕW)〉2+〈cos(ϕH−ϕW)〉2, where ɸ_H and ɸ_W are the instantaneous phase of the hippocampal and whisking signals, respectively, and averaging is over all trials and animals. (C) Summary indices of the increase in coherence between the band-limited hippocampal θ-rhythm and the spiking signal in the vibrissa primary sensory cortex (“barrel cortex”). The magnitude of the index for each neuron is plotted versus phase in the θ-rhythm. The arrows show the concentration of units around the mean phase—black arrows for the vector average across only neurons with significant phase locking (solid circles) and gray arrows for the vector average across all neurons (open and closed circles). The concurrent positions of the vibrissae are indicated. The vector average is statistically significant only for the approach (p < 0.0001) and touch (p = 0.04) epochs.The second finding by Grion, Akrami, Zuo, Stella, and Diamond [32] addresses the relationship between spiking activity in the vibrissa primary sensory cortex and the hippocampal θ-rhythm. The authors find that spiking is essentially independent of the θ-rhythm outside of the task (foraging in Fig 2C), similar to the result for whisking and the θ-rhythm (Fig 2B). They observe strong coherence between spiking and the θ-rhythm during the 0.5 s epoch when the animal approaches the textures (approach in Fig 2C), yet reduced (but still significant) coherence during the touch epoch (touch in Fig 2C). The latter result is somewhat surprising, given past work from a number of laboratories that observe spiking in the primary sensory cortex and whisking to be weakly yet significantly phase-locked during exploratory whisking [33–37]. Perhaps overtraining leads to only a modest need for the transfer of sensory information to the hippocampus. Nonetheless, these data establish that phase locking of hippocampal and sensory cortical activity is essentially confined to the epoch of sensory gathering.Given the recent finding of a one-to-one locking of whisking and sniffing [24], we expect to find direct evidence for the phase locking of sniffing and the θ-rhythm. Early work indeed reported such phase locking [38] but, as in the case of whisking [29], this may have been a consequence of too small a sample and, thus, inadequate statistical power. However, Macrides, Eichenbaum, and Forbes [39] reexamined the relationship between sniffing and the hippocampal θ-rhythm before, during, and after animals sampled an odorant in a forced-choice task. They found evidence that the two rhythms phase-lock within approximately one second of the sampling epoch. We interpret this locking to be similar to that seen in the study by Diamond and colleagues (Fig 2B) [32]. All told, the combined data for sniffing and whisking by the aroused rodent, as accumulated across multiple laboratories, suggest that two oscillatory circuits—the supramammillary nucleus and medial septum complex that drives the hippocampal θ-rhythm and the preBötzinger complex that drives inspiration and paces the whisking oscillator during sniffing (Fig 1)—can phase-lock during epochs of gathering sensory information and likely sustain working memory.What anatomical pathway can lead to phase locking of these two oscillators? The electrophysiological study of Tsanov, Chah, Reilly, and O’Mara [9] supports a pathway from the medial septum, which is driven by the supramammillary nucleus, to dorsal pontine nuclei in the brainstem. The pontine nucleus projects to respiratory nuclei and, ultimately, the preBötzinger oscillator (Fig 1). This unidirectional pathway can, in principle, entrain breathing and whisking. Phase locking is not expected to occur during periods of basal breathing, when the breathing rate and θ-rhythm occur at highly incommensurate frequencies. However, it remains unclear why phase locking occurs only during a selected epoch of a discrimination task, whereas breathing and the θ-rhythm occupy the same frequency band during the epochs of approach, as well as touch-based target selection (Fig 2A). While a reafferent pathway provides the rat with information on self-motion of the vibrissae (Fig 1), it is currently unknown whether that information provides feedback for phase locking.A seeming requirement for effective communication between neocortical and hippocampal processing is that phase locking must be achieved at all possible phases of the θ-rhythm. Can multiple phase differences between sensory signals and the hippocampal θ-rhythm be accommodated? Two studies report that the θ-rhythm undergoes a systematic phase-shift along the dorsal–ventral axis of the hippocampus [40,41], although the full extent of this shift is only π radians [41]. In addition, past work shows that vibrissa input during whisking is represented among all phases of the sniff/whisk cycle, at levels from primary sensory neurons [42,43] through thalamus [44,45] and neocortex [33–37], with a bias toward retraction from the protracted position. A similar spread in phase occurs for olfactory input, as observed at the levels of the olfactory bulb [46] and cortex [47]. Thus, in principle, the hippocampus can receive, transform, and output sensory signals that arise over all possible phases in the sniff/whisk cycle. In this regard, two signals that are exactly out-of-phase by π radians can phase-lock as readily as signals that are in-phase.What are the constraints for phase locking to occur within the observed texture identification epochs? For a linear system, the time to lock between an external input and hippocampal theta depends on the observed spread in the spectrum of the θ-rhythm. This is estimated as Δf ~3 Hz (half-width at half-maximum amplitude), implying a locking time on the order of 1/Δf ~0.3 s. This is consistent with the approximate one second of enhanced θ-rhythm activity observed in the study by Diamond and colleagues (Fig 2A) [32] and in prior work [39,48] during a forced-choice task with rodents.Does the θ-rhythm also play a role in the gating of output from the hippocampus to areas of the neocortex? Siapas, Lubenov, and Wilson [48] provided evidence that hippocampal θ-rhythm phase-locks to electrical activity in the medial prefrontal cortex, a site of sensory integration as well as decision-making. Subsequent work [49–51] showed that the hippocampus drives the prefrontal cortex, consistent with the known unidirectional connectivity between Cornu Ammonis area 1 (CA1) of the hippocampus and the prefrontal cortex [11] (Fig 1). Further, phase locking of hippocampal and prefrontal cortical activity is largely confined to the epoch of decision-making, as opposed to the epoch of sensory gathering. Thus, over the course of approximately one second, sensory information flows into and then out of the hippocampus, gated by phase coherence between rhythmic neocortical and hippocampal neuronal activity.It is of interest that the medial prefrontal cortex receives input signals from sensory areas in the neocortex [52] as well as a transformed version of these input signals via the hippocampus (Fig 1). Yet it remains to be determined if this constitutes a viable hub for the comparison of the original and transformed signals. In particular, projections to the medial prefrontal cortex arise from the ventral hippocampus [2], while studies on the phase locking of hippocampal θ-rhythm to prefrontal neocortical activity were conducted in dorsal hippocampus, where the strength of the θ-rhythm is strong compared to the ventral end [53]. Therefore, similar recordings need to be performed in the ventral hippocampus. An intriguing possibility is that the continuous phase-shift of the θ-rhythm along the dorsal to the ventral axis of the hippocampus [40,41] provides a means to encode the arrival of novel inputs from multiple sensory modalities relative to a common clock.A final issue concerns the locking between sensory signals and hippocampal neuronal activity in species that do not exhibit a continuous θ-rhythm, with particular reference to bats [54–56] and primates [57–60]. One possibility is that only the up and down swings of neuronal activity about a mean are important, as opposed to the rhythm per se. In fact, for animals in which orofacial input plays a relatively minor role compared to rodents, such a scheme of clocked yet arrhythmic input may be a necessity. In this case, the window of processing is set by a stochastic interval between transitions, as opposed to the periodicity of the θ-rhythm. This may imply that up/down swings of neuronal activity may drive hippocampal–neocortical communications in all species, with communication mediated via phase-locked oscillators in rodents and via synchronous fluctuations in bats and primates. The validity of this scheme and its potential consequence on neuronal computation remains an open issue and a focus of ongoing research.     

Walk on the Wild Side: Estimating the Global Magnitude of Visits to Protected Areas

How often do people visit the world’s protected areas (PAs)? Despite PAs covering one-eighth of the land and being a major focus of nature-based recreation and tourism, we don’t know. To address this, we compiled a globally-representative database of visits to PAs and built region-specific models predicting visit rates from PA size, local population size, remoteness, natural attractiveness, and national income. Applying these models to all but the very smallest of the world’s terrestrial PAs suggests that together they receive roughly 8 billion (8 x 109) visits/y—of which more than 80% are in Europe and North America. Linking our region-specific visit estimates to valuation studies indicates that these visits generate approximately US $600 billion/y in direct in-country expenditure and US $250 billion/y in consumer surplus. These figures dwarf current, typically inadequate spending on conserving PAs. Thus, even without considering the many other ecosystem services that PAs provide to people, our findings underscore calls for greatly increased investment in their conservation.Enjoyment of nature, much of it in protected areas (PAs), is recognised as the most prominent cultural ecosystem service [1–3], yet we still lack even a rough understanding of its global magnitude and economic significance. Large-scale assessments have been restricted to regional or biome-specific investigations [4–8] (but see [9]). There are good reasons for this. Information on visit rates is limited, widely scattered, and confounded by variation in methods [10,11]. Likewise, estimates of the value of visits vary greatly—geographically, among methods, and depending on the component of value being measured [12–14]. Until now, these problems have prevented data-driven analysis of the worldwide scale of nature-based recreation and tourism. But with almost all the world’s governments committed (through the Aichi Biodiversity Targets [15]) to integrating biodiversity into national accounts, policymakers require such gaps in our knowledge of natural capital to be filled.We tackled this shortfall in our understanding of a major ecosystem service by focusing on terrestrial PAs, which cover one-eighth of the land [16] and are a major focus of nature-based recreation and tourism. We compiled data on visit rates to over 500 PAs and built region-specific models, which predicted variation in visitation in relation to the properties of PAs and to local socioeconomic conditions. Next, we used these models to estimate visit rates to all but the smallest of the world’s terrestrial PAs. Last, by summing these estimates by region and combining the totals with region-specific medians for the value of nature visits obtained from the literature, we derived approximate estimates of the global extent and economic significance of PA visitation.Given the scarcity of data on visits to PAs, our approach was to use all available information (although we excluded marine and Antarctic sites, and International Union for Conservation of Nature (IUCN) Category I PAs where tourism is typically discouraged; for further details of data collection and analysis see Materials and Methods). This generated a database of visitor records for 556 PAs spread across 51 countries and included 2,663 records of annual visit numbers over our best-sampled ten-year period (1998–2007) (S1 Table). Mean annual visit rates for individual PAs in this sample ranged from zero to over 10 million visits/y, with a median across all sampled PAs of 20,333 visits/y.We explored this variation by modelling it in relation to a series of biophysical and socioeconomic variables that might plausibly predict visit rates (after refs [6,7,17]): PA size, local population size, PA remoteness, a simple measure of the attractiveness of the PA’s natural features, and national income (see Materials and Methods for a priori predictions). For each of five major regions, we performed univariate regressions (S2 Table) and then built generalised linear models (GLMs) in an effort to predict variation in observed visit rates. While the GLMs had modest explanatory power within regions (S3 Table), together they accounted for 52.9% of observed global variation in visit rates. Associations with individual GLM variables—controlling for the effects of other variables—differed regionally in their strength but broadly matched our predictions (S1 Fig.). Visit rates increased with local population size (in Europe), decreased with remoteness (everywhere apart from Asia/Australasia), increased with natural attractiveness (in North and Latin America), and increased with national income (everywhere else). Controlling for these variables, visit rates were highest in North America, lower in Asia/Australasia and Europe, and lowest in Africa and Latin America.To quantify how often people visit PAs as a whole, we used our region-specific GLMs to estimate visit rates to 94,238 sites listed in the World Database on Protected Areas (WDPA) [18]). We again excluded marine, Antarctic, and Category I PAs, as well as almost 40,000 extremely small sites which were below the size (10 ha) of the smallest PA in our sample (S2 Fig.). The limited power of our GLMs and significant errors in the WDPA mean our estimates of visit rates should be treated with caution for individual sites or (when aggregated to national level) for smaller countries. However, the larger-scale patterns they reveal are marked. Estimated median visit rates per PA (averaged within countries) are lowest in Africa (at around 3,000/y) and Latin America (4,000/y), and greatest in North America (350,000/y) (S3 Table). When visit rates are aggregated across all PAs within a country, pronounced regional differences in the numbers of PAs (with relatively few in Africa and Latin America) magnify these patterns and indicate that while many African countries have <100,000 PA visits/y, PAs in the United States receive a combined total of over 3 billion visits/y (Fig. 1). This variation is underscored when aggregate PA visit rates are standardised by the annual number of non-workdays and total population size of each region: across Europe we reckon there are ~5 PA visits/100 non-work person-days; for North America, the figure is ~10 visits/100 non-work person-days respectively, while for each other region our estimates are <0.3 visits/100 non-work person-days.Open in a separate windowFig 1Estimated total PA visit rates for each country.Totals (which are log₁₀-transformed) were derived by applying the relevant regional GLM (S3 Table) to all of a country’s terrestrial PAs (excluding those <10 ha, and marine and IUCN Category I PAs) listed in the WDPA [18]. Asterisks show countries for which we had visit rate observations.Summing our aggregate estimates of PA visits suggests that between them, the world’s terrestrial PAs receive approximately 8 billion visits/y. Of these, we estimate 3.8 billion visits/y are in Europe (where more than half of the PAs in the WDPA are located) and 3.3 billion visits/y are in North America (S3 Table). These numbers are strikingly large. However, given our confidence intervals (95% CIs for the global total: 5.4–18.5 billion/y) and considering several conservative aspects of our calculations (e.g., the exclusion of ~40,000 very small sites and the incomplete nature of the WDPA), we consider it implausible that there are fewer than 5 billion PA visits worldwide each year. Three national estimates support this view: 2.5 billion visitdays/y to US PAs in 1996 [4], >1 billion visits/y (albeit many of them cultural rather than nature-based) to China’s National Parks in 2006 [19], and 3.2–3.9 billion visits/y to all British “ecosystems” (most of which are not in PAs) in 2010 [7].Finally, what can be inferred about the economic significance of visits on this scale? Economists working on tourism distinguish two main, non-overlapping components of value [12]: direct expenditure by visitors (an element of economic impact, calculated from spending on fees, travel, accommodation, etc.); and consumer surplus (a measure of economic value which arises because many visitors would be prepared to pay more for their visit than they actually have to, and which is defined as the difference between what visitors would be prepared to pay for a visit and what they actually spend; consumer surplus is typically quantified using travel cost or contingent valuation methods). We conducted an extensive literature search to derive median (but conservative) figures for each type of value for each region (S4 Table). Applying these to our corresponding estimates of visit rates and summing across regions yields an estimate of global gross direct expenditure associated with PA visits (within-country only, and excluding indirect and induced expenditure) of ~US $600 billion/y worldwide (at 2014 prices). The corresponding figure for global consumer surplus is ~US $250 billion/y.Such numbers are unavoidably imprecise. Uncertainty in our modelled visit rates and the wide variation in published estimates of expenditure and consumer surplus mean that they could be out by a factor of two or more. However, comparison with calculations that visits to North American PAs alone have an economic impact of $350–550 billion/y [4] and that direct expenditure on all travel and tourism worldwide runs at $2,000 billion/y [20] suggests our figures are of the correct order of magnitude, and that the value of PA visitation runs into hundreds of billions of dollars annually.These results quantify, we believe for the first time, the scale of visits to the world’s PAs and their approximate economic significance. We currently spend <$10 billion/y in safeguarding PAs [21]—a figure which is widely regarded as grossly insufficient [21–25]. Even without considering the many other benefits which PAs provide [22], our estimates of the economic impact and value of PA visitation dwarf current expenditure—highlighting the risks of underinvestment in conservation, and suggesting substantially increased investments in protected area maintenance and expansion would yield substantial returns.     

Harnessing Evolution to Elucidate the Consequences of Symbiosis

Many organisms harbor microbial associates that have profound impacts on host traits. The phenotypic effect of symbionts on their hosts may include changes in development, reproduction, longevity, and defense against natural enemies. Determining the consequences of associating with a microbial symbiont requires experimental comparison of hosts with and without symbionts. Then, determining the mechanism by which symbionts alter these phenotypes can involve genomic, genetic, and evolutionary approaches; however, many host-associated symbionts are not amenable to genetic approaches that require cultivation of the microbe outside the host. In the current issue of PLOS Biology, Chrostek and Teixeira highlight an elegant approach to studying functional mechanisms of symbiont-conferred traits. They used directed experimental evolution to select for strains of Wolbachia wMelPop (a bacterial symbiont of fruit flies) that differed in copy number of a region of the genome suspected to underlie virulence. Copy number evolved rapidly when under selection, and wMelPop strains with more copies of the region shortened the lives of their Drosophila hosts more than symbionts with fewer copies. Interestingly, the wMelPop strains with more copies also increase host resistance to viruses compared to symbionts with fewer copies. Their study highlights the power of exploiting alternative approaches when elucidating the functional impacts of symbiotic associations.Symbioses, long-term and physically close interactions between two or more species, are central to the ecology and evolution of many organisms. Though “Symbiosis” is more often used to define interactions that are presumed to be mutually beneficial to a host and its microbial partner, a broader definition including both parasitic and mutualistic interactions recognizes that the fitness effects of many symbioses are complex and often context dependent. Whether an association is beneficial can depend on ecological conditions, and mutation and other evolutionary processes can result in symbiont strains that differ in terms of costs and benefits to hosts (Fig. 1).Open in a separate windowFig 1The symbiosis spectrum.The costs and benefits of symbiosis for hosts are not bimodal but span a continuum. The benefit to cost ratio is mediated both by environmental conditions and by the strain of symbiont. For example, the bacteria Hamiltonella defensa increases aphid resistance to parasitoid wasps. When Hamiltonella loses an associated bacteriophage, protection is lost. Also, in aphids, Buchnera aphidicola is a bacterial symbiont that provisions its hosts with critical nutritional resources. However, alterations of the heat shock promoter in Buchnera lessen the fitness benefit of symbiosis for the hosts under elevated temperatures. Amplification of a region of the Wolbachia genome known as Octomom causes the bacteria to shorten the lifespan of its Drosophila fly hosts.Elucidating the effects of host-associated microbes includes, when possible, experiments designed to assay host phenotypes when they do and do not have a particular symbiont of interest (Fig. 2). In systems in which hosts acquire symbionts from the environment, hosts can be reared in sterile conditions to prevent acquisition [1]. If symbionts are passed internally from mother to offspring, antibiotic treatments can sometimes be utilized to obtain lineages of hosts without symbionts [2]. The impacts of symbiont presence on survival, development, reproduction, and defense can be quantified, with the caveat that these impacts may be quite different under alternative environmental conditions. While such experiments are sometimes more tractable in systems with simple microbial consortia, the same experimental processes can be utilized in systems with more complex microbial communities [3,4].Open in a separate windowFig 2Approaches to functionally characterize symbiont effects.The first step in functionally characterizing the phenotypic impacts of a symbiont on its host is to measure phenotypes of hosts with and without symbionts. Any effects need to be considered in the light of how they are modified by environmental conditions. Understanding the mechanisms underlying symbiont alteration of host phenotype can involve, and often combines, genomic, genetic, and evolutionary approaches. Solid arrows indicate the path leading to results highlighted in Chrostek and Teixeira’s investigation of Wolbachia virulence in this issue of PLoS Biology.Once a fitness effect of symbiosis is ascertained, determining the mechanistic basis of this effect can be challenging. A genomics approach sometimes provides informative insight into microbial function. Sequencing of many insect-associated symbionts, for example, has confirmed the presence of genes necessary for amino acid and vitamin synthesis [5–8]. These genomic revelations, in some cases, can be linked to phenotypic effects of symbiosis for the hosts. For example, aphids reared in the absence of their obligate symbiotic bacteria, Buchnera aphidicola, can survive when provisioned with supplemental amino acids but cannot survive without supplementation, suggesting that Buchnera’s provisioning of amino acids is critical for host survival [9,10]. The Buchnera genome contains many of the genes necessary for amino acid synthesis [5].Linking genotype to phenotype, however, can be complicated. Experiments are necessary to functionally test the insights garnered from genome sequencing. For example, just because a symbiont has genes necessary for synthesis of a particular nutrient does not mean that the nutrient is being provisioned to its host. Furthermore, in many systems we do not know what genetic mechanisms are most likely to influence a symbiont-conferred phenotype. For example, if hosts associated with a given microbe have lower fitness than those without the microbe, what mechanism mediates this phenotype? Is it producing a toxin? Is it using too many host resources? In these cases, a single genome provides even less insight.Comparative genomics can be another approach. This requires collection of hosts with alternative symbiont strains and then testing these strains in a common host background to demonstrate that they have different phenotypic effects. Symbiont genomes can then be sequenced and compared to identify differences. This approach was utilized to compare genomes of strains of the aphid bacterial symbiont Regiella insecticola that confer different levels of resistance to parasitoid wasps [11]; the protective and nonprotective Regiella genome differed in many respects. Comparing the genomes of Wolbachia strains with differential impacts on fly host fitness [12,13] revealed fewer differences, though none involved a gene with a function known to impact host fitness. Comparative genomics rarely uncovers a holy grail as the genomes of symbiont strains with alternative phenotypic effects rarely differ at a single locus of known function.Another approach, which is at the heart of studies of microbial pathogens, is to use genetic tools to manipulate symbionts at candidate loci (or randomly through mutagenesis) and compare the phenotypic effects of genetically-manipulated and unmanipulated symbionts. Indeed, this approach has provided insights into genes underlying traits of both pathogenic [14] and beneficial [15,16] microbes. There is one challenge. Many host-associated symbionts are not cultivable outside of their hosts, which precludes utilization of most traditional genetic techniques used to modify microbial genomes.An alternative approach to studying symbiont function leverages evolution. Occasionally, lineages that once conferred some phenotypic effect, when tested later, no longer do. If symbiont samples were saved along the way, researchers can then determine what in the genome changed. For example, pea aphids (Acyrthosiphon pisum) harboring the bacteria Hamiltonella defensa are more resistant to parasitoid wasps than those without the bacteria [17,18]. Toxin-encoding genes identified in the genome of a Hamiltonella-associated bacteriophage were hypothesized to be central to this defense [18,19]. However, confirmation of the bacteriophage’s role required comparing the insects’ resistance to wasps when they harbored the same Hamiltonella with and without the phage. No Hamiltonella isolates were found in nature without the phage, but bottleneck passaging of the insects and symbionts generation after generation in the laboratory led to the loss of phage in multiple host lineages. Experimental assays confirmed that in the absence of phage, there was no protection [20]. Similarly, laboratory passaging of aphids and symbionts serendipitously led to spread of a mutation in the genome of Buchnera aphidicola, the primary, amino acid-synthesizing symbiont of pea aphids. The mutation, a single nucleotide deletion in the promoter for ibpA, a gene encoding for a heat-shock protein, lowers aphid fitness under elevated temperature conditions [21]. The mutation is found at low levels in natural aphid populations, suggesting that laboratory conditions facilitate maintenance of the genotype.In the above cases, evolution was a fortunate coincidence. In this issue of PLoS Biology, Chrostek and Teixeira (2014) illustrate another alternative, directed experimental evolution. Previous work demonstrated that a strain of the symbiotic bacterium Wolbachia, wMelPop, is virulent to its Drosophila melanogaster hosts, considerably shortening lifespan while overproliferating inside the flies [22]. To investigate the mechanism of virulence, researchers compared the genomic content of an avirulent Wolbachia strain to that of the virulent wMelPop [12,13]. These comparisons revealed that the wMelPop genome contains a region with eight genes that is amplified multiple times; in avirulent strains there is only a single copy. This eight gene region was nicknamed “Octomom.” To functionally test whether Octomom mediates Wolbachia virulence, over successive generations, Chrostek and Teixeira selected for females with either high or low Octomom copy numbers to start the next generations. They found that copy number could evolve rapidly and was correlated with virulence. Flies harboring wMelPop with more copies of Octomom had shorter lifespans. This cost was reversed in the presence of natural enemies; flies harboring wMelPop with more copies of Octomom had higher resistance to viral pathogens. Thus, selection provided a functional link between genotype and phenotype in a symbiont recalcitrant to traditional microbial genetics approaches.In many respects, this is similar to the research on aphids and their symbionts, where protective phenotypes were lost through passaging of aphids and symbionts generation after generation, as part of standard laboratory maintenance. Chrostek and Teixeira simply used the tools of experimental evolution to select for altered symbionts in a controlled fashion. Comparison of the studies also highlights two potential approaches—select for a phenotype and determine the genotypic change, or select for a genotype of interest and determine the phenotypic effect.Why do we need to know the genetic mechanisms underlying symbiont-conferred traits? In terms of evolutionary dynamics, the maintenance of a symbiont’s effect in a population is predicated on the likelihood of it being maintained in the presence of mutation, drift, and selection. Symbiosis research often considers how ecological conditions influence symbiont-conferred traits but less often considers the instability of those influences due to evolutionary change. From the perspective of applied applications to human concerns, symbiont alteration of insect phenotypes are potential mechanisms to reduce vectoring of human and agricultural pathogens, either through directly reducing insect fitness or reducing the capacity of vectors to serve as pathogen reservoirs [23–28]. Short term field trials, for example, have demonstrated spread and persistence of Wolbachia in mosquito populations [29,30]. Because Wolbachia reduce persistence of viruses, including human pathogens, in insects [26,31–33], this is a promising pesticide-free and drug-free control strategy for insect-vectored diseases. Can we assume that Wolbachia and other symbionts will always confer the same phenotypes to their hosts? If the conferred phenotype is based on a region of the genome where mutation is likely (e.g., the homopolymeric track within the heat shock promoter of aphid Buchnera, the Octomom region in Drosophila wMelPop), then we have clear reason to suspect that the genotypic and phenotypic makeup of the symbiont population could change over time. We need to investigate how populations of bacterial symbionts evolve in host populations under natural ecological conditions, carefully screening for both changes in phenotype and changes in genotype over the course of such experimental observations. We then need to incorporate evolutionary changes when modeling symbiont maintenance and when considering the use of symbionts in applied applications.     

Adenosine Signaling and the Energetic Costs of Induced Immunity

Life history theory predicts that trait evolution should be constrained by competing physiological demands on an organism. Immune defense provides a classic example in which immune responses are presumed to be costly and therefore come at the expense of other traits related to fitness. One strategy for mitigating the costs of expensive traits is to render them inducible, such that the cost is paid only when the trait is utilized. In the current issue of PLOS Biology, Bajgar and colleagues elegantly demonstrate the energetic and life history cost of the immune response that Drosophila melanogaster larvae induce after infection by the parasitoid wasp Leptopilina boulardi. These authors show that infection-induced proliferation of defensive blood cells commands a diversion of dietary carbon away from somatic growth and development, with simple sugars instead being shunted to the hematopoetic organ for rapid conversion into the raw energy required for cell proliferation. This metabolic shift results in a 15% delay in the development of the infected larva and is mediated by adenosine signaling between the hematopoietic organ and the central metabolic control organ of the host fly. The adenosine signal thus allows D. melanogaster to rapidly marshal the energy needed for effective defense and to pay the cost of immunity only when infected.While sitting around the campfire, evolutionary biologists may tell tales of the Darwinian Demon [1], a mythical being who is reproductively mature upon birth, lives forever, and has infinite offspring. In the morning, though, we know that no such creature can exist. All organisms must make compromises, and fitness is determined by striking the optimal balance among traits with competing demands. This is the central premise of life history theory: adaptations are costly, and increasing investment in one trait often forces decreased investment in others [2].Phenotype plasticity provides a partial solution to this evolutionary conundrum. If traits can be called upon only when required, the costs can be mitigated during periods of disuse. There are many examples of plastic costly adaptations, including the defensive helmets grown by Daphnia species in response to the presence of predators or abiotic factors that signal risk of predation ([3,4] and references therein), the flamboyant plumage that male birds exhibit during breeding season [5], and the inducible immune systems of higher plants and animals [6,7].The very inducibility of immune systems implicitly argues for their cost. If immune defense was cost-free, it would be constantly deployed for maximum protection against pathogenic infection. However, immune reactions have frequently been inferred to be energetically demanding (e.g., [8,9]) and carry the risk of autoimmune damage (e.g., [10,11]). It may therefore be evolutionarily adaptive to minimize immune activity in the absence of infection, to rapidly ramp up immunity in response to infection, and then to quickly shut down the immune response after the infection has been managed [12,13]. A paper by Bajgar et al. in the current issue of PLOS Biology [14] uses the Drosophila–Leptopilina host–pathogen system to quantitatively measure the energetic expense of induced up-regulation of immunity, demonstrating plastic metabolic reallocation toward immune cell proliferation at the expense of nutrient storage, growth, and development.Parasitoid wasps such as Leptopilina species infect their insect larval hosts by laying an egg inside the host body cavity [15]. Unimpeded, the egg hatches into a wasp larva that feeds off and develops inside the still-living host. In the case of Leptopilina boulardi infecting Drosophila melanogaster, the infected host larva survives to enter pupation, but if the parasitization is successful, an adult wasp will ultimately emerge from the pupal case instead of an adult fruit fly. This clearly is against the interest of the D. melanogaster host, so the larval fly attempts to encapsulate the L. boulardi egg in a sheath of specialized blood cells called lamellocytes that collaborate with other cell types to suffocate the wasp egg, deprive it of nutrients, and kill it with oxidative free radicals [16]. The struggle between fly and wasp is of the highest stakes, with guaranteed death and complete loss of evolutionary fitness for the loser.Natural D. melanogaster populations are rife with genetic variation for resistance to parasitoids, and laboratory selection for as few as five generations can increase host survivorship from 1%–5% to 40%–60% (e.g., [17,18]). This evolved resistance comes at a cost, though. Larvae from evolved resistant strains have decreased capacity to compete with their unselected progenitors under crowded or nutrient-poor conditions [17,18]. The general mechanism for enhanced resistance has been recurrently revealed to be an increase in the number of circulating blood cells (hemocytes). But why are the resistant larvae outcompeted by susceptible larvae? One compelling hypothesis is that the extra investment in blood cell proliferation comes at an energetic cost to development of other tissues, a cost which may be exacerbated by a decrease in feeding rate of the selected lines [19] and that leads to impaired development under nutrient-limiting conditions. The cost can be limited, however, by producing the defensive blood cells in large numbers only when the host is infected and has need of them. Effectively achieving this requires the capacity to rapidly signal cell proliferation and to recruit the energy required for hematopoiesis from other physiological processes.Bajgar et al. [14] use a series of careful experiments to document energetic redistributions and costs associated with hemocyte proliferation and lamellocyte differentiation in D. melanogaster infected by L. boulardi. They find that parasitoid infection results in a 15% delay in host development, and that at least a fraction of this delay can be plausibly attributed to a metabolic reallocation that supports blood cell proliferation over somatic growth. Specifically, they find that dietary carbohydrates are shunted away from energetic storage and tissue development, and instead are routed to the lymph gland, where hemocytes are being produced and differentiated. The signal to activate this reallocation emanates from the lymph gland and developing hemocytes themselves in the form of secreted adenosine, which is then received and interpreted by the central metabolic control organ, the fat body. Bajgar et al. [14] are able to show in unprecedented quantitative precision, that secreted adenosine acts as a signal to rapidly trigger inducible immune defense, that this immune induction is costly at the level of individual tissues and the whole organism, and that the cost of induced defense is paid only upon infection.The D. melanogaster metabolic rearrangement after infection by L. boulardi is profound [14]. Within 6–18 hours of infection, host larvae show a strong reduction in the incorporation of dietary carbon into stored lipids and protein and an overall reduction in glycogen stores. The levels of circulating glucose and trehalose spike, with those saccharides seemingly directed to the hemocyte-producing lymph gland. Expression of glycolytic genes is suppressed in the fat body, while fat body expression of a trehalose transporter is up-regulated to promote trehalose secretion into circulation. Glucose and trehalose transporters are concomitantly up-regulated in the lymph gland and developing hemocytes to facilitate import of the circulating saccharides. Genes required for glycolysis—but not the TCA cycle—are simultaneously up-regulated in the lymph gland and nascent hemocytes to turn those sugars into quick energy under the Warburg effect [20]. The totality of the data is consistent with an interpretation that the induced hemocyte proliferation is directly costly to host larval growth as an immediate consequence of substantial energetic reallocation.This same research group had previously shown that extracellular adenosine can be used by Drosophila as a signal in regulating inflammation-like responses [21]. In the present study, they use RNAi to knock down expression of the adenosine transporter ENT2 specifically in the lymph gland and developing hemocytes. This prevention of adenosine export eliminates the spike in circulating glucose and trehalose after infection. Infected larvae with knocked down expression of ENT2 continue to allocate dietary carbon to lipid and proteins as though they were uninfected, they fail to differentiate an adequate number of lamellocytes, and they suffer reduced resistance to the parasitoid. These data firmly implicate extracellular adenosine as a critical trigger mediating the switch in energetic allocation from growth and development to induced immunity. In further support of this interpretation, larvae that are mutant for the adenosine receptor AdoR show a nearly 70% reduction relative to wild type in the number of differentiated lamellocytes after infection and consequently encapsulate the parasitoid egg at nearly 4-fold lower rates. Like the ENT2 knockdowns, AdoR mutant larvae fail to exhibit elevated levels of circulating glucose or trehalose when infected by L. boulardi, although surprisingly, the AdoR mutants do show an appropriate inhibition of glycogen storage when infected. Overall, the genetic results indicate that ENT2 protein allows secretion of adenosine from the lymph gland. This adenosine signal is received in the fat body, which then dampens energetic storage in favor of secretion and circulation of simple saccharides. These sugars are taken up by the lymph gland to rapidly facilitate hemocyte proliferation, lamellocyte differentiation, and immune defense against the parasitoid. In satisfying consistency with this model, Bajgar et al. [14] show that lamellocyte differentiation and host resistance can be partially rescued even in the absence of ENT2 or AdoR by supplementing the D. melanogaster diet with extra glucose, thereby providing the boost in the circulating saccharides required for hematopoiesis.There are key differences between studies of evolutionary tradeoffs, such as those conducted by Kraaijeveld and colleagues (e.g., [17–19]), and studies of physiological costs such as the one conducted by Bajgar et al. [14]. The evolutionary tradeoffs exposed by experimental selection are experienced even in the absence of parasitoid infection. Blood cell number is constitutively higher in the evolved resistant strains, which presumably allows a faster and more robust defense against a wasp egg. However, the resistant larvae always suffer the cost when reared in competitive conditions. Because the costs arise from a constitutive investment in defense, regardless of whether parasitoids are present, these are sometimes referred to as “maintenance” costs or fixed costs [22]. In contrast, Bajgar et al. have measured a physiological “deployment” cost [22], which is conditionally experienced only once the host activates an immune response.It remains an open question the extent to which maintenance costs and deployment costs share mechanistic bases. In the present example, Bajgar et al. [14] show clearly that the machinery is in place to route energetic investment away from growth and development in favor of hemocyte proliferation. Extrapolating from the laboratory selection experiments [17–19], a natural increase in the epidemiological risk of parasitization in the wild might favor greater constitutive investment in hemocyte production. In such a scenario, it is easy to imagine the adaptive value of a mutation or genetic variant that results in higher expression of ENT2 in the lymph gland in the absence of infection, driving constitutively higher hemocyte number and protection against infection at the expense of constitutively lower nutrient storage and growth. In this way, a plastic switch could be converted into a hardwired trait, a deployment cost would become a maintenance cost, and a physiological tradeoff would become an evolutionary one. However, it is important to appreciate that there typically are many solutions to any biological problem, and constitutively elevated blood cell number could also evolve via mechanisms unrelated to adenosine signaling. A key challenge for life history biologists will be to bridge physiological studies such as that by Bajgar and colleagues [14] with the evolutionary studies that preceded it, testing whether physiological and evolutionary tradeoffs share a mechanistic basis.While in the bright light of day, evolutionary biologists can agree that the Darwinian Demon is just a ghost story, we have precious few examples of evolutionary or physiological tradeoffs where the mechanistic bases are well understood. More careful quantitative and genetic studies like that of Bajgar et al. [14] are necessary to carry us beyond reliance on abstract concepts like “resource pools” [2] and into mechanistic understanding of how life history tradeoffs operate on both the physiology of individuals and the evolution of species.     

The Reticular Cell Network: A Robust Backbone for Immune Responses

Lymph nodes are meeting points for circulating immune cells. A network of reticular cells that ensheathe a mesh of collagen fibers crisscrosses the tissue in each lymph node. This reticular cell network distributes key molecules and provides a structure for immune cells to move around on. During infections, the network can suffer damage. A new study has now investigated the network’s structure in detail, using methods from graph theory. The study showed that the network is remarkably robust to damage: it can still support immune responses even when half of the reticular cells are destroyed. This is a further important example of how network connectivity achieves tolerance to failure, a property shared with other important biological and nonbiological networks.Lymph nodes are critical sites for immune cells to connect, exchange information, and initiate responses to foreign invaders. More than 90% of the cells in each lymph node—the T and B lymphocytes of the adaptive immune system—only reside there temporarily and are constantly moving around as they search for foreign substances (antigen). When there is no infection, T and B cells migrate within distinct regions. But lymph node architecture changes dramatically when antigen is found, and an immune response is mounted. New blood vessels grow and recruit vast numbers of lymphocytes from the blood circulation. Antigen-specific cells divide and mature into “effector” immune cells. The combination of these two processes—increased influx of cells from outside and proliferation within—can make a lymph node grow 10-fold within only a few days [1]. Accordingly, the structural backbone supporting lymph node function cannot be too rigid; otherwise, it would impede this rapid organ expansion. This structural backbone is provided by a network of fibroblastic reticular cells (FRCs) [2], which secrete a form of collagen (type III alpha 1) that produces reticular fibers—thin, threadlike structures with a diameter of less than 1 μm. Reticular fibers cross-link and form a spider web–like structure. The FRCs surrounding this structure form the reticular cell network (Fig 1), which was first observed in the 1930s [3]. Interestingly, experiments in which the FRCs were destroyed showed that the collagen fiber network remained intact [4].Open in a separate windowFig 1Structure of the reticular cell network.The reticular cell network is formed by fibroblastic reticular cells (FRCs) whose cell membranes ensheathe a core of collagen fibers that acts as a conduit system for the distribution of small molecules [5]. In most other tissues, collagen fibers instead reside outside cell membranes, where they form the extracellular matrix. Inset: graph structure representing the FRCs in the depicted network as “nodes” (circles) and the direct connections between them as “edges” (lines). Shape and length of the fibers are not represented in the graph.Reticular cell networks do not only support lymph node structure; they are also important players in the immune response. Small molecules from the tissue environment or from pathogens, such as viral protein fragments, can be distributed within the lymph node through the conduit system formed by the reticular fibers [5]. Some cytokines and chemokines that are vital for effective T cell migration—and the nitric oxide that inhibits T cell proliferation [6]—are even produced by the FRCs themselves. Moreover, the network is thought of as a “road system” for lymphocyte migration [7]: in 2006, a seminal study found that lymphocytes roaming through lymph nodes were in contact with network fibers most of the time [8]. A few years before, it had become possible to observe lymphocyte migration in vivo by means of two-photon microscopy [9]. Movies from these experiments strikingly demonstrated that individual cells were taking very different paths, engaging in what appeared to be a “random walk.” But these movies did not show the structures surrounding the migrating cells, which created an impression of motion in empty space. Appreciating the role of the reticular cell network in this pattern of motion [8] suggested that the complex cell trajectories reflect the architecture of the network along which the cells walk.Given its important functions, it is surprising how little we know about the structure of the reticular cell network—compared to, for instance, our wealth of knowledge on neuron connectivity in the brain. In part this is because the reticular cells are hard to visualize. In vivo techniques like two-photon imaging do not provide sufficient resolution to reliably capture the fine-threaded mesh. Instead, thin tissue sections are stained with fluorescent antibodies that bind to the reticular fibers and are imaged with high-resolution confocal microscopy to reveal the network structure. One study [10] applied this method to determine basic parameters such as branch length and the size of gaps between fibers. Here, we discuss a recent study by Novkovic et al. [11] that took a different approach to investigating properties of the reticular cell network structure: they applied methods from graph theory.Graph theory is a classic subject in mathematics that is often traced back to Leonhard Euler’s stroll through 18th-century Königsberg, Prussia. Euler could not find a circular route that crossed each of the city’s seven bridges exactly once, and wondered how he could prove that such a route does not exist. He realized that this problem could be phrased in terms of a simple diagram containing points (parts of the city) and lines between them (bridges). Further detail, such as the layout of city’s streets, was irrelevant. This was the birth of graph theory—the study of objects consisting of points (nodes) connected by lines (edges). Graph theory has diverse applications ranging from logistics to molecular biology. Since the beginning of this century, there has been a strong interest in applying graph theory to understand the structure of networks that occur in nature—including biological networks, such as neurons in the brain, and more recently, social networks like friendships on Facebook. Various mathematical models of network structures have been developed in an attempt to understand network properties that are relevant in different contexts, such as the speed at which information spreads or the amount of damage that a network can tolerate before breaking into disconnected parts. Three well-known network topologies are random, small-world, and scale-free networks (Box 1). Novkovic et al. modeled reticular cell networks as graphs by considering each FRC to be a node and the fiber connections between FRCs to be edges (Fig 1).

Box 1. Graph Theory and the Robustness of Real Networks

After the publication of several landmark papers on network topology at the end of the previous century, the science of complex networks has grown explosively. One of these papers described “small-world” networks [16] and demonstrated that several natural networks have the amazing property that the average length of shortest paths between arbitrary nodes is unexpectedly small (making it a “small world”), even if most of the network nodes are clustered (that is, when neighbors of neighbors tend to be neighbors). The Barabasi group published a series of papers describing “scale-free” networks [17,18] and demonstrated that scale-free networks are extremely robust to random deletions of nodes—the vast majority of the nodes can be deleted before the network falls apart [15]. In scale-free networks, the number of edges per node is distributed according to a power law, implying that most nodes have very few connections, and a few nodes are hubs having very many connections. Thus, the topology of complex networks can be scale-free, small-world, or neither, such as with random networks [19]. Novkovic et al. [11] describe the clustering of the edges of neighbors and the average shortest path–length between arbitrary nodes, finding that reticular cell networks have small-world properties. Whether or not these networks have scale-free properties is not explicitly examined in the paper, but given that they are embedded in a three-dimensional space, that they “already” lose functionality when about 50% of the FRCs are ablated, and that the number of connected protrusions per FRC is not distributed according to a power law (see the data underlying their Figure 2g), reticular cell networks are not likely to be scale-free. Thus, the enhanced robustness of reticular cell networks is most likely due to their high local connectivity: Networks lose functionality when they fall apart in disconnected components, and high clustering means that the graph is unlikely to split apart when a single node is removed, because the neighbors of that node tend to stay connected [14]. Additionally, since the reticular cell network has a spatial structure (unlike the internet or the Facebook social network), its high degree of clustering is probably due to the preferential attachment to nearby FRCs when the network develops, which agrees well with Novkovic et al.’s recent classification as a small-world network with lattice-like properties [11].Some virus infections are known to damage reticular cell networks [12], either through infection of the FRCs or as a bystander effect of inflammation. It is therefore important to understand to what extent the network structure is able to survive partial destruction. Novkovic et al. first approached this question by performing computer simulations, in which they randomly removed FRC nodes from the networks they had reconstructed from microscopy images. They found that they had to remove at least half of the nodes to break the network apart into disconnected parts. To study the effect of damage on the reticular cell network in vivo rather than in silico, Novkovic et al. used an experimental technique called conditional cell ablation. In this technique, a gene encoding the diphtheria toxin receptor (DTR) is inserted after a specific promoter that leads it to be expressed in a particular cell type of interest. Administration of diphtheria toxin kills DTR-expressing cells, leaving other cells unaffected. By expressing DTR under the control of the FRC-specific Ccl19 promoter, Novkovic et al. were able to selectively destroy the reticular cell network and then watch it grow back over time. Regrowth took about four weeks, and the resulting network properties were no different from a network formed naturally during development. Thus, it seems that the reticular cell network structure is imprinted and reemerges even after severe damage. This finding ties in nicely with previous data from the same group [13], showing that reticular cell networks form even in the absence of lymphotoxin-beta receptor, an otherwise key player in many aspects of lymphoid tissue development. Together, these data make a compelling case that network formation is a robust fundamental trait of FRCs.Next, Novkovic et al. varied the dose of diphtheria toxin such that only a fraction of FRCs were destroyed, effectively removing a random subset of the network nodes. They measured in two ways how FRC loss affects the immune system: they tracked T cell migration using two-photon microscopy and they determined the amount of antiviral T cells produced by the mice after an infection. Remarkably, as predicted by their computer simulations, lymph nodes appeared capable of tolerating the loss of up to half of FRCs with little effect on either T cell migration or the numbers of activated antiviral T cells. Only when more than half of the FRCs were destroyed did T cell motion slow down significantly and the mice were no longer able to mount effective antiviral immune responses. Such a tolerance of damage is impressive—for comparison, consider what would happen if one were to close half of London’s subway stations!Robustness to damage is of interest for many different networks, from power grids to the internet [14]. In particular, the “scale-free” architecture that features rare, strongly connected “hub” nodes is highly robust to random damage [15]. Novkovic et al. did not address whether the reticular cell network is scale-free, but it is likely that it isn’t (Box 1). Instead, the network’s robustness probably arises from its high degree of clustering, which means that the neighbors of each node are likely to be themselves also neighbors. If a node is removed from a clustered network, then there is still likely a short detour available by going through two of the neighbors. Therefore, one would have to randomly remove a large fraction of the nodes before the network structure breaks down. High clustering in the network could be a consequence of the fact that multiple fibers extend from each FRC and establish connections to many FRCs in its vicinity. A question not yet addressed by Novkovic et al. is how robust reticular cell networks would be to nonrandom damage, such as a locally spreading viral infection. In fact, scale-free networks are drastically more vulnerable to targeted rather than random damage: the United States flight network can come to a grinding halt by closing a few hub airports [15]. Less is known about the robustness to nonrandom damage for other network architectures, and the findings by Novkovic et al. motivate future research in this direction.Novkovic et al. did not yet explicitly identify all mechanisms that hamper T cell responses when more than half of the FRCs are depleted. But given the reticular cell network’s many different functions, this could occur in several ways. For instance, severe depletion might prevent the secretion of important molecules, halt the migration of T cells, prevent the anchoring of antigen-presenting dendritic cells (DCs) on the network, or cause structural disarray in the tissue. In addition to the effects on T cell migration, Novkovic et al. also showed that the amount of DCs in fact decreased when FRCs were depleted, emphasizing that several mechanisms are likely at play. Disentangling these mechanisms will require substantial additional research efforts.The current reticular cell network reconstruction by Novkovic et al. is based on thin tissue slices. It will be exciting to study the network architecture when it can be visualized in the whole organ. Some aspects of the network may then look different. For instance, those FRCs that are near the border of a slice will have their degree of connectivity underestimated, as not all of their neighbors in the network can be seen. Further refinements of the network analysis may also consider that reticular fibers are real physical objects situated in a three-dimensional space (unlike abstract connections such as friendships). Migrating T cells may travel quicker via a short, straight fiber than on a long, curved one, but the network graph does not make this distinction. More generally, it would be interesting to understand conceptually how reticular cell networks help foster immune cell migration. While at first it appears obvious that having a “road system” should make it easier for cells to roam lymph node tissue, three different theoretical studies have in fact all concluded that effective T cell migration should also be possible in the absence of a network [20–22]. A related question is whether T cells are constrained to move only on the network or are merely using it for loose guidance. For instance, could migrating T cells be in contact with two or more network fibers at once or with none at all? This would make the relationship between cell migration and network structure more complex than the graph structure alone suggests.There is also some evidence that T cells can migrate according to what is called a Lévy walk [23]—a kind of random walk where frequent short steps are interspersed with few very long steps, a search strategy that appears to occur frequently in nature (though this is debated [24]). While there is so far no evidence that T cells perform a Lévy walk when roaming the lymph node [25], this may be in part due to limitations of two-photon imaging, and one could speculate that reticular cell networks might in fact be constructed in a way that facilitates this or another efficient kind of “search strategy.” Resolving this question will require substantial improvements in imaging technology, allowing individual T cells to be tracked across an entire lymph node.No doubt further studies will address these and other questions, and provide further insights on how reticular cell networks benefit immune responses. Such advances may help us design better treatments against infections that damage the network. It may also help us understand how we can best administer vaccines or tumor immune therapy treatments in a way that ensures optimal delivery to immune cells in the lymph node. As is nicely illustrated by the study of Novkovic et al., mathematical methods may well play key roles in this quest.     

From Dinosaurs to Modern Bird Diversity: Extending the Time Scale of Adaptive Radiation

What explains why some groups of organisms, like birds, are so species rich? And what explains their extraordinary ecological diversity, ranging from large, flightless birds to small migratory species that fly thousand of kilometers every year? These and similar questions have spurred great interest in adaptive radiation, the diversification of ecological traits in a rapidly speciating group of organisms. Although the initial formulation of modern concepts of adaptive radiation arose from consideration of the fossil record, rigorous attempts to identify adaptive radiation in the fossil record are still uncommon. Moreover, most studies of adaptive radiation concern groups that are less than 50 million years old. Thus, it is unclear how important adaptive radiation is over temporal scales that span much larger portions of the history of life. In this issue, Benson et al. test the idea of a “deep-time” adaptive radiation in dinosaurs, compiling and using one of the most comprehensive phylogenetic and body-size datasets for fossils. Using recent phylogenetic statistical methods, they find that in most clades of dinosaurs there is a strong signal of an “early burst” in body-size evolution, a predicted pattern of adaptive radiation in which rapid trait evolution happens early in a group''s history and then slows down. They also find that body-size evolution did not slow down in the lineage leading to birds, hinting at why birds survived to the present day and diversified. This paper represents one of the most convincing attempts at understanding deep-time adaptive radiations.

“It is strikingly noticeable from the fossil record and from its results in the world around us that some time after a rather distinctive new adaptive type has developed it often becomes highly diversified.” – G. G. Simpson ([1], pp. 222–223)

George Gaylord Simpson was the father of modern concepts of adaptive radiation—the diversification of ecological traits in a rapidly speciating group of organisms (Figure 1; [2]). He considered adaptive radiation to be the source of much of the diversity of living organisms on planet earth, in terms of species number, ecology, and body form [1]–[3]. Yet more than 60 years after Simpson''s seminal work, the exact role of adaptive radiation in generating life''s extraordinary diversity is still an open and fundamental question in evolutionary biology [3],[4].Open in a separate windowFigure 1An example of adaptive radiation and early bursts in rates of speciation and phenotypic evolution.(a) The adaptive radiation of the modern bird clade Vanginae, which shows early rapid speciation, morphological diversity, and diversity in foraging behavior and diet [15],[32]. (b) Hypothetical curve of speciation rates through time that would be expected in adaptive radiation. The exponential decline in speciation rates shows that there was an “early burst” of speciation at the beginning of the clade''s history. (c) Hypothetical curve of rates of phenotypic evolution through time that would be expected in adaptive radiation, also showing an early burst of evolution with high initial rates. Part (a) is reproduced from [32] with permission (under CC-BY) from the Royal Society and the original authors.To address this question, researchers have looked for signatures of past adaptive radiation in the patterns of diversity in nature. In particular, it has been suggested that groups that have undergone adaptive radiation should show an “early-burst” signal in both rates of lineage diversification and phenotypic evolution through time—a pattern in which rates of speciation and phenotypic evolution are fast early in the history of groups and then decelerate over time (Figure 1; [3]–[5]). These predictions arise from the idea that clades should multiply and diversify rapidly in species number, ecology, and phenotype in an adaptive radiation and that rates of this diversification should decrease later as niches are successively occupied [2].Early bursts have been sought in both fossils and phylogenies. Few fossil studies have discussed their results in the context of adaptive radiation (but see [6]), but they often have found rapid rises in both taxonomic and morphological diversity early in the history of various groups [7], ranging from marine invertebrates [8] to terrestrial mammals [9]. However, fossils often lack the phylogeny needed to model how evolution has proceeded [7]. On the other hand, studies that test for early bursts in currently existing (extant) species typically use phylogenies, which allow us to model past evolution in groups with few or no fossils [5]. Phylogenies have most often been used to test early bursts in speciation (see, e.g., [10]). However, such tests may be misled by past extinction, which will decay the statistical signal of rapid, early diversification [11]. Furthermore, diverse evolutionary scenarios beyond adaptive radiation can give rise to early bursts in speciation [12]. By contrast, studies of phenotypic diversification may be more robust to extinction [13] and they test the distinguishing feature that separates adaptive from nonadaptive radiation [2],[12].Thus, studies of adaptive radiation in extant organisms increasingly have focused on phylogenetic tests of the early-burst model of phenotypic evolution. Some studies show strong support for this prediction in both birds [14],[15] and lizards [5],[16]. However, the most extensive study to date showed almost no support for the early-burst model. In this study, Harmon et al. [17] examined body size in 49 (and shape in 39) diverse groups of animals, including invertebrates, fishes, amphibians, reptiles, birds, and mammals. They found strong support for the early-burst model in only two of these 88 total datasets.This result raises an important question: if adaptive radiation explains most of life''s diversity [1], how is it possible that there is so little phylogenetic evidence for early bursts of phenotypic evolution? One possibility is that early bursts are hard to detect. This can be due to low statistical power in the most commonly employed tests [18]. It may also be due to a lack of precision in the way “early burst” is defined (and thus tested), as the ecological theory of adaptive radiation suggests that the rate of phenotypic evolution will decrease as species diversity increases in a group, not just over time [14],[16]. Indeed, recent studies [14],[16] detected a decline in rates with species diversity in clades that were also in the Harmon et al. [17] study, yet for which no decline over time was detected.A second possible reason for why early-burst patterns are uncommon is more fundamental: the patterns of phenotypic diversity that result from adaptive radiation may be different at large time scales. Many of the best examples of adaptive radiation are in groups that are relatively young, including Darwin''s finches (2.3 million years old [myr]; [19]) and Lake Malawi and Victoria cichlids (2.3 myr; [20]), whereas most groups that are examined for early bursts in phenotypic evolution are much older (e.g., 47 of 49 in Harmon et al. [17]; mean ± sd = 23.8±29.2 myr). So there may be an inherent difference between what unfolds over the relatively short time scales emphasized by Schluter [2] and what one sees at macroevolutionary time scales (see [21] for an in-depth discussion of this idea as it relates to speciation).The time scale over which adaptive radiations unfold has been little explored. As a result, the link between extant diversity and major extinct radiations remains unclear. Simpson [1] believed that adaptive radiation played out at the population level, but that it should manifest itself at larger scales as well—up to phyla (e.g., chordates, arthropods). He suggested that we should see signals of adaptive radiations in large, old clades because they are effectively small-scale adaptive radiation writ large [1]. Under this view, we should see the signal of adaptive radiation even in groups that diversified over vast time scales, particularly if adaptive radiation is as important for explaining life''s diversity as Simpson [1] thought it was.Part of the reason why potential adaptive radiations at deep time scales remain poorly understood is that studies either focus on fossils or phylogenies, but rarely both. In this issue, Benson et al. [22] combine these two types of data to address whether dinosaurs show signs that they adaptively radiated. Unlike most other studies, the temporal scale of the current study is very large—in this case, over 170 million years throughout the Mesozoic era, starting at 240 million years ago in the Triassic period. This characteristic allowed Benson et al. to shed light on deep-time adaptive radiation.The authors estimated body mass from fossils by using measurements of the circumference of the stylopodium shaft (the largest bone of the arm or leg, such as the femur), which shows a consistent scaling relationship with body mass in extant reptiles and mammals [23]. They then combined published phylogenies to obtain a composite phylogeny for the species in their body-size dataset. The authors finally conducted two types of tests of the rate of body-size evolution—tests of early bursts in phenotypic evolution that are the same as those of Harmon et al. [17], as well as an additional less commonly used test that estimates whether differences between estimated body size at adjacent phylogenetic nodes decreases over time.Benson et al. [22] found two striking results. First, in both of their analyses, the early-burst model was strongly supported for most clades of dinosaurs. This early burst began in the Triassic period, indicating that diversification in body size in dinosaurs began before the Triassic-Jurassic mass extinction event would have opened competition-free ecological space (as commonly hypothesized; [24],[25]). Rather, the authors [22] suggest that a key innovation led to this rise in dinosaurs, though it is not clear what this innovation was [26]. In general, though, the finding of an early burst in body-size evolution in most dinosaurs—if a consequence of adaptive evolution—suggests that adaptive radiation may play out over large evolutionary time scales, not just on the short time scales typical of the most well-studied cases of extant groups.Second, one clade—Maniraptora, which is the clade in which modern-day birds are nested—was the only part of the dinosaur phylogeny that did not show such a strong early burst in body-size evolution. Instead, this clade fit a model to a single adaptive peak—an optimum body size, if you will—but also maintained high rates of undirected body-size evolution throughout their history. Benson et al. [22] suggest that this last result connects deep-time adaptive radiation in the dinosaurs, which quickly exhausted the possibility of phenotypic space, with the current radiation in extant birds, which survived to the present day because their constant, high rate of evolution meant that they were constantly undergoing ecological innovation. This gives a glimpse into why modern birds have so many species (an order of magnitude higher than the nonavian dinosaurs) and so much ecological diversity.The use of fossils allowed Benson et al. [22] to address deep-time radiation in dinosaurs and its consequence on present-day bird diversity. Nevertheless, the promise of using fossils to understand adaptive radiation has its limits. The paleontological dataset presented here is exceptional, yet still insufficient to explore major components of adaptive radiations like actual ecological diversification. As in many paleontological studies, Benson et al. used body-size data to represent ecology because body size is one of the few variables that is available for most species. But it is unclear how important body size really is for ecological diversification and niche filling, because body size is important for nearly every aspect of organismal function. Consequently, evolutionary change in body size can result not only from the competition that drives adaptive radiation, but also from predation pressure, reproductive character displacement, and physiological advantages of particular body sizes in a given environment, among other reasons [27].Despite the broad coverage of extinct species presented in Benson et al. [22], the data were insufficient to study another major part of adaptive radiation: early bursts of lineage diversification. While new approaches are becoming available to study diversification with phylogenies containing extinct species [28],[29] or with incomplete fossil data [30], these approaches are limited when many taxa are known from only single occurrences. This is the case in the Benson et al. dataset, and more generally in most fossil datasets.Given that few fossils exist for many extant groups, a major goal for future studies will be the incorporation of incomplete fossil information into analyses primarily focused on traits and clades for which mostly neontological data are available. For example, Slater et al. [31] developed an approach to include fossil information in analyses of phenotypic evolution. They showed that adding just a few fossils (12 fossils in a study of a 135-species clade) drastically increased the power and accuracy of their analyses of extant taxa. Thus, the combination of fossil data and those based on currently living species is important for future studies, as are new approaches that allow analyzing early bursts of lineage diversification along with phenotypic evolution in fossils.So what answers do Benson et al. [22] bring to Simpson''s original question of the importance of adaptive radiation for explaining diversity on earth? The authors present an intriguing and unconventional link between adaptive radiation and the diversity of modern-day birds. They argue that bird diversification was possible because the dinosaur lineage leading to birds did not exhaust niche space, potentially thanks to small body sizes; in contrast, other dinosaur groups adaptively radiated, filled niche space, and thus could not produce the ecological innovation that may have been necessary to survive the Cretaceous-Paleogene mass extinction. This intriguing hypothesis suggests an important role for the relative starting points of successive adaptive radiations in explaining current diversity, giving a new spin to the pivotal question raised by Simpson more than 60 years ago.     

Peer-Led Team Learning Helps Minority Students Succeed

Active learning methods have been shown to be superior to traditional lecture in terms of student achievement, and our findings on the use of Peer-Led Team Learning (PLTL) concur. Students in our introductory biology course performed significantly better if they engaged in PLTL. There was also a drastic reduction in the failure rate for underrepresented minority (URM) students with PLTL, which further resulted in closing the achievement gap between URM and non-URM students. With such compelling findings, we strongly encourage the adoption of Peer-Led Team Learning in undergraduate Science, Technology, Engineering, and Mathematics (STEM) courses.Recent, extensive meta-analysis of over a decade of education research has revealed an overwhelming consensus that active learning methods are superior to traditional, passive lecture, in terms of student achievement in post-secondary Science, Technology, Engineering, and Mathematics (STEM) courses [1]. In light of such clear evidence that traditional lecture is among the least effective modes of instruction, many institutions have been abandoning lecture in favor of “flipped” classrooms and active learning strategies. Regrettably, however, STEM courses at most universities continue to feature traditional lecture as the primary mode of instruction.Although next-generation active learning classrooms are becoming more common, large instructor-focused lecture halls with fixed seating are still the norm on most campuses—including ours, for the time being. While there are certainly ways to make learning more active in an amphitheater, peer-interactive instruction is limited in such settings. Of course, laboratories accompanying lectures often provide more active learning opportunities. But in the wake of commendable efforts to increase rigorous laboratory experiences at the sophomore and junior levels at Syracuse University, a difficult decision was made for the two-semester, mixed-majors introductory biology sequence: the lecture sections of the second semester course were decoupled from the laboratory component, which was made optional. There were good reasons for this change, from both departmental and institutional perspectives. However, although STEM students not enrolling in the lab course would arguably be exposed to techniques and develop foundational process skills in the new upper division labs, we were concerned about the implications for achievement among those students who would opt out of the introductory labs. Our concerns were apparently warranted, as students who did not take the optional lab course, regardless of prior achievement, earned scores averaging a letter grade lower than those students who enrolled in the lab. However, students who opted out of the lab but engaged in Peer-Led Team Learning (PLTL) performed at levels equivalent to students who also took the lab course [2].Peer-Led Team Learning is a well-defined active learning model involving small group interactions between students, and it can be used along with or in place of the traditional lecture format that has become so deeply entrenched in university systems (Fig 1, adapted from [3]). PLTL was originally designed and implemented in undergraduate chemistry courses [4,5], and it has since been implemented in other undergraduate science courses, such as general biology and anatomy and physiology [6,7]. Studies on the efficacy of PLTL have shown improvements in students’ grade performance, attitudes, retention in the course [6–11], conceptual reasoning [12], and critical thinking [13], though findings related to the critical thinking benefits for peer leaders have not been consistent [14].Open in a separate windowFig 1The PLTL model.In the PLTL workshop model, students work in small groups of six to eight students, led by an undergraduate peer leader who has successfully completed the same course in which their peer-team students are currently enrolled. After being trained in group leadership methods, relevant learning theory, and the conceptual content of the course, peer leaders (who serve as role models) work collaboratively with an education specialist and the course instructor to facilitate small group problem-solving. Leaders are not teachers. They are not tutors. They are not considered to be experts in the content, and they are not expected to provide answers to the students in the workshop groups. Rather, they help mentor students to actively construct their own understanding of concepts.     

Aging in a Long-Lived Clonal Tree

From bacteria to multicellular animals, most organisms exhibit declines in survivorship or reproductive performance with increasing age (“senescence”) [1],[2]. Evidence for senescence in clonal plants, however, is scant [3],[4]. During asexual growth, we expect that somatic mutations, which negatively impact sexual fitness, should accumulate and contribute to senescence, especially among long-lived clonal plants [5],[6]. We tested whether older clones of Populus tremuloides (trembling aspen) from natural stands in British Columbia exhibited significantly reduced reproductive performance. Coupling molecular-based estimates of clone age with male fertility data, we observed a significant decline in the average number of viable pollen grains per catkin per ramet with increasing clone age in trembling aspen. We found that mutations reduced relative male fertility in clonal aspen populations by about 5.8×10⁻⁵ to 1.6×10⁻³ per year, leading to an 8% reduction in the number of viable pollen grains, on average, among the clones studied. The probability that an aspen lineage ultimately goes extinct rises as its male sexual fitness declines, suggesting that even long-lived clonal organisms are vulnerable to senescence.     

Designing and implementing a research integrity promotion plan: Recommendations for research funders

Various stakeholders in science have put research integrity high on their agenda. Among them, research funders are prominently placed to foster research integrity by requiring that the organizations and individual researchers they support make an explicit commitment to research integrity. Moreover, funders need to adopt appropriate research integrity practices themselves. To facilitate this, we recommend that funders develop and implement a Research Integrity Promotion Plan (RIPP). This Consensus View offers a range of examples of how funders are already promoting research integrity, distills 6 core topics that funders should cover in a RIPP, and provides guidelines on how to develop and implement a RIPP. We believe that the 6 core topics we put forward will guide funders towards strengthening research integrity policy in their organization and guide the researchers and research organizations they fund.

Research funders are prominently placed to foster research integrity by requiring that researchers make an explicit commitment to research integrity. This Consensus View suggests 6 core topics that funders should cover in a research integrity promotion plan and provides practical recommendations for how to implement one.

To improve research quality and validity, foster responsible research cultures, and maintain public trust in science, various stakeholders have put research integrity high on their agenda. Among them, research funders are increasingly acknowledging their pivotal role in contributing to a culture of research integrity. For example, the European Commission (EC) is mandating research organizations receiving funding from the €95 billion Horizon Europe program to have, at the institutional level, policies and processes in place for research integrity covering the promotion of good practice, prevention of misconduct and questionable practices, and procedures to deal with breaches of research integrity [1]. To meet these obligations, the EC requires beneficiaries to respect the principles of research integrity as set out in the European Code of Conduct for Research Integrity (ECoC) and suggests that research organizations develop and implement a Research Integrity Promotion Plan (RIPP) [2]. In this Consensus View, we have adopted the World Conference on Research Integrity’s approach to research integrity, by having “research integrity” refer to “the principles and standards that have the purpose to ensure validity and trustworthiness of research” [3]. More specifically, we mostly adhere to the principles outlined in the ECoC: reliability, honesty, respect, and accountability. While many definitions of research integrity exist [4,5], for example, those that distinguish between the integrity of a researcher, integrity of research, and integrity of the research record, the ECoC combines these approaches in a balanced way [1].We believe that funders are prominently placed to foster a culture of research integrity by requiring that the organizations and individual researchers they support make an explicit commitment to research integrity. At the same time, funders need to adopt appropriate research integrity practices themselves. Of late, attention to research integrity among funders has gathered pace, as reflected in several initiatives around the globe that demonstrate how funders can support a culture of research integrity. For example, the US National Science Foundation (NSF) [6] requires applicants’ research organizations to provide training and oversight in the responsible conduct of research, designate individuals responsible for research integrity, and have an institutional certification to testify of its commitment. Also, in 2016, 3 Canadian federal funders joined forces to support research integrity in the Canadian Tri-Agency Framework: Responsible Conduct of Research–Harmony and Responsibility [7]. The framework was subsequently updated in 2021. This framework sets out responsible practices that research organizations and researchers should follow, including rigor, record keeping, accurate referencing, responsible authorship, and the management of conflicts of interest. It also acknowledges the responsibilities of the funders, including “helping to promote the responsible conduct of research and to assist individuals and institutions with the interpretation or implementation of this Responsible Conduct of Research (RCR) Framework”.It is not only major funding organizations in highly developed research environments that are taking steps. Smaller funders are also acting to mandate compliance with research integrity standards. The constantly growing literature on the topic is another sign of development within this area [2,3]. In the USA, research integrity recently reached the political arena, when, following a call from researchers [8], President Biden’s administration published a memorandum on restoring trust [9] that highlights the importance of integrity in research. The memorandum will be supported by the reintroduction of the Scientific Integrity Act. This act will prohibit research misconduct and the manipulation of research findings. It talks of a “culture of research integrity” and demands that funding agencies adopt and enforce research integrity policies, appoint a research integrity officer, and provide regular research integrity and ethics training. The US are not alone in their endeavors. Governments in other countries are equally gearing up to support the integrity and reproducibility of research [10]. However, so far, there is only limited evidence about the effectiveness of such initiatives, although it is generally accepted that they raise awareness among various stakeholders concerning research integrity challenges, strengthen the sense of responsibility of those stakeholders to address those challenges, and thereby ultimately contribute to fostering a culture of research integrity.In a collective effort to foster research integrity, research organizations and funders have their own, complementary roles. The Standard Operating Procedures for Research Integrity (SOPs4RI) consortium has recommended that both research organizations and funders develop a RIPP. A RIPP outlines the key responsibilities of an organization concerning research integrity and details methods and procedures to foster it. For example, in the case of research organizations, a RIPP should facilitate and stimulate a healthy research environment, proper mentoring and supervision, research ethics structures, research integrity training, high-quality dissemination practices, research collaboration, effective data management, and open and fair procedures to deal with breaches of research integrity [11]. Funders have a different role. They can support, safeguard, and incentivise, or even mandate, responsible research practices from research organizations and researchers. Equally important, funders should make sure that their internal processes live up to the highest standards of research integrity. We recognize that funders are many and varied in their scale, portfolio, disciplinary focus, and the extent to which they have procedures and governance arrangements to support research integrity. For all funders, adopting a RIPP will structure and coordinate research integrity practices, giving clarity and transparency to applicant institutions and researchers.In this Consensus View, we highlight examples of best practice of funders worldwide to foster a research integrity culture. With these examples in mind, we suggest guidelines to support funders in taking a leading role in fostering research integrity. In so doing, we acknowledge the local contexts in which funders operate, but we believe that all funders, large and small, in all parts of the world, can and should contribute to improving research validity and building and maintaining trust in science through incentivising and mandating a culture of research integrity. Our core argument is that developing a tailored RIPP will contribute to building an institutional culture of research integrity, both within funding organizations and among the research organizations and individual researchers they fund. Based on empirical work from the SOPs4RI project, we have identified 6 key research integrity topics: researchers’ compliance with research integrity standards; expectations for research organizations; selection of grant applications; declaration of interests; monitoring funded research; and dealing with internal integrity breaches (Fig 1). We recommend that these topics should be included in a RIPP and provide guidelines on developing and implementing a RIPP.Open in a separate windowFig 1Topics to be covered in a RIPP for funders.An overview of the 6 most important topics identified by the SOPs4RI to be included in research funding organization’s RIPP. RIPP, Research Integrity Promotion Plan; SOPs4RI, Standard Operating Procedures for Research Integrity.     

Turning promise into practice: Crop biotechnology for increasing genetic diversity and climate resilience

As climate change increasingly threatens agricultural production, expanding genetic diversity in crops is an important strategy for climate resilience in many agricultural contexts. In this Essay, we explore the potential of crop biotechnology to contribute to this diversification, especially in industrialized systems, by using historical perspectives to frame the current dialogue surrounding recent innovations in gene editing. We unearth comments about the possibility of enhancing crop diversity made by ambitious scientists in the early days of recombinant DNA and follow the implementation of this technology, which has not generated the diversification some anticipated. We then turn to recent claims about the promise of gene editing tools with respect to this same goal. We encourage researchers and other stakeholders to engage in activities beyond the laboratory if they hope to see what is technologically possible translated into practice at this critical point in agricultural transformation.

Will gene editing contribute to improved crop diversity and climate resilience? In this Essay, the authors look at lessons from past biotechnology efforts to inform action for the future.

In 1970, a virulent fungal blight decimated the United States corn harvest. This southern corn leaf blight epidemic was linked to a subset of genes that made certain varieties more susceptible than others—genes that also happened to be shared across some 75% of commercial varieties [1,2]. The blight arrived just as scientists concerned about a more general loss of genetic diversity in crop plants, both in the US and abroad, were finally gaining the ear of governments and philanthropies. They called for more and better gene bank facilities and, brandishing blighted maize as the canary in the coal mine, a re-diversification of industrial crops [3–5].With these concerns as motivation, some researchers pointed to the possibility of increasing genetic diversity among cultivars of a given crop with a brand-new biotechnology: recombinant DNA [6]. These techniques could be used to introduce novel genes into the high yielding but genetically narrow lines dominating commercial markets. But this anticipated use of recombinant DNA technologies for expanding genetic diversity has yet to materialize.The need to diversify crops is coming back into focus due to increasingly urgent climate and nutrition challenges [7–9]. Diversified agricultural systems are more resilient to climate hazards and can stabilize food production [10]. Increasing genetic diversity, by both widening the genetic bases of commonly cultivated crop species and restoring a greater number of species to cultivation, is therefore a high priority for climate action.Biotechnology is once again offering a path forward. Today’s plant scientists are developing gene editing techniques that could facilitate genetic diversification of commodities like wheat, rice, and maize and potentially support the adoption or continued cultivation of “neglected” crops that have been less often subject to crop breeding and development activities. But will gene editing really generate a diversity boom? Can it upend a pattern of genetic narrowing that breeders and botanists have observed since the late 19th century—a pattern frequently pinpointed as a major source of vulnerability in global agricultural production systems?Excavating comments that reveal an often forgotten subset of early aspirations for recombinant DNA technologies provides insight on contemporary dialogue about gene editing. The history of these technologies illustrates the extent to which diversification depends on much more than a laboratory toolkit. Awareness of past efforts can inform today’s aspirations for and decision-making about the use of crop biotechnologies to enhance genetic diversity.