Gut bacteria mediate aggregation in the German cockroach

Aggregation of the German cockroach, Blattella germanica, is regulated by fecal aggregation agents (pheromones), including volatile carboxylic acids (VCAs). We demonstrate that the gut microbial community contributes to production of these semiochemicals. Chemical analysis of the fecal extract of B. germanica revealed 40 VCAs. Feces from axenic cockroaches (no microorganisms in the alimentary tract) lacked 12 major fecal VCAs, and 24 of the remaining compounds were represented at extremely low amounts. Olfactory and aggregation bioassays demonstrated that nymphs strongly preferred the extract of control feces over the fecal extract of axenic cockroaches. Additionally, nymphs preferred a synthetic blend of 6 fecal VCAs over a solvent control or a previously identified VCA blend. To test whether gut bacteria contribute to the production of fecal aggregation agents, fecal aerobic bacteria were cultured, isolated, and identified. Inoculation of axenic cockroaches with individual bacterial taxa significantly rescued the aggregation response to the fecal extract, and inoculation with a mix of six bacterial isolates was more effective than with single isolates. The results indicate that the commensal gut microbiota contributes to production of VCAs that act as fecal aggregation agents and that cockroaches discriminate among the complex odors that emanate from a diverse microbial community. Our results highlight the pivotal role of gut bacteria in mediating insect–insect communication. Moreover, because the gut microbial community reflects the local environment, local plasticity in fecal aggregation pheromones enables colony-specific odors and fidelity to persistent aggregation sites.Diverse microbial communities inhabit the alimentary tract and other tissues of many insect species. Their effects on the host vary, ranging from facultative provision of essential nutrients to stimulation of the immune system and exclusion of pathogenic microbes (1–6). Insect-symbiotic associations, some obligatory, are common, where hosts are nutritionally and immunologically dependent on their symbiotic microbes: Buchnera in aphids (7), nitrogen-fixing bacteria in termites (8), Blattabacterium in cockroaches (e.g., ref. 9), lactic acid bacteria in honey bees (10) and Wolbachia, which affects sex determination (11), immune function (e.g., ref. 12) and nutrition (13) in many insect species. The alimentary tract, and especially the hindgut of many (possibly all) insects, is persistently colonized by opportunistic, facultative, and commensal microbiota largely structured by exogenous (diet and local environment) and endogenous (gut environment) factors. The commensal gut microbiota can modulate various aspects of insect biology, including behavior (e.g., refs. 14–16), host–parasite and host–pathogen interactions (e.g., refs. 2 and 4), and various life history traits (1, 17).The German cockroach, Blattella germanica is a major pest of the built environment, where it can acquire and transmit pathogens, contaminate food, and produce allergenic proteins that cause human morbidity (18, 19). The German cockroach lives in aggregations (20), and contact with conspecifics accelerates nymphal development (21) and reproductive maturation in both sexes (22, 23). Younger nymphs benefit from coprophagy in aggregations (24), and gregarious behavior may also facilitate mate location, predator avoidance, thermoregulation, and prevention of water loss. Fidelity to the resting/aggregation site may also facilitate group foraging in the rapidly changing human environment. Aggregation behavior is mediated by at least two types of chemical cues: endogenous compounds produced by the insect and compounds contained in feces. Cuticular hydrocarbons facilitate aggregations (25), and salivary compounds contribute to dissolution of aggregations (26); both are examples of endogenous signals. Feces-associated compounds function as powerful attractants and arrestants in all life stages of the German cockroach (27, 28).Identification of the fecal aggregation pheromones of cockroaches has been fraught with controversy. Candidate pheromones are thought to be endogenously produced by rectal pads (29), with arrestment agents, including blattellastanoside A and B (30) and volatile carboxylic acids (VCAs) (31, 32), and attractants, including ammonia, alkyl amines, amino alcohols, alcohols (33), and VCAs (31, 32). However, the chemical profiles of aggregation-inducing agents vary greatly among reports. The structures of blattellastanosides may be an artifact of chemical isolation and fractionation (34). Some compounds are inconsistently detected in feces, and behavioral responses to them range from attraction to neutral to avoidance (32, 35). More than 150 compounds, including 57 carboxylic acids, have been identified from feces of the German cockroach (31). Because methylation decreased the aggregation response (31), a mix of VCAs was considered the likely aggregation stimuli (32).Symbiotic and commensal bacteria modulate the production of sex pheromones in grass grub beetles (36) and Drosophila (15) and the aggregation pheromone in locusts (37). We hypothesized that the fecal VCAs that mediate aggregation in the German cockroach originate from the bacterial community in the feces, and, because gut-associated bacteria are acquired from the environment, we posited that both the VCA profiles and the behavioral responses to them depend on environmental conditions. Our behavioral assays and chemical analysis revealed that the feces of axenically reared cockroaches (no microorganisms in the alimentary tract) contained many fewer VCAs and failed to elicit aggregation behavior. Inoculation with fecal aerobic bacteria rescued the aggregation activity of fecal extracts of axenic cockroaches. A synthetic blend of VCAs was an effective aggregation stimulus for German cockroaches. We propose that gut bacteria impact the production of fecal VCAs as aggregation agents and that cockroaches use fecal VCAs from commensal microbes as aggregation cues that reflect their colony odor.     

Combinatorial quorum sensing allows bacteria to resolve their social and physical environment

Quorum sensing (QS) is a cell–cell communication system that controls gene expression in many bacterial species, mediated by diffusible signal molecules. Although the intracellular regulatory mechanisms of QS are often well-understood, the functional roles of QS remain controversial. In particular, the use of multiple signals by many bacterial species poses a serious challenge to current functional theories. Here, we address this challenge by showing that bacteria can use multiple QS signals to infer both their social (density) and physical (mass-transfer) environment. Analytical and evolutionary simulation models show that the detection of, and response to, complex social/physical contrasts requires multiple signals with distinct half-lives and combinatorial (nonadditive) responses to signal concentrations. We test these predictions using the opportunistic pathogen Pseudomonas aeruginosa and demonstrate significant differences in signal decay between its two primary signal molecules, as well as diverse combinatorial responses to dual-signal inputs. QS is associated with the control of secreted factors, and we show that secretome genes are preferentially controlled by synergistic “AND-gate” responses to multiple signal inputs, ensuring the effective expression of secreted factors in high-density and low mass-transfer environments. Our results support a new functional hypothesis for the use of multiple signals and, more generally, show that bacteria are capable of combinatorial communication.Bacteria must often make regulatory decisions on the basis of limited information about their external world (1). In many bacteria, these decisions are aided by the secretion and detection of small diffusible molecules, in a process called quorum sensing (QS) (2, 3). QS controls a variety of traits, including pathogen virulence (3) leading to “QS interference” (2–6) emerging as a control strategy for several bacterial pathogens.The mechanisms underlying production, uptake, and response to these signal molecules are well-understood, but relatively little is known about how quorum sensing contributes to bacterial fitness. Put another way, why do bacteria use QS? The classic adaptive interpretation of QS is that cells produce signal molecules to serve as a proxy for cellular density: more signal implying more bacteria (7–10). Others have argued for a “diffusion-sensing” interpretation, with more signal implying lower rates of mass transfer (diffusion or flow) (11). However, low mass transfer and high cellular density both lead to high signal concentrations, and so these two unknowns—information on the social parameter of cellular density and the asocial mass-transfer rate—are inextricably linked when only one signal is used (12, 13). It is possible that a signal molecule can still provide a reliable indicator of the achievable density of a more costly secreted factor (“efficiency sensing”) (13). However, where investigated, the majority of QS-regulated genes encode nonsecreted gene products (14), and QS is known to control an array of traits in various bacteria that are not directly impacted by mass transfer, such as luminescence, conjugation, or type-3 secretion (10). Even among secretion-related phenotypes, accumulation of a single autoinducer cannot reliably predict the dynamics of secreted products differing in rates of mass transfer or chemical decay or in how they interact with the environment to form beneficial compounds. Here, we argue that, by combinatorially responding to multiple signal molecules with the appropriate molecular properties (differing in chemical decay rates), bacteria can potentially infer the properties of their social and physical environment simultaneously (Fig. S1).     

Commensal bacteria protect against food allergen sensitization

Environmentally induced alterations in the commensal microbiota have been implicated in the increasing prevalence of food allergy. We show here that sensitization to a food allergen is increased in mice that have been treated with antibiotics or are devoid of a commensal microbiota. By selectively colonizing gnotobiotic mice, we demonstrate that the allergy-protective capacity is conferred by a Clostridia-containing microbiota. Microarray analysis of intestinal epithelial cells from gnotobiotic mice revealed a previously unidentified mechanism by which Clostridia regulate innate lymphoid cell function and intestinal epithelial permeability to protect against allergen sensitization. Our findings will inform the development of novel approaches to prevent or treat food allergy based on modulating the composition of the intestinal microbiota.Life-threatening anaphylactic responses to food are an increasingly important public health problem (1). Rising disease prevalence over a short period cannot be explained by genetic variation alone, renewing interest in the role of the environment in shaping allergic sensitization to food (2, 3). First proposed more than 20 years ago, the hygiene hypothesis suggested that societal efforts to reduce exposure to infectious microbes early in life have deprived the immune system of immunoregulatory stimulation necessary for protection against allergic disease (4). As our understanding of the profound influence of commensal microbes on the maturation of the immune system has grown, more recent iterations of this hypothesis have supported the idea that alterations in the composition of the intestinal microbiota induced by environmental factors (e.g., antibiotics, diet, vaccination, sanitation) play a central role in the regulation of allergic sensitization (5–7). In particular, antibiotic use during infancy potently perturbs intestinal bacterial populations and has often been cited as a contributing factor to the rising prevalence of allergic disease (8). However, the mechanisms by which changes in the composition of the intestinal microbiota regulate allergic responses to food remain poorly understood.The gastrointestinal tract must maintain nonresponsiveness to both an enormous variety of food antigens and the trillions of bacteria that comprise the commensal microbiota (9). Mucosal IgA and regulatory T-cell (Treg) responses induced by commensal bacteria are critical for sustaining the homeostatic host–microbe relationship and preventing intestinal inflammation (10). In addition, recent work has revealed that a heterogeneous population of innate immune cells, known collectively as innate lymphoid cells (ILCs), plays a critical role in integrating signals from the commensal microbiota to maintain homeostasis at epithelial barriers and guide adaptive immunity (11). In this report we show that sensitization to a food allergen is enhanced in mice that have been treated with antibiotics (Abx) or are devoid of commensal microbes (germ free, GF). Selective colonization of gnotobiotic mice demonstrated that the allergy-protective capacity is contained within the Clostridia, a class of anaerobic spore-forming Firmicutes that reside in close proximity to the intestinal epithelium. Reintroduction of a Clostridia-containing microbiota to Abx-treated mice blocks sensitization to a food allergen. Using microarray analysis of intestinal epithelial cells from gnotobiotic mice, we identify an innate mechanism by which Clostridia protect against sensitization to dietary antigens. Defects in intestinal permeability have been implicated in aberrant allergic responses to food, but the mechanisms governing uptake of dietary antigen have not been clear. We show here that Clostridia colonization induces IL-22 production by both RAR-related orphan receptor gamma (RORγt)⁺ ILCs and T cells in the intestinal lamina propria (LP) and that this cytokine acts to reduce uptake of orally administered dietary antigen into the systemic circulation, contributing, in part, to protection against sensitization.     

Neither cyclic AMP nor cyclic GMP appear to mediate antral gastrin release in the dog.

To evaluate whether cyclic nucleotides play a role as mediators in antral gastrin release, the following in vivo experiments were performed in dogs. An antral pouch was constructed and the rest of the stomach, the pancreas and small and large intestine were resected. A gastric artery supplying the pouch was cannulated, and after a basal period with saline infusion either dibutyryl cyclic 3',5'-adenosine monophosphate (cyclic AMP, 0.2 mg per kg per min), dibutyryl cyclic 3',5'-guanosine monophosphate (cyclic GMP, 0.05 mg per kg per min) or saline were infused over a period of 60 min. To prove the viability of the pouch and to show its ability to release gastrin with proper stimulation, bethanechol chloride (urecholine) was infused into the gastric artery at the end of the experiment. Blood samples were taken from the portal vein and assayed for gastrin. Neither cyclic AMP nor cyclic GMP infusion was found to increase portal gastrin concentration to a significant degree. A marked increase in portal gastrin concentration however, was observed when bethanechol chloride was infused. These studies lend no support to the thesis that cyclic nucleotides mediate gastrin release in the dog.     

Gut bacteria signaling to mitochondria in intestinal inflammation and cancer

ABSTRACT

The gastrointestinal microbiome plays a pivotal role in physiological homeostasis of the intestine as well as in the pathophysiology of diseases including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Emerging evidence suggests that gut microbiota signal to the mitochondria of mucosal cells, including epithelial cells and immune cells. Gut microbiota signaling to mitochondria has been shown to alter mitochondrial metabolism, activate immune cells, induce inflammasome signaling, and alter epithelial barrier function. Both dysbiosis of the gut microbiota and mitochondrial dysfunction are associated with chronic intestinal inflammation and CRC. This review discusses mitochondrial metabolism of gut mucosal cells, mitochondrial dysfunction, and known gut microbiota-mediated mitochondrial alterations during IBD and CRC.     

From the Cover: Exploitative leaders incite intergroup warfare in a social mammal

Collective conflicts among humans are widespread, although often highly destructive. A classic explanation for the prevalence of such warfare in some human societies is leadership by self-serving individuals that reap the benefits of conflict while other members of society pay the costs. Here, we show that leadership of this kind can also explain the evolution of collective violence in certain animal societies. We first extend the classic hawk−dove model of the evolution of animal aggression to consider cases in which a subset of individuals within each group may initiate fights in which all group members become involved. We show that leadership of this kind, when combined with inequalities in the payoffs of fighting, can lead to the evolution of severe intergroup aggression, with negative consequences for population mean fitness. We test our model using long-term data from wild banded mongooses, a species characterized by frequent intergroup conflicts that have very different fitness consequences for male and female group members. The data show that aggressive encounters between groups are initiated by females, who gain fitness benefits from mating with extragroup males in the midst of battle, whereas the costs of fighting are borne chiefly by males. In line with the model predictions, the result is unusually severe levels of intergroup violence. Our findings suggest that the decoupling of leaders from the costs that they incite amplifies the destructive nature of intergroup conflict.

Humans are capable of astonishing feats of altruism and cooperation (1–3), but, at the same time, of violent and destructive conflicts (4–8). A key factor contributing to the latter may be that wars are often waged at the behest of leaders who do not share fully in the immediate risks of conflict, and stand to gain benefits in terms of resources and status that are not enjoyed by the majority of combatants (4, 9–11). Could such “warmongering” be a feature of animal conflicts too? Only recently have models of animal aggression begun to explore the impact of inequalities among combatants in collective conflict (12, 13), and the usual assumption of existing theory is that individuals who initiate intergroup conflicts also contribute most to group conflict effort and thereby confer fitness benefits on the rest of their group (a positive or “heroic” model of leadership) (14–17). Here, we explore the more sinister possibility that those who initiate conflict may actually harm their fellows in pursuit of their own interests by exposing them to the risks of conflict while contributing little to fighting themselves (a negative or “exploitative” model of leadership).     

From the Cover: Elephants can determine ethnicity,gender, and age from acoustic cues in human voices

Animals can accrue direct fitness benefits by accurately classifying predatory threat according to the species of predator and the magnitude of risk associated with an encounter. Human predators present a particularly interesting cognitive challenge, as it is typically the case that different human subgroups pose radically different levels of danger to animals living around them. Although a number of prey species have proved able to discriminate between certain human categories on the basis of visual and olfactory cues, vocalizations potentially provide a much richer source of information. We now use controlled playback experiments to investigate whether family groups of free-ranging African elephants (Loxodonta africana) in Amboseli National Park, Kenya can use acoustic characteristics of speech to make functionally relevant distinctions between human subcategories differing not only in ethnicity but also in sex and age. Our results demonstrate that elephants can reliably discriminate between two different ethnic groups that differ in the level of threat they represent, significantly increasing their probability of defensive bunching and investigative smelling following playbacks of Maasai voices. Moreover, these responses were specific to the sex and age of Maasai presented, with the voices of Maasai women and boys, subcategories that would generally pose little threat, significantly less likely to produce these behavioral responses. Considering the long history and often pervasive predatory threat associated with humans across the globe, it is likely that abilities to precisely identify dangerous subcategories of humans on the basis of subtle voice characteristics could have been selected for in other cognitively advanced animal species.The ability to recognize predators and assess the level of threat that they pose is a crucial cognitive skill for many wild animals that has very direct and obvious fitness consequences (1–5). Until recently, most research in this area focused on how a range of birds and mammals classify other animal predators, demonstrating complex abilities to differentiate between predators with different hunting styles and respond with appropriate escape tactics (2, 3, 6–8). However, for many wild populations, humans represent a significant predatory threat (5, 9, 10) and this threat is rapidly increasing as areas for wildlife decrease and human–animal conflict grows. Moreover, as it is typically the case that not all humans pose the same risk to prey species, distinguishing between different human subgroups to identify those associated with genuinely threatening situations could present a major cognitive challenge. The extent of behavioral flexibility that different species may exhibit in correctly classifying human predators—and the degree of sophistication possible in such abilities—is therefore of considerable interest.Most research on the abilities of animals to classify human predators has focused on discrimination through facial features or general differences in behavior and appearance (5, 11–13). This focus has demonstrated that a number of different species are able to use visual cues to distinguish between individual humans that present varying levels of threat (4, 5, 14–16). However, acoustic cues could potentially provide a more effective means of classifying human predators by virtue of enabling categories of particularly dangerous humans to be identified. Such cues have an advantage because they code information on sex and age as well as cultural divisions that may be associated with differing levels of predation risk. Furthermore, these cues are available when the predator is still out of sight, potentially providing an important early warning system. Until now, however, studies of animal responses to human voices have focused on demonstrating skills in recognizing individual humans (17–21) rather than investigating specific abilities to identify particular human subgroups that have functional relevance in the natural environment.African elephants present an ideal model for a study of this nature, as humans constitute their most significant predator other than lions (1) and different human subgroups present them with different threats. African elephants are also already known to make broad distinctions between human ethnic groups on the basis of visual and olfactory cues (15). In the Amboseli ecosystem in Kenya, Maasai pastoralists periodically come into conflict with elephants over access to water and grazing for their cattle, and this sometimes results in elephants being speared, particularly in retaliation when Maasai lives have been lost (15, 22). In contrast, Kamba men, with more agricultural lifestyles, do not typically pose a significant threat to elephants within the National Park, and where conflict occurs outside over crop raiding, this largely involves male rather than female elephants (see, for example, ref. 23). Previous research has demonstrated that elephant family groups exhibit greater fear-based reactions to the scent of garments previously worn by Maasai men than Kamba men and also show aggression to presentations of the red clothes that Maasai typically wear (15). However, experiments involving presentations of human artifacts are inevitably limited in the level of natural variation that they can realistically simulate, whereas playback of human voice stimuli offers the possibility of investigating abilities to make a much wider range of functionally important distinctions. The extent to which elephants can use human voice cues to determine not only ethnicity, but also finer-scaled differences in sex and age that can dramatically affect predation risk, is highly relevant not only for determining the cognitive abilities that underlie predator recognition but also for understanding the coevolution of humans and arguably their most cognitively advanced prey.We used controlled playback experiments to investigate whether elephant family groups in Amboseli National Park were able to make subtle distinctions between the varying levels of threat posed by different categories of human. Although the presence of Maasai typically represents the greatest threat, this is specific to Maasai men because Maasai women are not involved in elephant-spearing events (22). We were able to compare behavioral responses of 48 female family groups not only to a large sample of voice stimuli from adult Maasai men versus Kamba men saying “Look, look over there, a group of elephants is coming” in their own language, but also to Maasai men versus Maasai women giving this utterance. In this latter experiment we used both natural stimuli and stimuli that had been resynthesized to mimic sex differences while keeping other acoustic characteristics of the voice unchanged. In a final experiment, we contrasted elephant responses given to the voices of Maasai men versus Maasai boys (see Materials and Methods for details).     

Malaria-induced changes in host odors enhance mosquito attraction

Vector-borne pathogens may alter traits of their primary hosts in ways that influence the frequency and nature of interactions between hosts and vectors. Previous work has reported enhanced mosquito attraction to host organisms infected with malaria parasites but did not address the mechanisms underlying such effects. Here we document malaria-induced changes in the odor profiles of infected mice (relative to healthy individuals) over the course of infection, as well as effects on the attractiveness of infected hosts to mosquito vectors. We observed enhanced mosquito attraction to infected mice during a key period after the subsidence of acute malaria symptoms, but during which mice remained highly infectious. This attraction corresponded to an overall elevation in the volatile emissions of infected mice observed during this period. Furthermore, data analyses—using discriminant analysis of principal components and random forest approaches—revealed clear differences in the composition of the volatile blends of infected and healthy individuals. Experimental manipulation of individual compounds that exhibited altered emission levels during the period when differential vector attraction was observed also elicited enhanced mosquito attraction, indicating that compounds being influenced by malaria infection status also mediate vector host-seeking behavior. These findings provide important insights into the cues that mediate vector attraction to hosts infected with transmissible stages of malaria parasites, as well as documenting characteristic changes in the odors of infected individuals that may have potential value as diagnostic biomarkers of infection.Parasite manipulation of hosts is a widespread phenomenon with broad significance for ecology and human health (1–5). Increased attention has recently focused on manipulation by vector-borne parasites (2, 6, 7), which may enhance their own transmission via direct effects on vector behavior (6–10) or by altering traits of their primary hosts in ways that influence vector attraction and dispersal, as well as the likelihood of pathogen acquisition by vectors during interactions with the primary host (6, 7, 10–12). In the case of pathogens vectored by insects, host odors seem particularly likely targets for manipulation, as olfactory cues play a key role in host location and discrimination by both plant- and animal-feeding insects. And a number of recent studies have documented pathogen-induced effects on volatile mediated host-vector interactions (11–16). In addition to their ecological significance, pathogen-induced changes in host-derived olfactory cues have potential applied implications for efforts to disrupt vector transmission (e.g., via the development of chemical lures or repellents), as well as for disease diagnosis. Indeed, given that a key challenge for the development of volatile-based diagnostics lies in recognizing the “signal” of disease presence against the background “noise” of genetic and environmental variation (17), it is plausible that volatile biomarkers will prove particularly valuable for detecting pathogens that actively manipulate host odors, although little work to date has explored this possibility.The current study explores potential manipulation of host odors by protozoan parasites in the genus Plasmodium responsible for causing malaria, which remains among the deadliest of human diseases and a significant hindrance to economic development in regions where it occurs (18, 19). A good deal of previous research has documented effects of the plasmodium parasites on the physiology and behavior of mosquito vectors (8, 10, 20–24). There is reason to suspect that manipulation of host odors by these parasites also influences vector behavior. For example, a provocative study found that Kenyan children harboring the transmissible (gametocyte) stage of the malaria parasite Plasmodium falciparum were more attractive to mosquitoes than uninfected children or those harboring the nontransmissible stage of the parasite (25). The cues responsible for this enhanced attraction were not identified, but parasite-induced changes in host odors seem the likeliest explanation, as the attraction occurred at a distance and was apparently not explained by variation in body heat or activity (as all of the children involved in the study were asymptomatic). A subsequent study documented preferential blood feeding by the mosquito Culex pipiens on canaries (Serinus canaria) infected with the avian malaria parasite Plasmodium relictum, but the cues mediating this preference were again not determined (26).As noted above, the identification of pathogen-induced changes in host odors that influence vector behavior has potential applied implications, and this is particularly true for malaria. Minimizing transmission by mosquito vectors is a key focus of efforts to control this devastating disease, but resistance evolution poses a continual challenge for strategies that entail suppressing vector populations (27–30). An improved understanding of the ecological mechanisms mediating vector transmission may inform the development of more effective and sustainable control strategies. Furthermore, the ability to effectively direct drug treatments and other interventions to asymptomatic carriers of infection is a key issue for controlling disease spread and likely essential for the long-range goal of malaria eradication (31). Thus, the presence of volatile biomarkers capable of distinguishing asymptomatic individuals bearing the transmissible stage of the disease—as suggested by the findings of the Kenyan field study discussed above—could potentially have great diagnostic value.With these issues in mind, we initiated laboratory studies using a mouse model and the rodent malaria parasite Plasmodium chabaudii to confirm and elucidate the role of parasite-induced volatile cues in mediating preferential vector attraction to infected individuals. Our specific goals were to assess the relative attractiveness of infected individuals to mosquito vectors (compared with healthy controls) over the course of infection and to document associated differences in the volatile profiles of healthy and infected individuals.     

Plasmids in bacteria exposed to activated neutrophils mediate mutagenesis when transferred to new hosts

The plasmid pUC18 contains a lacZ alpha-complementation gene that codes for a small peptide that can complement the delta M15 mutation of the Escherichia coli lacZ (beta-galactosidase) gene, converting bacteria carrying that mutated gene from the lacZ- to the lacZ+ phenotype. This plasmid was used in experiments designed to study mutagenesis by human neutrophils. E coli carrying pUC18 were incubated with neutrophils under conditions in which little ingestion of the bacteria took place; the plasmid was then isolated and transformed into an E coli strain (BOZO) that carries the lacZ delta M15 mutation. Of these transformants, 11 of 205,000 were lacZ, suggesting that in these 11, alpha-complementation had been lost through a mutation. No lac- colonies were detected among several hundred thousand BOZO transformed with plasmid isolated from incubations in which phagocytosis could take place, nor from incubations from which neutrophils were omitted. Despite the lac- phenotype of these 11 transformants, plasmids reisolated from nine of them showed normal alpha-complementing ability when transformed into fresh BOZO. These findings indicated that in these nine, the mutations were located in the chromosomes of the transformed BOZO. It thus appears that on exposure to activated neutrophils, a plasmid may acquire a lesion (? mutation) that can somehow be transferred to the genome of a recipient microorganism, resulting in repair of the damaged plasmid accompanied by mutation of the recipient's chromosome. Restriction mapping of the DNA from four of these nine chromosomal mutants suggested that the mutations did not represent major insertions or deletions in the portion of the bacterial chromosome corresponding to the pUC18 lac operon insert, nor in the remainder of the lacZ delta M15 gene. These results confirm previous work showing that exposure to activated neutrophils can induce mutations in biological systems, and provides an experimental model in which the mechanism of neutrophil-mediated mutagenesis may be examined.     

From the Cover: Cross-modal individual recognition in domestic horses (Equus caballus)

Individual recognition is considered a complex process and, although it is believed to be widespread across animal taxa, the cognitive mechanisms underlying this ability are poorly understood. An essential feature of individual recognition in humans is that it is cross-modal, allowing the matching of current sensory cues to identity with stored information about that specific individual from other modalities. Here, we use a cross-modal expectancy violation paradigm to provide a clear and systematic demonstration of cross-modal individual recognition in a nonhuman animal: the domestic horse. Subjects watched a herd member being led past them before the individual went of view, and a call from that or a different associate was played from a loudspeaker positioned close to the point of disappearance. When horses were shown one associate and then the call of a different associate was played, they responded more quickly and looked significantly longer in the direction of the call than when the call matched the herd member just seen, an indication that the incongruent combination violated their expectations. Thus, horses appear to possess a cross-modal representation of known individuals containing unique auditory and visual/olfactory information. Our paradigm could provide a powerful way to study individual recognition across a wide range of species.     

The influence of sex,handedness, and washing on the diversity of hand surface bacteria

Bacteria thrive on and within the human body. One of the largest human-associated microbial habitats is the skin surface, which harbors large numbers of bacteria that can have important effects on health. We examined the palmar surfaces of the dominant and nondominant hands of 51 healthy young adult volunteers to characterize bacterial diversity on hands and to assess its variability within and between individuals. We used a novel pyrosequencing-based method that allowed us to survey hand surface bacterial communities at an unprecedented level of detail. The diversity of skin-associated bacterial communities was surprisingly high; a typical hand surface harbored >150 unique species-level bacterial phylotypes, and we identified a total of 4,742 unique phylotypes across all of the hands examined. Although there was a core set of bacterial taxa commonly found on the palm surface, we observed pronounced intra- and interpersonal variation in bacterial community composition: hands from the same individual shared only 17% of their phylotypes, with different individuals sharing only 13%. Women had significantly higher diversity than men, and community composition was significantly affected by handedness, time since last hand washing, and an individual''s sex. The variation within and between individuals in microbial ecology illustrated by this study emphasizes the challenges inherent in defining what constitutes a “healthy” bacterial community; addressing these challenges will be critical for the International Human Microbiome Project.     

UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota

The composition of the human microbiota is recognized as an important factor in human health and disease. Many of our cohabitating microbes belong to phylum-level divisions for which there are no cultivated representatives and are only represented by small subunit rRNA sequences. For one such taxon (SR1), which includes bacteria with elevated abundance in periodontitis, we provide a single-cell genome sequence from a healthy oral sample. SR1 bacteria use a unique genetic code. In-frame TGA (opal) codons are found in most genes (85%), often at loci normally encoding conserved glycine residues. UGA appears not to function as a stop codon and is in equilibrium with the canonical GGN glycine codons, displaying strain-specific variation across the human population. SR1 encodes a divergent tRNA^Gly_UCA with an opal-decoding anticodon. SR1 glycyl-tRNA synthetase acylates tRNA^Gly_UCA with glycine in vitro with similar activity compared with normal tRNA^Gly_UCC. Coexpression of SR1 glycyl-tRNA synthetase and tRNA^Gly_UCA in Escherichia coli yields significant β-galactosidase activity in vivo from a lacZ gene containing an in-frame TGA codon. Comparative genomic analysis with Human Microbiome Project data revealed that the human body harbors a striking diversity of SR1 bacteria. This is a surprising finding because SR1 is most closely related to bacteria that live in anoxic and thermal environments. Some of these bacteria share common genetic and metabolic features with SR1, including UGA to glycine reassignment and an archaeal-type ribulose-1,5-bisphosphate carboxylase (RubisCO) involved in AMP recycling. UGA codon reassignment renders SR1 genes untranslatable by other bacteria, which impacts horizontal gene transfer within the human microbiota.     

Specific modulation of the root immune system by a community of commensal bacteria

Plants have an innate immune system to fight off potential invaders that is based on the perception of nonself or modified-self molecules. Microbe-associated molecular patterns (MAMPs) are evolutionarily conserved microbial molecules whose extracellular detection by specific cell surface receptors initiates an array of biochemical responses collectively known as MAMP-triggered immunity (MTI). Well-characterized MAMPs include chitin, peptidoglycan, and flg22, a 22-amino acid epitope found in the major building block of the bacterial flagellum, FliC. The importance of MAMP detection by the plant immune system is underscored by the large diversity of strategies used by pathogens to interfere with MTI and that failure to do so is often associated with loss of virulence. Yet, whether or how MTI functions beyond pathogenic interactions is not well understood. Here we demonstrate that a community of root commensal bacteria modulates a specific and evolutionarily conserved sector of the Arabidopsis immune system. We identify a set of robust, taxonomically diverse MTI suppressor strains that are efficient root colonizers and, notably, can enhance the colonization capacity of other tested commensal bacteria. We highlight the importance of extracellular strategies for MTI suppression by showing that the type 2, not the type 3, secretion system is required for the immunomodulatory activity of one robust MTI suppressor. Our findings reveal that root colonization by commensals is controlled by MTI, which, in turn, can be selectively modulated by specific members of a representative bacterial root microbiota.

Plants are inhabited by hundreds of species of commensals, many of which have beneficial effects on the host (1). These microbes often express the same immunogenic microbe-associated molecular patterns (MAMPs) that are found in pathogens, highlighting their potential to trigger immune responses in their hosts (2–4). How plants mount effective defenses against pathogens while allowing the colonization of commensals remains a mystery. Plant-associated microbial communities are much less diverse than those of the surrounding environment (1), indicating that the host exerts selection pressure over their microbiota and that some microbes are better adapted to colonize plant tissues than others. While multiple environmental and genetic factors likely orchestrate microbiota assembly and structure, recent research indicates that the plant immune system operates as a major gatekeeper. Arabidopsis thaliana plants (hereafter Arabidopsis) compromised in the signaling of the defense phytohormones salicylic acid and jasmonic acid harbor altered microbiota (5, 6). Similarly, mutants impaired in MAMP-triggered immunity (MTI) and in the MIN7-vesicle trafficking pathway carry altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis under conditions of high humidity (7). Recent evidence demonstrates that perception of flg22 is usually low in most root cells, but up-regulated following tissue wounding associated with infection and potentially colonization (8, 9).The ability to suppress the host immune response is a hallmark of successful pathogens (10). In animals, both specific and redundant immunomodulatory effects have been defined for taxonomically diverse commensal gut bacteria (11). Likewise, plant-associated commensals have been shown to modulate MTI (12–19). Nevertheless, studies in plants have so far focused on single microbes and the specific immunomodulatory effects of different strains have not been integrated in the context of complex communities. Furthermore, the significance of immunomodulation for community assembly remains unexplored. In this work, we investigated how a community of root-associated commensal bacteria interacts with the Arabidopsis immune system. We verified that the ability to suppress MTI is common and taxonomically widespread among these commensals. High-throughput gene-expression analyses of plants colonized by synthetic communities (SynComs) or by single microbes led to the identification of a set of defense-related genes that were commonly manipulated by phylogenetically distinct suppressor strains, highlighting a sector of the plant immune system that may control plant colonization by the microbiota. Notably, suppressors could promote the growth of other nonsuppressor strains, indicating that certain microbes may benefit from co-occurring suppressor strains. Furthermore, a genetic screening revealed that the type 2 secretion system (T2SS) is required for the robust suppressor Dyella japonica MF79 to interfere with root MTI, while the type 3 secretion system (T3SS) was dispensable. Our results expand our understanding of how the plant immune system functions in roots colonized by commensals, underscoring the role of MTI and of its suppression in microbiota assembly.     

Characteristics of intestinal bacteria with fatty liver diseases and cirrhosis

Non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD) are significant health burdens worldwide with a substantial rise in prevalence. Both can progress to liver cirrhosis. Recent studies have shown that the gut microbiome was associated with NAFLD/AFLD development and progression. The present review focuses on the characteristics of bacteria in NAFLD, AFLD and liver cirrhosis. The similarities and differences of intestinal bacteria are discussed.This study reviews the existing literatures on the microbiota, fatty liver disease, and liver cirrhosis based on Pubmed database.The study showed NAFLD was characterized by increased amounts of Lachnospiraceae from the phylum Firmicutes and Roseburia from the Lachnospiraceae family, and the proportion of Enterobacteria and Proteobacteria was increased after alcohol intake. Reduced Bacteroidetes was observed in cirrhosis. Microbiota can improve or aggravate the above liver diseases through several mechanisms, like increasing liver lipid metabolism, increasing alcohol production, increasing intestinal permeability, bacterial translocation, intestinal bacterial overgrowth, enteric dysbiosis, and impairing bile secretion.Different hepatic diseases owned different intestinal bacterial characters. Microbiota can improve or aggravate three kinds of liver diseases through several mechanisms. However, the depletion of these bacteria is needed to verify their role in liver disease.     

Adaptive immunity to murine skin commensals

The adaptive immune system provides critical defense against pathogenic bacteria. Commensal bacteria have begun to receive much attention in recent years, especially in the gut where there is growing evidence of complex interactions with the adaptive immune system. In the present study, we observed that commensal skin bacteria are recognized by major populations of T cells in skin-draining lymph nodes of mice. Recombination activating gene 1 (Rag1)^−/− mice, which lack adaptive immune cells, contained living skin-derived bacteria and bacterial sequences, especially mycobacteria, in their skin-draining lymph nodes. T cells from skin-draining lymph nodes of normal mice were shown, in vitro, to specifically recognize bacteria of several species that were grown from Rag1^−/− lymph nodes. T cells from skin-draining lymph nodes, transferred into Rag1^−/− mice proliferated in skin-draining lymph nodes, expressed a restricted T-cell receptor spectrotype and produced cytokines. Transfer of T cells into Rag1^−/− mice had the effect of reducing bacterial sequences in skin-draining lymph nodes and in skin itself. Antibacterial effects of transferred T cells were dependent on IFNγ and IL-17A. These studies suggest a previously unrecognized role for T cells in controlling skin commensal bacteria and provide a mechanism to account for cutaneous infections and mycobacterial infections in T-cell–deficient patients.Mammalian epithelium is host to a myriad of microbial species, and this requires that the epithelium provides barrier functions. The intestinal epithelial barrier is especially complex because, whereas it must be permeable to nutrients, it must also block microbial invasion via mechanical and innate immune mechanisms. Therefore, the adaptive immune system of the gut contributes to blocking microbial invasion and yet has a delicate tolerance mechanism that attenuates reactions to enteric antigens.The skin epithelial barrier, in contrast to the intestinal barrier, would seem to require simpler immunological mechanisms for blocking entry of commensals and pathogens because the skin is relatively impermeable. The innate immune system has been ascribed a role in skin barrier function through release of antimicrobial peptides (1); defects in the innate immune system are implicated in atopic dermatitis (2). The adaptive immune system, in contrast, has received less attention regarding its contribution to barrier function of the skin. Although tolerance would seem less necessary than for the more permeable gut, there is some evidence for tolerance (3); excess T-cell reactions are implicated in several common skin diseases, including atopic dermatitis and psoriasis.In humans deficient in T and B cells, about a third show infectious cutaneous manifestations (4). T and B cell deficiency (TB⁻) that is specifically due to recombination activating gene (Rag) mutations similarly show a high frequency of cutaneous manifestations (5). These clinical findings implicate adaptive immunity in skin host defense in man. Susceptibility to disseminated mycobacterial infections is often seen in TB⁻ severe combined immunodeficiency patients (SCID) with disseminated bacillus Calmette–Guérin (4). A Rag2-deficient SCID was reported with disseminated bacillus Calmette–Guérin (6) and a Rag1 hypomorphic patient was reported with disseminated nontuberculous mycobacteria (7). These reports support a role for adaptive immunity in defense against mycobacteria in man.T cells within normal human dermis are predominantly of an effector memory (T_EM) phenotype with a diverse repertoire (8) and have been suggested to be involved in immune surveillance (9). T-cell homing to skin is directed by E selectin and chemokines chemokine (C-C motif) ligand (CCL)20, -22, and -27 expressed by keratinocytes acting on cutaneous lymphocyte-associated antigen (CLA), chemokine receptor (CCR)6, -4, and -10, respectively, expressed by the subset of T cells that home to skin. There is implied interaction between the skin flora and the adaptive immune system, suggested by the defect in Th17 development in mice lacking skin flora (10). The adaptive immune system is implicated in skin barrier function by recent studies suggesting Th17 cells play a significant role in defense against cutaneous pathogens (11). Autoantibodies against Th17 cytokines, including IL-17A and F, IL-22 (12), and mutations in the receptor for IL-17 (13) have been implicated in skin infections. It remains to be determined whether skin “commensals,” i.e., bacteria that are normally not pathogenic, elicit T-cell immunity in a normal host and whether these T cells could contribute to skin barrier function.The present study derives from experiments showing that transfer of OT-1 cells, monoclonal CD8 T cells, into Rag1^−/− mice required host IL-7 for survival and proliferation of the transferred cells (14), a phenomenon termed “homeostatic” proliferation. However, our group (15) and others (16) subsequently observed that, unexpectedly, normal polyclonal CD8 T cells transferred into Rag1^−/− survived and proliferated independently of IL-7; moreover, their proliferation was much faster than homeostatic proliferation observed with OT-1 cells. This phenomenon, the rapid, IL-7–independent proliferation of normal T cells transferred into Rag1^−/− mice, resembled the reaction of memory cells to antigens. Here we investigate this reaction to determine the nature of the postulated stimulatory antigens and to characterize the responding T cells and their physiological role. We will show that much of this T-cell reaction is triggered by skin commensal bacterial antigens and demonstrate a protective role of these T cells in barrier function that requires IFNγ and IL-17.     

Antibiotics,gut microbiome and obesity

Antibiotics have been hailed by many as “miracle drugs” that have been effectively treating infectious diseases for over a century, leading to a marked reduction in morbidity and mortality. However, with the increasing use of antibiotics, we are now faced not only with the increasing threat of antibiotic resistance, but also with a rising concern about potential long‐term effects of antibiotics on human health, including the development of obesity. The obesity pandemic continues to increase, a problem that affects both adults and children alike. Disruptions to the gut microbiome have been linked to a multitude of adverse conditions, including obesity, type 2 diabetes, inflammatory bowel diseases, anxiety, autism, allergies, and autoimmune diseases. This review focuses on the association between antibiotics and obesity, and the role of the gut microbiome. There is strong evidence supporting the role of antibiotics in the development of obesity in well‐controlled animal models. However, evidence for this link in humans is still inconclusive, and we need further well‐designed clinical trials to clarify this association.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Neural correlates of behavior in the moth Manduca sexta in response to complex odors

With Manduca sexta as a model system, we analyzed how natural odor mixtures that are most effective in eliciting flight and foraging behaviors are encoded in the primary olfactory center in the brain, the antennal lobe. We used gas chromatography coupled with multiunit neural-ensemble recording to identify key odorants from flowers of two important nectar resources, the desert plants Datura wrightii and Agave palmeri, that elicited responses from individual antennal-lobe neurons. Neural-ensemble responses to the A. palmeri floral scent, comprising >60 odorants, could be reproduced by stimulation with a mixture of six of its constituents that had behavioral effectiveness equivalent to that of the complete scent. Likewise, a mixture of three floral volatiles from D. wrightii elicited normal flight and feeding behaviors. By recording responses of neural ensembles to mixtures of varying behavioral effectiveness, we analyzed the coding of behaviorally “meaningful” odors. We considered four possible ensemble-coding mechanisms—mean firing rate, mean instantaneous firing rate, pattern of synchronous ensemble firing, and total net synchrony of firing—and found that mean firing rate and the pattern of ensemble synchrony were best correlated with behavior (R = 41% and 43%, respectively). Stepwise regression analysis showed that net synchrony and mean instantaneous firing rate contributed little to the variation in the behavioral results. We conclude that a combination of mean-rate coding and synchrony of firing of antennal-lobe neurons underlies generalization among related, behaviorally effective floral mixtures while maintaining sufficient contrast for discrimination of distinct scents.