Much as Mark Twain famously stated that reports of his death were greatly exaggerated, it turns out that land animals in tropical protected areas may be better off than we thought.Current assessments warn of widespread biodiversity loss in the tropics-which is home to half of species globally-and put declines as high as 56% over the last four decades.But now, in this issue of PLOS Biology, Lydia Beaudrot, Jorge Ahumada, and colleagues report that tropical forest preserves around the world may be helping after all: at the community level, ground-dwelling mammals and birds are holding their own.Why the difference?Tropical forests are understudied, and most of the animals living there are rare and elusive, so existing biodiversity assessments rely heavily on aggregated secondary data and expert opinion.In contrast, the new findings are based on standardized primary data from camera traps in the Tropical Ecology Assessment and Monitoring (TEAM) Network, which give close-to-real-time snapshots of wildlife status and trends (Fig 1).Beaudrot, Ahumada, and colleagues determined biodiversity trends for more than 500 populations of nearly 250 species of mammals and birds, using half a million images per year from tropical forest preserves on three continents.The distribution of the 15 study sites reflected the proportion of tropical forest cover on each continent: half were in South and Central America, and the remainder were split between Africa and Southeast Asia.Each of the protected areas studied had a camera trap every square kilometer or two, for a total of 60 to 90 per site.Even so, camera sightings were infrequent-five per year for a given population was not uncommon-and the researchers used occupancy as a proxy for abundance.Analysis of the sightings showed that occupancy declined in 22%, increased in 17%, and did not change in 22% of the populations during the study period, which ranged from three to eight years depending on the site.Further analysis of the camera trap images revealed encouraging news: despite the variability in occupancy trends, biodiversity did not decline systematically at the community level in the protected areas studied.Importantly, occupancy trends did not vary significantly between mammals and birds, or by International Union for Conservation of Nature (IUCN) Red List status.The latter suggests that, at least in the study sites, populations of Threatened and Near Threatened species are doing as well as those of Least Concern.However, sightings were too low to determine trends for nearly two-fifths of the populations studied, leading the researchers to caution that the observed community-level stability could mask wildlife losses in protected areas.The researchers also underscore that their findings do not apply to the many unprotected tropical forests worldwide.Today's extinction rates are estimated to be 1,000 times faster than the average over the history of life on Earth, and this catastrophic species loss occurs disproportionately in the tropics.These caveats aside, this work gives hope that species loss in protected tropical forests is less severe than we feared.Moreover, for the first time, conservation decisions can be based on real-time changes in wildlife populations.Some 200 nations have committed to a suite of

A new study reveals how flies and their larvae avoid parasitoid wasps by eavesdropping on their chemical communications. Read the associated Research Article.

A new study reveals that bats use the inertia of their unusually heavy wings—rather than their aerodynamic properties—to help them perform acrobatic maneuvers like landing upside down to roost. Read the Research Article.

In many parts of the world, groundwater contains so much arsenic that it builds up in irrigated crops. Linked to cancer and heart disease, this toxic element is particularly worrisome in rice, which absorbs arsenic more readily than other grains and is a staple for billions of people. Countries with the double whammy of arsenic-laced groundwater and heavy rice consumption include Bangladesh, India, and China. While plants can detoxify arsenic, we don't know precisely how they do it. In this issue of PLOS Biology, the collaborative team of Dai-Yin Chao, Fang-Jie Zhao, and David E. Salt identify an arsenic-reducing enzyme in the plant Arabidopsis thaliana and show that this protein is critical to arsenic elimination (Figure 1). Figure 1 Arsenic is toxic, and its elimination from plants requires it to be converted into arsenite, a form of arsenic that can be released back into the soil from roots. Inorganic arsenic (arsenate) resembles phosphate and, once taken up by roots, likely loads via phosphate transporters into the xylem, which delivers water and nutrients to the shoots. Plants get rid of arsenate by reducing it to arsenite, a form that no longer mimics phosphate and is readily extruded from the roots back into the soil. To find the enzyme that transforms arsenate into arsenite in plants, the researchers used genome-wide association mapping, which links phenotypes—in this case, arsenic levels in leaves—to genes. They grew 349 types of A. thaliana collected from around the world at an environmentally relevant concentration of arsenic, and found that leaf arsenic levels varied more than 20-fold and that this variation was associated with a region of chromosome 2. Comparison of strains with high and average arsenic levels (Kr-0 and Col-0, respectively) showed that the former has a cytosine at a specific nucleotide in this region, while the latter has a thymine in the same spot. Crossing the two strains showed that arsenic was high in about 25% of the offspring, suggesting that leaf arsenic levels are controlled primarily by a single gene. Named High Arsenic Content 1 (HAC1), this gene has a predicted amino acid domain characteristic of arsenate reductases. To verify that this newly discovered enzyme reduces arsenate to the easily eliminated arsenite, Chao and colleagues expressed HAC1 in an Escherichia coli mutant that lacks its own arsenate reductase. As expected, HAC1 restored arsenic elimination in this E. coli mutant. In addition, the team found that HAC1 is expressed in the roots and that root expression rises in A. thaliana exposed to arsenate. Moreover, in A. thaliana mutants that lack HAC1, arsenic stunted both root and overall plant growth. The latter is important because it shows that HAC1 also keeps arsenic low in shoots, which are often the edible part of a plant. Another important finding is that arsenite extrusion is dramatically reduced in an HAC1 mutant A. thaliana exposed to arsenate, suggesting that this arsenate-reducing enzyme may be coupled with the arsenite efflux transporter. Besides making a compelling case that HAC1 is part of a major defense against arsenic in plants, Chao and colleagues cleared up a mystery over a previous candidate for this job. Yeast reduces arsenate with an enzyme called ACR2, and initial studies had suggested that plants use a similar enzyme to detoxify arsenic. It turned out, however, that this ACR2-like enzyme reduces arsenate only in vitro and not in living plants. This apparent discrepancy is resolved by the fact that the two plant arsenate reductases (ACR2 and the newly discovered HAC1) share a similar DNA sequence, suggesting that experiments meant to knock out ACR2 actually knocked out HAC1. To dispel any lingering doubts that ACR2 may still play a role in plant arsenic detoxification by interacting with HAC1, the researchers compared arsenic in an A. thaliana mutant that lacks HAC1 to a double mutant that lacks both HAC1 and ACR2. As expected, arsenic metabolism was similar in the two mutants, confirming that ACR2 has no impact on arsenic detoxification and elimination in plants. Using a different method, Eduardo Sanchez-Bermejo and colleagues also recently identified the same gene, which they called ATQ1, showing that it encodes an arsenate reductase enzyme involved in plant tolerance to arsenate. However, Chao and colleagues went further by revealing the functional role of HAC1 in arsenic accumulation and arsenate resistance. The researchers caution that when highly active, this arsenate reducing gene may come at a cost to plants, which could diminish production. That said, their work has tremendous potential to benefit human health. Their key finding—that HAC1 keeps arsenic low in shoots of plants grown at real-world arsenic levels—is a much needed first step toward developing crops that can be grown in high-arsenic regions and still be safe to eat. Chao D-Y, Chen Y, Chen J, Shi S, Chen Z, et al. (2014) Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants. doi:10.1371/journal.pbio.1002009

New Developmental Evidence Clarifies the Evolution of Wrist Bones in the Dinosaur-Bird Transition

Tiny sap-sucking insects that are a scourge to gardeners also have the upside of helping trees survive in seasonally dry forests in Central America. How? Scale insects use carbon they get from Cordia alliodora trees to make sugar-rich “honeydew” for Azteca pittieri ants, which in turn defend the trees against leaf-munching insects. Mutualism is often stronger when resources are scarce, but this interdependence usually involves a commodity that is traded directly between species. Now, in this issue of PLOS Biology, Pringle and colleagues show that lack of a resource that is not traded—water—intensifies the bonds between C. alliodora, scale insects, and ants. An A. pittieri ant patrols the surface of a C. alliodora tree stem in Mexico. Pringle et al. show that the strength of the defensive mutualism between A. pittieri ants and C. alliodora trees increases with water stress across Mesoamerica. Found from southern Mexico through South America, C. alliodora has stem hollows where ants nest and tend flocks of scale insects. Named for their protective coverings, scale insects are vampires to the vegetable kingdom, piercing plants with tubular mouths to drink straight from the vascular system. As they imbibe, they secrete honeydew for ants to harvest and eat. Rounding out this mutualistic circle, the ants patrol their C. alliodora host for beetle larvae, caterpillars, and other herbivores, biting them until they leave. Previous studies suggested that plants may invest more carbon in ant defense during water stress. This scenario is particularly taxing for C. alliodora, which drops its leaves during the dry season and must make its carbon stores last long enough to grow new leaves during the next rainy season. But ant colonies must be maintained year-round to ensure defense of leaves during the growing season, safeguarding the production of carbon to get the trees through the next dry season. This led Pringle and colleagues to hypothesize that when C. alliodora trees get less rain, they incur the cost of supplying more carbon to scale insects but recoup their investment in the currency of better leaf protection by ants. To test their hypothesis, the researchers compared trees at 26 sites from Mexico to Costa Rica where rainfall varied four-fold. As expected, trees at drier sites had both more scales and bigger ant colonies. Amazingly, ants in drier areas also mounted stronger defenses of their trees, finding and chasing away herbivores more vigorously than ants in wetter areas. In keeping with this observation, excluding ants increased leaf herbivory at drier sites but made no difference at a wetter site. Next, the researchers assessed whether trees in drier areas had less carbon near the end of the dry season. Analysis of carbohydrates stored in stems confirmed that carbon pools were smaller in trees at drier sites than at wetter sites. Further, trees at drier sites showed early signs of carbon stress: more starch had been converted to the sucrose that helps maintain plant turgor during water stress. When are the carbon costs of ant defense worth it to carbon-stressed C. alliodora? To find out, the researchers modeled carbon trading among the players in this mutualistic system under rainy seasons of varying lengths. Comparison of two herbivory models—chronic but low level versus rare but catastrophic—showed that the latter “insurance” model fit the researchers' observations. The insurance model correctly predicted that shorter rainy seasons reduce trees' carbon pools while increasing their carbon investment in ants. Likewise, ants invest more in leaf protection when rain is scanty, enlarging colonies and allocating more carbon to producing the workers that defend against herbivores. Bolstering the case for the catastrophic insurance model, it also fits the real world. Over the course of six years of field work in the study area, the researchers say most C. alliodora trees were fine, but in a few cases all the leaves had been eaten down to the nubs. And, as they point out, even a rare event could pose a real risk to long-lived trees like C. alliodora. Tying their findings together, the researchers propose that mutualism in this system is driven by the combination of an environmental stress (low rainfall) and a biological stress (the risk of catastrophic herbivory), both of which affect carbon futures in plants. They further suggest that the changes in ant behavior at drier sites may reflect genetic adaptation to local conditions. Previous work has divided A. pittieri into northern and southern lineages that occupy different precipitation niches, raising the question of whether the rainfall-related differences in ant defensive behaviors revealed in this study also track these two lineages. Besides showing that water scarcity strengthens mutualism among C. alliodora, scale insects, and ants, this work suggests that the adage “hard times make us closer” may apply broadly to mutualisms between plants and animals, with carbon as the common currency. According to the carbon trading model in this study, trees' bet-hedging against irregular rainy seasons may drive the evolution of variable carbon investments. Pringle EG, Akcay E, Raab TK, Dirzo R, Gordon DM (2013) Water Stress Strengthens Mutualism Among Ants, Trees, and Scale Insects. doi:10.1371/journal.pbio.1001705

​MostMost of us know our families back a few generations but, beyond that, have little idea who our ancestors were or where they lived. Jumping further back, all of us alive today likely share most of our ancestors from 3,000 to 4,000 years ago. What happened between then and now? We've pieced together a broad picture of human kinship based on disciplines from archeology to linguistics to history. In Europe, for example, several relatively recent migrations have helped shape links and gaps amongst today's populations. Now, in this issue of PLOS Biology, Peter Ralph and Graham Coop use genomic data to give us a closer look at the recent roots of modern Europeans. The distribution of distant cousins of modern-day people in the UK, at three different levels of geneological distance (circle size proportional to numbers of cousins; units are numbers of shared ancestors). Previous work has shown that genotypes in Europe vary with latitude and longitude, and that genetic diversity tends to increase from north to south. To get a sharper picture of the interplay between recent relatedness and geography in Europe, Ralph and Coop compared genome-wide sequencing data from 2,257 people across the continent. The researchers used sharing of long genome segments between individual people as well as populations as a measure of common ancestry. Shared segments become progressively shorter back through history as they have undergone more generations of recombination. Thus, the longer a shared segment, the more recent the common ancestor. As might be expected, comparison of these long shared segments by country typically showed that recent relatedness is highest amongst people who live near each other. There are, however, some noteworthy departures from this norm. People in the UK share more recent ancestors with people in Ireland than with others living in their own country. Likewise, people in Germany share more recent ancestors with the Polish than with other Germans. This pattern could reflect the migrations of smaller populations into a larger one. Similarly, while recent relatedness generally drops evenly across geographic distances in Europe, a few exceptions stand out. Regardless of physical proximity, recent relatedness is low between the Italian peninsula and the rest of the continent. At the other end of the scale, recent relatedness is high within northern Europe as well as across eastern Europe – three times that within other regions at similar distances. Ralph and Coop also used long shared segments to gauge how many recent ancestors are common to people across modern Europe, as well as roughly how long ago they lived. Because a person does not inherit genetic material from every single ancestor, this analysis only reveals a small fraction of the shared genealogical relationships; the researchers call this fraction “genetic common ancestors.” The distributions of long segments revealed that genetic common ancestors from about 500 years ago are typically shared only by people who live in the same country today. Albanian speakers are at the high end, with about 90 genetic common ancestors within the last 500 years, and about 600 genetic common ancestors between the last 500 and 1,500 years . In contrast, just about any two people from almost anywhere across Europe today share hundreds of genetic ancestors from more than 1,500 years ago. The outliers are the Italian and Iberian (Spain and Portugal) peninsulas, where people have only about two genetic ancestors in common with populations elsewhere on the continent over the last 1,500 years. Ralph and Coop then take into account that these genetic common ancestors are only a small fraction of the genealogical ancestors. Based on this, the researchers extrapolate that, conservatively, even people living in opposite ends of Europe today are likely to have a shared ancestry that includes everyone who both lived a thousand years ago and had descendants. The conclusion that all Europeans are related over such a short time period lends credence to the theory that everyone in the world is related over just the last few millennia. Indeed, the researchers speculate that Europe's common ancestors over the past millennium may also be shared worldwide. Another intriguing finding is that the numbers and timing of common ancestors among different parts of Europe may reflect major events in the continent's history. Notably, the number of common ancestors within the last 1,000 to 2,000 years is particularly high within eastern Europe — similar to those in Ireland despite spanning far greater distances — and the timing fits with the series of migrations that began with the Huns in the 4th century and ended with the Slavs between the 6th and 10th centuries. In support of linking this spike in common ancestry with these migrations, many of today's eastern Europeans who share long segments also speak Slavic languages. Furthermore, the regions with the fewest common ancestors (France, and the Italian and Iberian peninsulas) are also thought to have been largely untouched by the migrations of Huns and Slavs. This work both corroborates and extends our understanding of Europe's recent past, adding another dimension to what other disciplines tell us about historical events. Besides giving us a fuller picture of the close ties between people around the world, delving into our recent past with population genomics could ultimately help answer these most basic of human questions: Where did we come from and how did we get here? Ralph P, Coop G (2013) The Geography of Recent Genetic Ancestry across Europe. doi:10.1371/journal.pbio.1001555

Bumblebees are remarkable navigators. While their flight paths may look scattered to the casual eye, all that buzzing about is anything but random. Like the travelling salesman in the famous mathematical problem of how to take the shortest path along multiple stops, bumblebees quickly find efficient routes among flowers. And once they find a good route, they stick to it. The same goes for other animals from hummingbirds to bats to primates that depend on patchy resources such as nectar and fruit. Perhaps this is not such a surprising feat for animals with relatively high brain power. But how do bumblebees, with their tiny brains, manage it? As new research in this issue of PLOS Biology by Lars Chittka and colleagues shows, a simple strategy may be enough for a realworld solution to this complex problem. For computers, solving the travelling salesman problem means methodically calculating and comparing the lengths of all possible routes. But such an exhaustive approach isn’t feasible in practice, and indeed animals can find a near-optimal foraging route, or trapline, without trying them all. Determining exactly how they do this, however, has been stymied by the difficulties of tracking animals as they forage in the wild. Chittka and colleagues got around this problem by tracking bumblebees (Bombus terrestris) on five artificial flowers set in a mown pasture. The ‘‘flowers’’ had landing platforms with drops of sucrose in the middle, and were fitted with motion-triggered webcams. To keep the bees’ focus on the artificial flowers, the experiments were done in October, when natural sources of nectar and pollen were scarce. To make the bees want to find all five flowers, each sucrose drop was only enough to fill one-fifth of a bumblebee’s crop. And to keep the bees from finding one foraging site from another visually, the flowers were arranged in a pentagon that was 50 m on each side, which is more than three times as far as bumblebees can see them. The researchers released bees individually from a nest box that was about 60 m from the nearest flower, and used the webcams to track the sequence of flower visits during consecutive foraging bouts. The bees found the closest flowers first and added new flowers during subsequent bouts. With experience, they repeated segments of the visitation sequence that shortened the overall route while abandoning those that did not. Traplines linking all five flowers in a short route were established after an average of 26 foraging bouts, which entailed trying only about 20 of the 120 possible routes. In addition, the researchers fitted five bees with transponders and tracked them with radar as they developed traplines. This revealed that flight paths between trapline segments were relatively straight and that between their first and last bouts, bees cut their total travel distance by 80% (from 1,953 to 458 m). In contrast to computers, bees did not find the absolute shortest route of 312 m even in this simple experimental arrangement. But they came very close, especially considering that they explored only a small fraction of the possible routes, and established traplines relatively rapidly. This tradeoff between perfection and speed highlights the differences between mathematical and biological solutions to the travelling salesman problem. How do bees develop such efficient routes so fast? The researchers assessed three possibilities—that bees optimize foraging routes by visiting flowers in the order of discovery, by shuffling them randomly, or by visiting those that are closest together—but found that the first two failed to fit their observations while the third did not fully explain them. Rather, the researchers propose that bees optimize foraging routes through trial and error, combining exploration with learning from previous bouts to progressively adjust their routes as they find shorter paths. Based on the bees’ movements during trapline establishment, the researchers developed a model linking experience to the likelihood of visiting particular flowers. Bees are well-known to be able to compute and memorize distances between locations, and the model assumes that they remember the length of the shortest route so far, compare it to the length of the current route, and then choose the shorter of the two. Over time, choosing the more efficient route favors shorter segments over longer ones. The model is a good fit with the researchers’ observations, predicting, for example, that bees will develop and stick to optimal routes in 20–25 bouts. Besides shedding light on how bees develop traplines, this work suggests that small-brained animals can use simple methods to solve complex routing problems without the need for cognitive maps of spatial relationships, as has been suggested. It remains to be seen whether big-brained animals can also develop traplines with such elementary tools. But if so, that would free up their brain power for other tasks.

A new experimental approach reveals a bet hedging strategy in unstressed, clonal yeast cells, whereby they adopt a range of growth states that correlate with expression of a trehalose-synthesis regulator and predict resistance to future stress.

​BirdsBirds do it, bees do it—and now it appears that even plants do it. No, it's not what you're thinking. Sexual reproduction is obviously well-known in plants. Rather, the phenomenon in question is equalizing sex chromosome expression in males and females. In animals where males are XY and females are XX, the Y chromosome has lost most of its genes, so X chromosome expression is adjusted to keep the balance between sexes. In people, females inactivate one of their X chromosomes. The fruit fly Drosophila melanogaster takes the opposite approach, with males hyper-expressing their X-linked genes. Called dosage compensation, such balancing is found in many animals but was thought to be absent in plants. Now, however, in this issue of PLoS Biology, Gabriel Marais and colleagues report the first evidence of dosage compensation in the plant Silene latifolia, or white campion, along with insights into how this phenomenon evolves. Pictures of female and male flowers (top) and the X and Y chromosomes (bottom) in Silene latifolia. As in mammals and fruit flies, S. latifolia males are XY and females are XX. The classical view holds that there are three steps in the evolution of dosage compensation: recombination between the X and Y chromosomes is suppressed, the Y degenerates, and this massive loss of Y chromosome genes is balanced by dosage compensation of the X chromosome. But animal sex chromosomes began evolving so long ago that the process is too advanced to trace its course. Human sex chromosomes originated about 150 million years ago, for example, and the Y chromosome has now lost some 97% of its genes. In contrast, sex chromosomes are still in the early stages of evolution in S. latifolia, making it a good system for studying this process. Like animal sex chromosomes, S. latifolia X and Y chromosomes have gradually stopped recombining and the Y chromosome is degenerating. However, work on sex chromosome evolution in this plant has been limited by the small number of known sex-linked genes. To identify more S. latifolia sex-linked genes, the researchers used a new technique called RNA sequencing, which both sequences and estimates the abundance of mRNAs. They identified more than 1,700 sex-linked genes, a 100-fold increase on the number previously known. Next, the researchers assessed degeneration of the S. latifolia Y chromosome by comparing expression levels of X- and Y-linked genes in males. In keeping with research on the previously known sex-linked genes, the results confirmed that the average expression levels of Y-linked genes were lower than those of their X-linked counterparts. Reduced expression fits with the on-going degeneration of the Y chromosome, raising the question of whether S. latifolia also has dosage compensation. The researchers tested this by comparing the expression of sex chromosome-linked genes in males and females. If S. latifolia lacked dosage compensation, the expression of X-linked genes in males would be half that seen in females. Indeed, this is true for genes where the Y-linked copy is still expressed. However, for genes with reduced Y expression, expression of the X-linked copy was nearly as high in males as in females. This suggests that, like fruit flies, S. latifolia compensates for reduced Y expression by increasing X expression in males. The researchers then excluded sex chromosome genes that are expressed more in males than in females. Such male-biased genes are not subject to dosage compensation in other species, and comprised 25% of the newly identified sex-linked genes in S. latifolia. Analysis of expression levels of the remaining 75% revealed that in males, expression of the X-linked version of a gene rises in proportion to the drop in expression of the Y-linked version, bolstering the conclusion that dosage compensation is gradually evolving in S. latifolia in piecemeal fashion. The finding that S. latifolia balances X chromosome expression between the sexes contradicts a recent study by another research team (Chibalina and Filatov [2011] Curr Biol 21: 1475), which concluded that S. latifolia lacks dosage compensation. But Marais and colleagues argue that, if analyzed differently, the results of the previous study may actually support dosage compensation as well. The other team based their conclusion on the fact that X expression levels were not equal in males and females. However, Marais and colleagues point out that the X expression level in males was still considerably higher than would be expected in the absence of dosage compensation. Instead of being half as high, the average X expression level in males was nearly 70% that of females, suggesting that there is dosage compensation for many genes. Moreover, partial dosage compensation is to be expected in a system where it is still evolving. Besides being the first to support the existence of dosage compensation in plants, this work provides the evolutionarily earliest example of balanced X expression between the sexes. In animals, dosage compensation has been found only in sex chromosome systems that are more than 100 million years old, while S. latifolia's sex chromosomes are just 10 million years old. The discovery of dosage compensation in such young sex chromosomes affords a unique and exciting opportunity to learn how this phenomenon might evolve. Muyle A, Zemp N, Deschamps C, Mousset S, Widmer A, et al. (2012) Rapid De Novo Evolution of X Chromosome Dosage Compensation in Silene latifolia, a Plant with Young Sex Chromosomes. doi:10.1371/journal.pbio.1001308

Obesity and type 2 diabetes have risen tremendously over the last 20 years, and the causes of these epidemics are complex. In both diseases, insulin resistance manifests early due to a combination of genetic and environmental factors, with gut bacteria and the immune system playing key roles. For example, weight gain and insulin resistance are linked to a group of gut bacteria called Firmicutes, which provide a source of extra calories by breaking down polysaccharides that are otherwise indigestible in mammals. Insulin sensitivity is also affected by immune system proteins called Toll-like receptors (TLRs) that recognize microbial compounds. When raised in germ-free environments, mice that lack TLR2 are protected against obesity-induced insulin resistance. Intriguingly, the immune system helps regulate gut bacteria, and previous work suggests that TLRs may affect insulin sensitivity by altering the composition of enteric microbes. Now, in this issue of PLoS Biology, Andréa Caricilli and colleagues present compelling evidence that gut bacteria can nullify the genetic protection against insulin resistance in TLR2-deficient mice. To investigate the relationship between gut bacteria and insulin sensitivity, the researchers raised TLR2-deficient mice under conditions that were not germ-free. In contrast to previous findings, these mice became insulin resistant within 8 weeks, and were fatter at 12 weeks. Genetic analysis of their gut bacteria revealed that the abundance of Firmicutes was three times higher than that of wild-type mice, and the researchers suggest that this explains why these TLR2-deficient mice were not protected against insulin resistance. Because the composition of enteric microbes varies with the environment and diet, mice with the same genetic background can have different gut bacteria, and presumably this was the case for the TLR2-deficient mice in previous studies. How could gut bacteria counteract the innate insulin sensitivity of TLR2-deficient mice? Insulin resistance can be caused by bacterial cell membrane compounds called lipopolysaccharides, and several lines of evidence suggest that this is a likely mechanism for the development of insulin resistance in the TLR2-deficient mice studied. Notably, they had higher serum levels of lipopolysaccharides, and absorbed more of them after oral administration. This suggests that these mice had more permeable guts, which is supported by the finding that their intestines had less of a tight junction protein. The link between gut microbe composition and insulin resistance was further strengthened by a number of findings. In particular, treating TLR2-deficient mice with antibiotics brought their Firmicutes down to normal levels, reduced their fat, decreased their serum lipopolysaccharides, and increased their insulin sensitivity. This suggests that changing the composition of gut microbes reversed their insulin resistance. Moreover, when gut microbes were transplanted from TLR2-deficient mice into wild-type mice with only the genus Bacillus in their guts, the latter got fatter, had higher lipopolysaccharide levels, and were less sensitive to insulin. This suggests that the Firmicutes-rich gut bacteria from these TLR2-deficient mice were enough to cause insulin resistance. Because obesity and insulin resistance may be promoted by fatty foods in people, the researchers compared the effects of high-fat diets on TLR2-deficient and wildtype mice. The TLR2-deficient mice got much fatter and glucose tolerance tests revealed that they also developed diabetes, indicating that the high-fat diet exacerbated their insulin resistance. By showing that changes in gut bacteria can cause insulin resistance in mice that are genetically protected against this condition, this work suggests that the composition of enteric microbes may cause obesity and diabetes in animals that are predisposed to be lean. This work also sheds light on the interplay of genetic and environmental factors that cause metabolic syndrome in people, which is characterized by obesity and insulin resistance, and increases the risks of stroke and coronary artery disease along with type 2 diabetes.

​BacteriaBacteria can change course almost instantaneously, zipping towards food or away from toxins. How do such simple organisms do something so complex? It's all in the flagella, a tail-like structure with rotating helical filaments. The flagella work in unison to propel the cell forward by rotating counterclockwise and thus bundling together. When the flagella reverse their rotation to clockwise, they disrupt the bundle and make the cell tumble in place. When the flagella shift back to counterclockwise again, the bacteria set off on a new course. The motion of helix E caused by the conformational change of the hinge between FliGM and helix E is key to the cooperative switching of flagellar motor rotation. This description of bacterial locomotion is well known, but the mechanisms that allow the flagella to shift gears from counterclockwise to clockwise have proven difficult to identify. Now, in a new study in this issue of PLoS Biology, Katsumi Imada, Tohru Minamino and colleagues bring us closer to answering this fundamental question and propose a new model describing how flagella manage this switch. Filaments in the flagella are powered by rotary motors that span the cell membrane. Things of beauty, these motors are tooled so precisely that they are nearly 100% efficient, and their direction is set by a rotor that can turn thousands of revolutions per minute. The rotor shifts from the forward-propelling counterclockwise to the tumble-inducing clockwise when chemical gradients tell bacteria they've gone astray, for example, away from food. This activates a cytoplasmic signaling protein that binds proteins in the rotor switch, changing the orientation of another switch protein called FliG and thereby reversing the rotor's spin to clockwise. The details of the switch mechanism had been hypothesized but were as yet unproven. Previous X-ray crystallography studies of a FliG fragment had shown that two of its domains (FliGM and FliGC) are connected by a helical linker called helix E, and the 3-D structure of a FliG protein predicted from its DNA sequence suggested that helix E might be flexible enough to make a good molecular switch. This suggestion was further supported by a 2010 report that compared the structure of a full-length FliG to the fragment: helix E was tightly packed in closed conformation in the full-length structure, but was in open conformation and dissociated from FliGM in the fragment. To find out if helix E is indeed the molecular switch that sets the direction of rotor spin, the researchers compared wild-type and mutant FliG fragments containing the two domains linked by helix E. The wild-type motors were set to spin counterclockwise by experimental conditions, and the mutant had a type of amino acid deletion that sets the rotor spin to clockwise. As expected, X-ray crystallography revealed that the wild-type (counterclockwise) and mutant (clockwise) FliG fragments had different helix E conformations. The difference was in the hinge between helix E and FliGM, reorienting the former and exposing part of the latter in the mutant fragments, suggesting that this hinge may be the molecular switch that shifts FliG's orientation between counterclockwise and clockwise states. This conclusion was strengthened by the finding that while the FliG proteins studied came from several bacteria species and varied considerably, they shared a conserved FliGM-FliGC element. Based on their discovery, the researchers propose a new model for rotational switching in bacterial flagella. The rotor base has a ring of FliG subunits that switch cooperatively between counterclockwise and clockwise states. The model holds that besides affecting the orientation of its own subunit, the hinge between helix E and FliGM also affects the orientation of the neighboring FliG subunit. Thus, conformational change of this molecular switch rapidly spreads from subunit to subunit, thus propagating it all around the ring. This work provides the most direct evidence yet that helix E is the molecular switch underlying the flagellar motor's gear shift from counterclockwise to clockwise, as well as the most complete model of the cooperative flagellar switch. Besides advancing our understanding of the flagellar motor, which is a marvel of nature, this study could help lay the groundwork for developing drugs that target key motor proteins and so immobilize harmful bacteria. Minamino T, Imada K, Kinoshita M, Nakamura S, Morimoto YV, et al. (2011) Structural Insight into the Rotational Switching Mechanism of the Bacterial Flagellar Motor. doi:10.1371/journal.pbio.1000616

​AtAt first glance, diatoms, malaria parasites, and fungus-like plant pathogens called oomycetes look wildly different. But these organisms all have something in common: they belong to a group called the Chromalveolata that contains genes from algae. This diversity of lifestyles within a single group presents an opportunity to learn how the various strategies evolved. For example, oomycetes include both necrotrophs, which feed on dead plant tissue, and obligate biotrophs, which require living hosts. In addition, biotrophy evolved independently in two groups of oomycetes in the same lineage, the white rusts such as Albugo laibachii and the downy mildews such as the Irish potato famine pathogen Phytophthora infestans and Hyaloperonospora arabidopsidis, which infects the plant Arabidopsis thaliana. White blister rust of Arabidopsis thaliana, caused by the obligate biotroph oomycete Albugo laibachii, found in a field plot in Norwich, and later single-spore purified and sequenced. Recent research on H. arabidopsidis has linked biotrophy to massive gene loss in biosynthetic metabolic pathways. This makes sense because there is less selection to synthesize products when the host provides them. Biotrophy is also thought to result from gaining ways to circumvent host defenses. However, the evolution and molecular mechanisms of this lifestyle are poorly understood. In this issue of PLoS Biology, Kemen, Jones, and colleagues confirm that obligate biotrophy entails pathway loss in oomycetes, and report a new class of molecules that suppress host plant defenses. First, the researchers sequenced the genome of the white rust A. laibachii that, like the downy mildew H. arabidopsidis, is an obligate biotroph and parasitizes A. thaliana. Then they identified A. laibachii genes that code for proteins. This entailed extracting complementary DNA (which is made from the messenger RNAs that are translated into proteins) from A. thaliana leaves infected with A. laibachii, and then matching it to this white rust's genome sequences. The researchers also assigned A. laibachii genes to metabolic pathways using computational and manual prediction strategies. To investigate the origins of biotrophy, the researchers compared genes from the two biotrophs (A. laibachii and H. arabidopsidis) with those of a hemibiotroph (Phytophthora infestans) and a necrotroph (Pythium ultimum) in the same oomycete lineage. The two biotrophs were the least related, confirming that this lifestyle arose twice in this lineage. To identify gene losses linked to biotrophy, the researchers looked for genes that were present in the hemibiotroph and the necrotroph but missing in the biotrophs. Both biotrophs were missing genes in nitrogen and sulfur acquisition pathways. This is in keeping with the hypothesis that the evolution of biotrophy involves losing biosynthetic pathways. Likewise, the findings validate the other hypothesized component of biotroph evolution, suppression of host defenses. Biotrophic oomycetes form structures called haustoria that take up nutrients from and counteract defenses by their hosts. Outgrowths from intercellular hyphae enter plant cells and differentiate into haustoria, creating an intimate interface between host and parasite plasma membranes. Plants defend against invasion partly with disease resistance proteins that can recognize pathogen molecules. However, pathogens counteract plant defenses with secreted proteins called effectors, and in oomycetes some effectors have sequence motifs such as “RxLR”. Comparison of secreted proteins from two A. laibachii strains revealed a new class of effectors with a CHxC motif. The researchers validated the CHxC effectors with a translocation assay that confers avirulence, which involves fusing the N-terminus of potential effectors to a known avirulence protein. The findings showed that one of the new CHxC effectors conferred avirulence as effectively as a previously known class of A. laibachii effectors. To develop a model of how biotrophy evolves, the researchers determined the molecular divergence of A. laibachii from other Chromalveolata species with a variety of lifestyles: the other biotroph (H. arabidopsidis), the hemibiotroph (P. infestans), and the necrotroph (P. ultimum) in the same oomycete lineage, as well as a diatom (Thalassiosira pseudonana) and the malaria parasite Plasmodium falciparum. The researchers then assessed gene and metabolic pathway gains and losses amongst these species. For example, all of the oomycetes that form haustoria have also lost the pathway to make thiamine. In addition, like the oomycete biotrophs, the malaria parasite has lost molybdopterin-requiring pathways. This parasite also forms a structure that functions like a haustorium, taking up nutrients from the host and delivering secreted proteins to suppress host defenses. Based on the gene and metabolic pathway patterns amongst these Chromalveolata species of known relatedness, the researchers propose that the first step towards obligate biotrophy in oomycetes is suppressing host defenses. This facilitates haustoria formation, letting oomycetes get nutrients from their hosts and so ultimately leading to the loss of biosynthetic pathways. This pathway loss then results in absolute dependence on the host. This works sheds light on the origins and mechanisms of biotrophy, revealing a new class of avirulence proteins that can nullify plant defenses, and supporting the hypothesis that this lifestyle results from a combination of gaining ways to overcome host defenses and losing the ability to make nutrients. Understanding how organisms become obligate biotrophs that depend on specific hosts could also help lead to protections against human parasites in the Chromalveolata, including the malaria parasite and Toxoplasma gondii, which causes toxoplasmosis and can be fatal to fetuses and people with compromised immune systems. Kemen E, Gardiner A, Schultz-Larsen T, Kemen AC, Balmuth AL, et al. (2011) Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis. doi:10.1371/journal.pbio.1001094

No matter what you’re doing at any given moment, from walking to talking or even sleeping, your brain is doing its own thing. Networks of neurons constantly and often spontaneously generate rhythmic electrical activity in the cortex, the brain’s outermost layer and the seat of judgment, decision making, and other higher order functions. These cortical networks contain pyramidal cells that can excite inhibitory interneurons, which can in turn decrease the activity of pyramidal cells. This interplay of excitation and inhibition generates specific activity patterns that are critical to various cortical functions, like working memory and attention. However, it is not clear what cellular mechanisms maintain the proper balance between these two opposing inputs. Now, in this issue of PLoS Biology, Yousheng Shu and colleagues report that small changes in the electrical properties of pyramidal cells help maintain the excitation–inhibition balance that keeps these cortical networks humming along. In addition to exciting other neurons via ‘‘all or none’’ events (also known as digital mode) called action potentials, pyramidal cells may also have another way of communicating within a network. The researchers had previously found that pyramidal cells can use a ‘‘graded’’ method (analog mode) of exciting their targets via small changes in their membrane potential. Because pyramidal cells activate inhibitory interneurons and thus generate recurrent inhibition, the researchers asked whether this analog control of membrane potential could fine-tune the balance between excitation and inhibition in the cortex. The researchers began investigating recurrent network activity by recording activity between pairs of nearby pyramidal cells that presumably had an inhibitory interneuron between them. They first established that electrically stimulating one pyramidal cell in this microcircuit resulted in a late-onset, ‘‘slow’’ recurrent inhibition in the second pyramidal cell. They next made a positive (depolarizing) shift of the membrane potential by injecting current into the first pyramidal cell, and found that this increased the slow recurrent inhibition in the second cell. This modulation was sensitive to membrane potential shifts as small as 5 to 10 mV, considerably less than the shifts required to generate an all-or-none action potential. What about the other connections in this microcircuit? Knowing that lowthreshold spiking (LTS) interneurons can mediate slow recurrent inhibition, the authors next asked whether modulation of pyramidal cells can directly influence these inhibitory cells. Indeed, they found that small membrane potential shifts in pyramidal cells can modulate LTS interneuron activity. Importantly, they also observed these analog effects for fast spiking interneurons, which mediate ‘‘fast’’ recurrent inhibition. Taken together, these findings demonstrate that the membrane potential of pyramidal cells modulates recurrent inhibition, which helps balance the excitation and inhibition that lend stability to cortical network rhythms. Finally, the researchers examined the possible mechanisms of this membrane potential effect, and found a role for a type of potassium current called the D-current that helps control the duration of axonal action potentials. Blocking the D-current with drugs increased the inhibitory effect between pairs of pyramidal cells as well as the excitatory effect of pyramidal cells on LTS interneurons. Based on these findings, the researchers proposed the following model: depolarization in the first pyramidal cell inactivates D-current and so prolongs axonal action potentials, thereby enhancing synaptic transmission to the interneuron that then causes more inhibition to the second pyramidal cell. By showing that membrane potential helps balance excitation and inhibition in microcircuits, this work suggests a key role of analog communication in the rhythmic activity of cortical networks. Notably, recent work has implicated disruptions in the balance of excitation–inhibition in neurological disorders such as epilepsy and schizophrenia. Whether analog modulation may prove relevant to such diseases, however, is unknown. Many questions must be investigated before such possibilities can be addressed, including whether analog modulation applies to all cortical circuits, and whether it occurs during behaviorally relevant processes.

There’s a surprisingly fine line between bacterial symbiosis and chronic infection. While one is beneficial and the other detrimental, recent findings suggest that they share mechanisms for sidestepping host defenses. Plants in the pea family (legumes) have symbiotic nitrogen-fixing bacteria living in root nodule compartments that also contain antimicrobial compounds. For example, the nitrogenfixer Sinorhizobium meliloti survives antimicrobial compounds called nodule-specific, cysteine-rich (NCR) peptides that are produced by alfalfa and related legumes. Intriguingly, S. meliloti is closely related to Brucella abortus, which causes abortions in cattle and can also cause debilitating chronic infections in people. Moreover, the host legume antimicrobial peptides that S. meliloti evades are similar to defensins, small proteins that help plants and animals kill bacteria. Once inside root nodule compartments, S. meliloti differentiates from the free-living form into elongated bacteroids that fix nitrogen. This differentiation is mediated by antimicrobial NCR peptides on the plant side, but the corresponding factors on the bacterial side are unknown. An S. meliloti cytoplasmic membrane protein called BacA has long been known to be essential for bacteroid development in alfalfa relatives, but its role has remained uncertain. Recently, however, BacA has been linked to NCR peptides: while BacA is required for S. meliloti differentiation in legumes that produce NCR peptides (such as alfalfa and peas), this protein is not required for bacteroid development in legumes that lack these peptides (such as beans and lotus). Now, in this issue of PLoS Biology, Mergaert, Ferguson, and colleagues present compelling evidence that BacA is key to protecting S. meliloti from antimicrobial NCR peptides produced by host legumes. To explore the role of BacA further, the researchers synthesized an antimicrobial peptide (NCR247) of the alfalfa relative Medicago truncatula and compared its effects on cultured S. meliloti with and without BacA. They found that while NCR247 decreases wild-type S. meliloti colony formation, this inhibition is more pronounced in a mutant strain that lacks BacA. In addition, this mutant’s growth was restored by a plasmid carrying the bacA gene. Taken together, these findings suggest that BacA protects cultured S. meliloti against NCR peptides. To test the effects of BacA in living plants, the researchers compared the viability of the wild-type and BacA-deficient S. meliloti strains. Nitrogen-fixing bacteria use infection threads to enter root nodule compartments, which contain NCR peptides that damage the cytoplasmic membrane of S. meliloti. Using a fluorescent dye test that stains dead bacteria red and stains living bacteria green, the researchers showed that both the wild-type and BacA-deficient S. meliloti were alive in the infection threads. In contrast, while the wild-type bacteria were still alive once inside the host root nodule compartments, most of the BacA-deficient mutants were dead. This suggests that without the protection of BacA, S. meliloti is rapidly killed by host antimicrobial NCR peptides in the compartments. To confirm the hypothesis that BacA protects S. meliloti against antimicrobial NCR peptides, the researchers also applied the fluorescent dye test to an M. truncatula mutant that blocks NCR peptide transport into root nodule compartments. As expected, both wild-type and BacAdeficient S. meliloti thrived within this mutant’s host compartments. Extending the similarities between the symbiotic S. meliloti and bacteria that cause chronic infections, BacA-like proteins are also found in B. abortus and Mycobacterium tuberculosis. In addition, the B. abortus BacA is essential for chronic infections in mammals. Previous work had shown that B. abortus BacA protects S. meliloti mutants that lack this protein against antimicrobial peptides in alfalfa relatives, and the researchers likewise found that B. abortus BacA protected these S. meliloti mutants against the synthesized antimicrobial peptide (NCR247) in culture. Altogether, these findings suggest a functional similarity between the S. meliloti and B. abortus BacA proteins, and show that the latter protects against antimicrobial activity like that of the defensins found in its mammalian hosts. This work suggests that by protecting S. meliloti against antimicrobial NCR peptides, BacA is key to establishing the symbiosis between these nitrogen-fixing bacteria and their legume hosts. How does BacA confer protection and limit the damage these compounds cause to the S. meliloti cytoplasmic membrane? Possibilities include a direct role for BacA in NCR transport or an indirect effect of BacA on outer membrane lipids. Because BacA is also key to establishing pathogenic bacteria including B. abortus in animal hosts, the answers could have widespread implications for understanding and ultimately treating such chronic infections in mammals.

​TinyTiny worms with simple genetics, nematodes have almost everything it takes to be a favored model system to study viral infections. Recent work has shown that the nematode Caenorhabditis elegans, long known to enlist innate immune pathways against bacteria, can also muster such resistance to viruses. And because these immune responses function much the same way in vertebrates, the worm could help answer questions about the role of these pathways in other species, including humans. So what's missing? FISH staining demonstrating infection of intestinal epithelial cell of C. elegans by Orsay virus. Because no viruses were known to infect nematodes naturally, biologists had to rely on artificial means of introducing viruses or viral RNA. But these studies had the drawback of assessing immunity only to viral genome replication. Thus, the question of how the immune system guards against other stages of the virus life cycle, from cell entry to egress, has remained a mystery. Now, biologists may finally get a more comprehensive look at viral infection and immunity in nematodes. In this issue of PLoS Biology, Marie-Anne Felix, Eric Miska, David Wang, and colleagues report the first viruses known to infect nematodes naturally in the wild. The researchers started by collecting nematodes from rotting fruit in French orchards and vineyards. Light microscopy revealed that the worms had striking intestinal symptoms, including cells with degenerated nuclei and cell fusion, suggesting that they were infected. As is true for other animals, the intestine is exposed to ingested microbes and is a main entry point for pathogens in nematodes. However, there were no obvious pathogens, suggesting that the worms might be infected by viruses, which are too small to be seen with light microscopes. Surprisingly, despite the severity of their affliction, the worms led rather normal lives and kept moving, eating, and reproducing, albeit at lower levels. The researchers set off on their virus hunt by investigating two of the afflicted strains: a C. elegans strain from a rotting apple in Orsay and a Caenorhabditis briggsae strain from a snail on a rotting grape in Santeuil. Multiple lines of evidence suggested that their intestinal symptoms were viral. For example, afflicted nematodes were cured by bleaching and then reinfected when mixed with filtrates of symptomatic worms. Moreover, electron microscopy revealed virus-sized particles in intestinal cells of symptomatic nematodes but not in those that had been bleach-cured, bolstering the hypothesis that the affliction was viral. Molecular analysis then indicated that each of the two nematode strains contained RNA sequences that were similar to those of nodaviruses, a family of viruses that infect invertebrates and vertebrates. Based on phylogenetic analysis of the proteins predicted from these RNA sequences, the researchers concluded that the sets of sequences comprised two novel viruses, one from each strain, that are distantly related to known nodaviruses and most closely related to each other. These new viruses were named for the places where they were found, with the “Orsay” virus infecting only C. elegans and the “Santeuil” virus infecting only C. briggsae. Previous studies of nematode immunity to artificially introduced viral RNA had shown that nematodes counter infection via the RNA interference (RNAi) pathway, which suppresses replication of viral genomes by destroying RNA sequences. This is a key defense mechanism against RNA viruses common to plants and animals. To see if this also holds for natural viral infections, the researchers determined whether Orsay virus–infected C. elegans produced the small RNAs—the short non-coding sequences that mediate and are products of the RNAi pathway. Of the nearly 1.5 million unique small RNAs identified in the infected C. elegans strain, about 21,000, or nearly 2%, mapped to the Orsay virus RNA. After bleach-curing, however, the strain no longer produced this subset of small RNAs. These findings suggest that, in keeping with the earlier works that used artificially introduced viral RNA, nematodes do indeed fight naturally occurring viral infections with RNAi. This conclusion was further strengthened by the finding that RNAi pathway mutations affected viral replication in another C. elegans strain. Called N2, this strain can also be infected by the Orsay virus but at far lower levels and with no discernable intestinal symptoms. However, the researchers found that both viral RNA levels and intestinal symptoms were bumped up in N2 worms that harbor a mutant rde-1 gene, which codes for a protein required for activation of the RNAi pathway. Likewise, Orsay virus–infected N2 worms that had mutations in other RNAi pathway genes, including rde-2, rde-4, and mut-7, also had higher levels of viral RNA. This work opens the way to using Orsay virus–infected mutants to identify additional genes that are instrumental in this mode of anti-viral immunity in nematodes. The researchers have also found evidence suggestive of other anti-viral defenses in nematodes. Comparison of wild C. elegans collected from around the world showed that even though some of the strains had defective RNAi pathways, they still resisted Orsay virus infection. This means the infected strains could be used to identify additional anti-viral pathways in nematodes. The discovery of virus-infected nematodes in nature proves the worm's value as a model system for studying viral infection. In addition to enabling exploration of defenses against viruses at all stages of their life cycle, the worm will help researchers understand how anti-viral immunity evolves as well as how hosts and pathogens coevolve with each other. And with a wealth of genetic tools already in place, this tiny, transparent model organism will surely help uncover more mechanisms of innate immunity. All eyes in the world of molecular immunology will be watching to see what discoveries this system yields next. Felix M-A, Ashe A, Piffaretti J, Wu G, Nuez I, et al. (2011) Natural and Experimental Infection of Caenorhabditis Nematodes by Novel Viruses Related to Nodaviruses. doi:10.1371/journal.pbio.1000586

Unlike those in the periphery, nerve fibers in the central nervous system (brain and spinal cord) do not recover from traumatic injury. This makes disabilities from spinal cord damage permanent, with the severity depending on the location of the cord injury. Neck injuries can paralyze the torso and limbs, while lower back injuries can impair movement below the waist. The spinal cord is a bundle of long, thin fibers called axons that connect the brain to the body, and regrowth of these axons can be spurred by a variety of pharmacological treatments. In an effort to understand the basic mechanisms of axon regrowth, Ping Yip and colleagues report how a protein called neuronal calcium sensor-1 (NCS1) can help repair central nervous system (CNS) damage in rats. Previous studies have linked NCS1 to neuron survival, as well as to the outgrowth or sprouting of neuronal processes. This sprouting can occur on axons, which transmit neuronal signals, and on dendrites, which receive signals. Yip and colleagues have previously shown that axons can regenerate in CNS neurons that overexpress retinoic acid receptor β2, a protein that regulates cell growth, and also observed that this regeneration is accompanied by a rise in NCS1 protein. Here, Yip and colleagues further test the relevance of NCS1 to axonal regrowth in both cultured neurons and living rats. They begin by developing a method of increasing the expression of NCS1, and confirming this overexpression triggers sprouting in cultured neurons from adult rat brains. After treatment with a viral vector carrying NCS1 and green fluorescent protein (GFP), these neurons expressed five times the normal amount of NCS1 and sprouted abundantly. Labeling with a dendritic marker called microtubule associated protein 2 revealed sprouting on both axonal and dendritic projections. In contrast, untreated neurons hardly sprouted at all. To gain insight into the mechanism of NCS1-induced sprouting, the researchers investigated the role of a pathway (P13K/Akt) that regulates cell growth and proliferation and increases neuron survival. They found that cultured neurons that overexpress also have high levels of phospho-Akt protein, indicating activation of the P13K/Akt pathway. Furthermore, blocking this pathway in NCS1-transduced neurons led to a drop in both phospho-Akt levels and neurite sprouting. To see if these findings also hold for CNS neurons in living rats, the researchers injected the NCS1-GFP vector into the region of the cerebral cortex that controls limb movements for only one side of the body. The right half (or hemisphere) of the cortex contains neurons that control movement on the left side of the body, while the left hemisphere controls the right side of the body. Axons from each hemisphere of the cortex form the pyramidal tract in the base of the brain and then enter the spinal column. Like the brain, the spinal cord also has two halves, each of which controls one side of the body. Three weeks after the NCS1-GFP vector injection, the neurons and axons expressing high NCS1 levels were completely GFP-labeled all the way from the cortex to the spinal cord. The researchers then severed the pyramidal tract on the other side, denervating the untreated half of the spinal cord while leaving the treated half intact. Six weeks later, nerve fibers from the intact side of the spinal cord had extended into the injured side, confirming that NCS1 overexpression boosts axon sprouting in whole animals as well as in cell culture. To determine whether this new sprouting actually translated into rescue of motor behavior, the researchers next asked how well NCS1-transduced rats could use their limbs on the injured side. One behavioral test required rats to use their forelimbs to reach for and grasp food pellets. Two days after their spinal cord injuries, the NCS1-transduced rats could hardly get food pellets with their affected forelimbs. But within 21 days, they grabbed food pellets as handily as uninjured rats. The other behavioral test assessed how well rats could walk on the wires of a mesh. Soon after their spinal cord injuries, NCS1-transduced rats were quite clumsy at navigating the grid with their affected limbs. But within 21 days, they were as surefooted as uninjured rats. Finally, the reserachers tested whether NCS1 transduction after spinal injury would also lead to substantial axon regrowth and behavioral recovery. They found that NCS1 overexpression has much the same benefits when begun two days after injury as when begun beforehand. Encouragingly, these benefits include new fibers extending from the intact side of the spinal cord into the injured side as well as regeneration of fibers on the injured side. The combination of anatomical and behavioral recovery makes this work particularly promising, suggesting that therapies to increase NCS1 levels may someday help people recover from spinal cord injuries. Yip PK, Wong L-F, Sears TA, Yanez-Munoz RJ, McMahon SB (2010) Cortical Overexpression of Neuronal Calcium Sensor 1 Induces Functional Plasticity in Spinal Cord Following Unilateral Pyramidal Tract Injury in Rat. doi:10.1371/journal.pbio.1000399

​PlantsPlants grow in a wondrous assortment of patterns, from simple to complex, with near mathematical precision. Honeysuckles sprout leaf pairs at regular intervals along their stems, for instance, whereas some succulents form double spirals turning in opposite directions. The big question is how do they do this? New research reported in this issue of PLoS Biology suggests that mechanical stress is at the root of plant patterns, challenging the longstanding theory that the plant hormone auxin acts alone to direct this patterning. In the Arabidopsis shoot meristem, the orientation of cellular growth and the direction of auxin transport are coupled through a common orientation of interphase microtubules (green) and PIN1 auxin efflux carrier localization (red). Regardless of the growth pattern, new leaves and flowers arise from blobs of pluripotent cells at shoot tips (the shoot apical meristem, or SAM). Auxin triggers the transformation of these shoot tip cells into new organs and is also linked to plant growth patterns—when a given SAM cell has high auxin levels, the neighboring cells form clusters of an auxin export protein (PINFORMED1 or PIN1) in the parts of their plasma membranes that border the auxin-rich cell. This sets up an auxin circulatory system in which the auxin-rich cells get even more of this hormone, further boosting their growth. But although this chemical signaling could drive plant patterning in theory, it is difficult to see how it would work in actuality. Because auxin lacks directional effects in cells, it is unlikely to cause PIN1 clustering, leaving the origin of plant designs a mystery. Recently, however, Marcus Heisler and colleagues showed that mechanical stress is linked to the direction of organ growth, and here they reveal that stress is also linked to plant patterning. Whereas auxin initiates leaf and flower development, the direction of this new growth depends on the orientation of microtubules in cell cytoskeletons. Plant plasma membranes are encased in sturdy cell walls that contain load-bearing components, such as cellulose, and chemical treatments that modify the mechanical properties of these walls can disrupt plant patterning. The researchers had previously found that when cell walls of the small flowering plant Arabidopsis thaliana are perturbed mechanically, the orientation of shoot tip microtubules reflects the resulting mechanical stress patterns. To see if stress also directs PIN1 clustering in shoot tips, the researchers treated Arabidopsis with isoxaben, which weakens cell walls by inhibiting cellulose synthesis. Treated shoot tip cells keep growing despite the absence of additional cellulose, which presumably increases the stress on their cell walls. The researchers then visualized the distributions of microtubules and PIN1. Before isoxaben treatment, microtubules were randomly oriented in many shoot tip cells, whereas after treatment, these cytoskeletal components formed thick bundles that were aligned as expected relative to increased stress. Likewise, PIN1 clustering in isoxaben-treated cells also matched the predicted stress patterns. For example, PIN1 was predominantly in cell corners, and corners are associated with high stress in structures. As an additional test of whether mechanical forces could generate these observed PIN1 patterns, the researchers developed a mathematical model accounting for stress, auxin transport and PIN1 dynamics. The model confirmed that cell wall stress could generate PIN1 distribution patterns, which could in turn generate plant growth patterns that, like those found in nature, are periodic. The researchers propose that patterning depends on the following chain of events: as a given shoot tip cell expands, stress levels in the adjacent cell walls of neighboring cells increase. This causes PIN1 clustering in the adjacent plasma membranes of neighboring cells, which then export auxin to the expanding cell, which then grows even larger. Periodic PIN1 distribution patterns would then give rise to periodic shoot tip cell growth, thus creating the overall pattern of leaf or flower growth. Besides accounting for plant patterning just as well as the chemical signaling model, this new mechanical signaling model has the added benefit of explaining additional observations. For example, root formation can be induced mechanically in Arabidopsis. And flower primordium initiation can be induced by pectin methyl-esterase, which likely changes the viscoelastic properties of cell walls by altering the degree of pectin cross-linking. Although it will be important to test this mechanical model further experimentally, this work makes a compelling case that a common mechanism—stress—drives both the microtubule orientations and the PIN1 polarities critical to the beautiful patterns of leaves and flowers in the world around us. It will be interesting to investigate other possible roles for mechanical signaling in development as well as the coordination of growth localization and growth direction in both normal development and wound repair. Heisler MG, Hamant O, Krupinski P, Uyttewaal M, Ohno C, et al. (2010) Alignment between PIN1 Polarity and Microtubule Orientation in the Shoot Apical Meristem Reveals a Tight Coupling between Morphogenesis and Auxin Transport. doi:10.1371/journal/pbio.1000516

​TremblingTrembling aspens (Populus tremuloides) are among the world's most remarkable trees, forming vast clones that can live an improbably long time. These clones share a common root system, from which new trees arise, cover up to 43 hectares and persist up to a million years. The vigor of these ancient plants belies their years, leading to speculation that they defy aging or senescence. But demonstrating senescence or its absence in plants that live longer than we do is tricky. Now, in new research reported in this issue of PLoS Biology, Dilara Ally, Kermit Ritland, and Sarah Otto have overcome this obstacle partly by using declining fertility as a proxy for senescence. A forest stand of aspen. Populus tremuloides, like many other clonal plants, is capable of growing and reproducing throughout its life. Because clonal plants continually renew themselves, the question raised by these organisms is: do they age? Aging is thought to be a byproduct of natural selection acting most effectively earliest in life. Because species typically reproduce early in life, they can accumulate mutations that are deleterious late in life without jeopardizing their production of offspring. In contrast, clonal plants can also grow and reproduce sexually throughout their lives, raising the question of whether they senesce as they get older. Ally and colleagues thought they might for two reasons. First, longer-lived clones have had more cell divisions, increasing the accumulation of mutations in their “body” (somatic) cells. Second, because plant reproductive cells come from somatic cells, plants can pass mutations from their shoots and roots on to their offspring. To investigate whether trembling aspens do senesce, Ally and colleagues asked if advanced age was linked to diminished fertility in clones. Age was estimated using a molecular clock, which was based on the amount of genetic diversity aspen clones had accumulated from the time since they were seeds. Aspen clones are either male or female, and fertility was based on the amount of viable pollen produced by male clones. To rule out the effects of environmental factors on fertility, the researchers accounted for influences such as disease, herbivory, and soil quality. The aspen clones studied ranged from roughly 70 to 10,000 years old, and pollen analysis revealed a slow but steady loss of fertility with age. Fertility was cut by more than three-quarters in the oldest clone and, based on extrapolation, would likely have dwindled away entirely by 20,000 years. Thus, though it may take millennia, even plants that grow indefinitely will eventually succumb to old age. Next the researchers investigated whether vegetative growth (asexual reproduction) comes at the expense of sexual reproduction in aspen clones. However, they found no evidence of tradeoffs between asexual and sexual reproduction. New shoots did not grow faster in aspen clones that were less fertile, older clones were not necessarily bigger than younger ones, and bigger clones were not less fertile than smaller ones. Alternative explanations for the age-related fertility drop include heritable epigenetic factors such as DNA methylation, which can affect reproductive functions from flowering to self-fertility. These findings show that male trembling aspen clones are at risk of extinction as they lose fertility with age, because without sex they cannot disperse and start new clones. This work also raises a number of intriguing questions. Do male and female aspen clones lose fertility at different rates? Previous work suggests they might: sperm transmit more deleterious mutations than do eggs. And do other plants that form clones also lose fertility as they get older? Such astonishingly long-lived plants are found around the world, in habitats from land to sea, and include the Tasmanian tree Lagarostrobos franklinii, the Mediterranean sea grass Posidonia oceanica, and the western US fungus Armillaria ostoyae. The researchers' technique of using molecular clocks to estimate clone age opens up the possibility of answering these questions for a variety of ancient clonal plants in the wild. Ally D, Ritland K, Otto SP (2010) Aging in a Long-Lived Clonal Tree. doi:10.1371/journal.pbio.1000454

The maxim ‘‘you are what you eat’’ goes only so far. The bacteria inhabiting our guts, which outnumber our own cells by perhaps 10 to 1, are commonly thought to reflect our diets. But other factors from geography to host physiology can also affect gut microbes, and sorting out their provenance is critical because they can affect our health for good or ill, from enhancing immune function to increasing the risk of stomach cancer. New research in this issue of PLoS Biology by Howard Ochman and colleagues counters the prevailing view that diet shapes the makeup of gut microbes, revealing that the host animal is a stronger determinant of these bacterial worlds in our digestive tracts. On the face of it, gut microbe composition could be determined by either the host or its environment. Mammals are born with sterile digestive tracts and usually get their first gut microbes from their mothers, but thereafter acquire new ones from their environment. Moreover, there is evidence on both sides of the host versus environment debate. The evolutionary relationships of some gut bacteria are known to match those of their hosts, notably Helicobacter pylori, which is found in about half of people and is linked to stomach ulcers and cancer. However, work on great apes had suggested that diet is the major determinant of gut microbe composition. To sort out whether gut microbe composition depends primarily on the host or its environment, Ochman and colleagues compared fecal samples from five closely related hominid species: Eastern and Western lowland gorillas, bonobos, and three subspecies of chimpanzees from several African countries as well as people from Africa and North America. By using fecal samples from locations separated by such considerable distances, the researchers effectively knocked out any effects of geography and the local environmental on gut microbes. The researchers constructed two phylogenetic trees from the fecal samples: one of the great ape hosts, which was based on mitochondrial DNA, and the other of their gut microbe communities. Gut microbe diversity and abundance were based on small subunit ribosomal RNA genes, which are unique to bacteria and other prokaryotes. In turn, the ‘‘gut microbe’’ tree was based on the relative abundance of the various microbe species in each fecal sample, much as physical measures such as femur lengths from, say, mouse to elephant are used to infer evolutionary relationships. The resulting gut microbe tree mirrored that for hominids; the patterns of relationships among the gut microbe communities were identical to those among the five great ape species. This congruence neatly establishes hosts as the major force behind the makeup of gut microbe communities. Further, the concordance of the two phylogenetic trees suggests that the gut microbe communities were shaped by divergences in host physiology over the course of great ape evolution. The disparity with previous findings is partly because this new work entailed surveying many more fecal bacteria sequences per host, with a median of 28,000 sequences. In contrast, in previous studies only 100 to 200 bacterial sequences were surveyed per host, which is insufficient for gauging the diversity and abundance of species in the complex microbe communities living in our digestive tracts. This work brings us closer to understanding the acquisition and evolution of the gut flora that can affect our health for better or for worse. Even though new microbes are constantly arriving in digestive tracts, their makeup is largely determined by the distinctive physiologies of people and our closest relatives. Gut microbe communities are thus predictable rather than variable, as they would be if determined largely by the host’s external environment. Perhaps this is not altogether surprising, given that we hosts essentially are the environment for the worlds of enteric microbes within us. In this case at least, what we are trumps what we eat.