From the Cover: Osmotrophy in modular Ediacara organisms

The Ediacara biota include macroscopic, morphologically complex soft-bodied organisms that appear globally in the late Ediacaran Period (575–542 Ma). The physiology, feeding strategies, and functional morphology of the modular Ediacara organisms (rangeomorphs and erniettomorphs) remain debated but are critical for understanding their ecology and phylogeny. Their modular construction triggered numerous hypotheses concerning their likely feeding strategies, ranging from micro-to-macrophagus feeding to photoautotrophy to osmotrophy. Macrophagus feeding in rangeomorphs and erniettomorphs is inconsistent with their lack of oral openings, and photoautotrophy in rangeomorphs is contradicted by their habitats below the photic zone. Here, we combine theoretical models and empirical data to evaluate the feasibility of osmotrophy, which requires high surface area to volume (SA/V) ratios, as a primary feeding strategy of rangeomorphs and erniettomorphs. Although exclusively osmotrophic feeding in modern ecosystems is restricted to microscopic bacteria, this study suggests that (i) fractal branching of rangeomorph modules resulted in SA/V ratios comparable to those observed in modern osmotrophic bacteria, and (ii) rangeomorphs, and particularly erniettomorphs, could have achieved osmotrophic SA/V ratios similar to bacteria, provided their bodies included metabolically inert material. Thus, specific morphological adaptations observed in rangeomorphs and erniettomorphs may have represented strategies for overcoming physiological constraints that typically make osmotrophy prohibitive for macroscopic life forms. These results support the viability of osmotrophic feeding in rangeomorphs and erniettomorphs, help explain their taphonomic peculiarities, and point to the possible importance of earliest macroorganisms for cycling dissolved organic carbon that may have been present in abundance during Ediacaran times.     

From the Cover: A DNA barcode for land plants

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.     

From the Cover: PNAS Plus: Pan-neuronal imaging in roaming Caenorhabditis elegans

We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal’s posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.Understanding how brain dynamics creates behaviors requires quantifying the flow and transformation of sensory information to motor output in behaving animals. Optical imaging using genetically encoded calcium or voltage fluorescent probes offers a minimally invasive method to record neural activity in intact animals. The nematode Caenorhabditis elegans is particularly ideal for optical neurophysiology owing to its small size, optical transparency, compact nervous system, and ease of genetic manipulation. Imaging systems for tracking the activity of small numbers of neurons have been effective in determining their role during nematode locomotion and navigational behaviors like chemotaxis, thermotaxis, and the escape response (1–6). Recordings from large numbers of interconnected neurons are required to understand how neuronal ensembles carry out the systematic transformations of sensory input into motor patterns that build behavioral decisions.Several methods for fast 3D imaging of neural activity in a fixed imaging volume have been developed for different model organisms (7–14). High-speed light sheet microscopy, light field microscopy, multifocus microscopy, and two-photon structured illumination microscopy have proved effective for rapidly recording large numbers of neurons in immobilized, intact, transparent animals like larval zebrafish and nematodes (15–19). However, these methods are problematic when attempting to track many neurons within the bending and moving body of a behaving animal. Panneuronal recording in moving animals poses higher demands on spatial and temporal resolution. Furthermore, extracting neuronal signals from recordings in a behaving animal requires an effective analysis pipeline to segment image volumes into the activity patterns of discrete and identifiable neurons.Here, we use high-speed spinning disk confocal microscopy—modified for automated tracking using real-time image analysis and motion control software—to volumetrically image the head ganglia of behaving C. elegans adults at single-cell resolution. Our setup can simultaneously track ∼80 neurons with 0.45 × 0.45 × 2-μm resolution at 10 Hz. Activity was reported by the ultrasensitive calcium indicator GCaMP6s expressed throughout the cytosol under the control of the pan-neuronal rgef-1 promoter (a gift from D. Pilgrim, University of Alberta, Edmonton, Alberta, Canada) (20). To facilitate segmentation into individual identifiable neurons, nuclei were tracked using calcium-insensitive, nuclear-bound red fluorescent protein (RFP), TagRFP, under the control of another pan-neuronal rab-3 promoter (a gift from O. Hobert, Columbia University, New York) (21). We developed an image analysis pipeline that converts the gross movements of the head into the time-varying position and posture of the crawling worm, and converts fluorescence measurements into near simultaneous activity patterns of all imaged neurons.A similar approach to brainwide imaging in moving C. elegans using the same transgenic strain has recently been reported (22). Although both setups use customized spinning disk confocal microscopes, the strategies for tracking the moving neurons and analyzing behavioral and neural activity patterns are different. Nguyen et al. (22) use a low power objective to track the posture of the animal and a high power objective to locate and image the nerve ring. The advantage of our single objective setup is that it affords the flexibility, for example, to deliver thermosensory inputs using an opaque temperature controlled stage below the animal. The advantage of low-magnification imaging is that it provides a direct measurement of animal posture, which we must infer. These new technologies for pan-neuronal imaging in roaming animals now enables correlating brainwide dynamics to sensory inputs and motor outputs in transparent behaving animals like C. elegans and Drosophila larvae.     

From the Cover: A Trojan horse mechanism of bacterial pathogenesis against nematodes

Understanding the mechanisms of host–pathogen interaction can provide crucial information for successfully manipulating their relationships. Because of its genetic background and practical advantages over vertebrate model systems, the nematode Caenorhabditis elegans model has become an attractive host for studying microbial pathogenesis. Here we report a “Trojan horse” mechanism of bacterial pathogenesis against nematodes. We show that the bacterium Bacillus nematocida B16 lures nematodes by emitting potent volatile organic compounds that are much more attractive to worms than those from ordinary dietary bacteria. Seventeen B. nematocida-attractant volatile organic compounds are identified, and seven are individually confirmed to lure nematodes. Once the bacteria enter the intestine of nematodes, they secrete two proteases with broad substrate ranges but preferentially target essential intestinal proteins, leading to nematode death. This Trojan horse pattern of bacterium–nematode interaction enriches our understanding of microbial pathogenesis.     

From the Cover: Experimental replacement of an obligate insect symbiont

Symbiosis, the close association of unrelated organisms, has been pivotal in biological diversification. In the obligate symbioses found in many insect hosts, organisms that were once independent are permanently and intimately associated, resulting in expanded ecological capabilities. The primary model for this kind of symbiosis is the association between the bacterium Buchnera and the pea aphid (Acyrthosiphon pisum). A longstanding obstacle to efforts to illuminate genetic changes underlying obligate symbioses has been the inability to experimentally disrupt and reconstitute symbiont–host partnerships. Our experiments show that Buchnera can be experimentally transferred between aphid matrilines and, furthermore, that Buchnera replacement has a massive effect on host fitness. Using a recipient pea aphid matriline containing Buchnera that are heat sensitive because of an allele eliminating the heat shock response of a small chaperone, we reduced native Buchnera through heat exposure and introduced a genetically distinct Buchnera from another matriline, achieving complete replacement and stable inheritance. This transfer disrupted 100 million years (∼1 billion generations) of continuous maternal transmission of Buchnera in its host aphids. Furthermore, aphids with the Buchnera replacement enjoyed a dramatic increase in heat tolerance, directly demonstrating a strong effect of symbiont genotype on host ecology.Symbiosis has been key in the ecological and evolutionary diversification of eukaryotes (1, 2). In many invertebrates, bacterial symbionts have been maternally transmitted for millions of years and are required for the growth and reproduction of hosts (3). These symbionts approach organelles in their degree of genetic and physiological integration with hosts and in their extreme genomic reduction. A model for obligate symbiosis is that of the pea aphid (Acyrthosiphon pisum) and its nutrient-provisioning bacterial symbiont, Buchnera aphidicola. Buchnera features a tiny genome (4), restriction to a small number of specialized host cells (bacteriocytes), host-controlled transmission (5), and regulated exchange of molecules with hosts (6, 7). This tight integration creates challenges for studies that aim to elucidate how symbiont variation affects host fitness and ecology.The ability to transfer obligate symbionts between host matrilines could provide a tool for teasing apart the separate contributions of symbiotic partners. Facultative symbionts, such as those conferring defense against pathogens, have been transferred experimentally into novel host matrilines, where they are typically stably inherited, enabling direct measures of symbionts on hosts (8). The success of these transfers is presumably related to the fact that facultative symbionts possess their own machinery for invading host cells (9, 10) and typically persist in several locations in the insect body. In contrast, obligate symbionts such as Buchnera are packaged into specialized host cells during early development and do not survive in the hemocoel or in other cell types.Buchnera colonizes developing aphids before birth, through a specialized transmission process that has been studied in detail in A. pisum (5, 11). Aphids are parthenogenetic for much of their life cycle, during which embryos develop within maternal ovarioles. Bacteriocytes and ovarioles containing developing embryos are located near one another within the mother’s abdomen, and transmission occurs when Buchnera cells are exocytosed from a maternal bacteriocyte in the vicinity of a blastula-stage embryo (5). The Buchnera cells become extracellular within the hemocoel, and some are endocytosed by the posterior syncytial cytoplasm of the embryo in which they are later packaged into the embryonic bacteriocytes. Buchnera cells released into the hemocoel quickly deteriorate if they do not enter an embryo. The molecular underpinnings of the transfer process are unknown. This specialized transmission process presents a challenge for the experimental transfer of symbionts between hosts.We developed a strategy aimed at replacing resident Buchnera with genetically distinct Buchnera from a different host matriline (Fig. 1), using parthenogenetic (all-female) lines of aphids. We used heat tolerance as a selectable phenotype. A previous study showed that a single nucleotide deletion in the promoter of a small heat shock protein [inclusion body-associated protein A (IbpA)] of Buchnera results in a reduction in Buchnera numbers of >100-fold after exposure to 4 h of 35 °C heat (12). Buchnera lacking this mutation undergo only modest declines after heat exposure. Our strategy was to reduce the native Buchnera in recipient aphids using heat, and then to inject homogenate containing heat-tolerant Buchnera from another A. pisum matriline. Because transmission to embryos includes an extracellular stage in which Buchnera is free in the hemocoel, the injected Buchnera might colonize embryos in lieu of maternal Buchnera, which have been eliminated or depleted. After successful replacement of native Buchnera, we demonstrated a large effect on aphid ability to withstand heat exposure.Open in a separate windowFig. 1.Experimental approach for replacement of the native Buchnera symbionts within an A. pisum matriline. The recipient line (LSR1) contains a heat-sensitive Buchnera genotype, and the donor (5AY) contains a heat-tolerant Buchnera genotype. Native Buchnera are depleted by heat in the recipient line, and microinjection is used to flood the hemocoel with donor Buchnera. Most embryos are successfully colonized by the donor symbionts. In some cases, complete replacement occurs in the progeny of injected females. In other cases, progeny have a mixed Buchnera population, which can be shifted completely to the donor type through further heat exposure.     

From the Cover: Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes

A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with “fever of unknown origin” in malaria-endemic areas.In 2002, Rickettsia felis, an obligate intracellular bacterium that belongs to the spotted fever group of Rickettsia, was definitively described (1, 2). Over the past 2 decades, an increasing number of reports have implicated R. felis as a human pathogen, paralleling an increase in reports of the detection of R. felis in arthropod hosts throughout the world (1, 3).By 2011, more than 70 human cases of R. felis had been reported worldwide, including in Central and South America, Asia, northern Africa, and Europe (1). More cases have been published since then, including the first probable human cases in Australia (4). In sub-Saharan Africa, recent studies have challenged the importance of R. felis infection in patients with “fever of unknown origin,” with this bacterium detected in up to 15% of such patients (5–7). In 2011, a potential R. felis primary infection, called “yaaf,” was suspected in the case of an 8-mo-old girl in Senegal with polymorphous skin lesions similar to those seen in patients from Mexico (8). The epidemiologic and clinical picture of this emerging infection in Africa, including its potential vectors, is poorly understood, however.Various arthropods, but primarily fleas, have been associated with R. felis (1, 3). More specifically, the cat flea Ctenocephalides felis is the arthropod in which R. felis has been most frequently detected. To date, it is the sole confirmed biological vector of R. felis, with both horizontal and vertical transmission making this flea a potential reservoir for this bacterium (9–11). However, in some countries where R. felis appears to be highly prevalent, such as Senegal, neither cat fleas nor other arthropods have been implicated in its transmission (12).Mosquitoes are the most important vectors of infectious diseases in humans, with more than one-half of the global population at risk for exposure to mosquito-borne infections (13, 14). Anopheles gambiae is known to be the primary vector of malaria in Africa, whereas Aedes albopictus is a vector of dengue and chikungunya (15, 16). Interestingly, Ae. albopictus and An. gambiae mosquito cells support R. felis growth (1, 17). In 2012, Ae. albopictus from Gabon and An. gambiae molecular form S (the primary African malarial vector) from Ivory Coast tested positive for R. felis by species-specific real-time quantitative PCR (qPCR) (17, 18). More recently, several mosquito species from Senegal were found to harbor R. felis, including Ae. luteocephalus, An. arabiensis, An. ziemanni, An. pharoensis, An. funestus, and Mansonia uniformis (5). These data raise new issues with respect to the epidemiology of R. felis in Africa, including the degree of vector competence of mosquitoes. The objective of this work was to study the acquisition and transmission of R. felis by An. gambiae mosquitoes in an experimental model of infection.     

From the Cover: Anthropogenic changes in sodium affect neural and muscle development in butterflies

The development of organisms is changing drastically because of anthropogenic changes in once-limited nutrients. Although the importance of changing macronutrients, such as nitrogen and phosphorus, is well-established, it is less clear how anthropogenic changes in micronutrients will affect organismal development, potentially changing dynamics of selection. We use butterflies as a study system to test whether changes in sodium availability due to road salt runoff have significant effects on the development of sodium-limited traits, such as neural and muscle tissue. We first document how road salt runoff can elevate sodium concentrations in the tissue of some plant groups by 1.5–30 times. Using monarch butterflies reared on roadside- and prairie-collected milkweed, we then show that road salt runoff can result in increased muscle mass (in males) and neural investment (in females). Finally, we use an artificial diet manipulation in cabbage white butterflies to show that variation in sodium chloride per se positively affects male flight muscle and female brain size. Variation in sodium not only has different effects depending on sex, but also can have opposing effects on the same tissue: across both species, males increase investment in flight muscle with increasing sodium, whereas females show the opposite pattern. Taken together, our results show that anthropogenic changes in sodium availability can affect the development of traits in roadside-feeding herbivores. This research suggests that changing micronutrient availability could alter selection on foraging behavior for some roadside-developing invertebrates.The development of fitness-related traits is closely tied to nutrition—from fecundity being influenced by protein availability (1, 2) to ornament coloration being linked to carotenoid abundance (3, 4). However, humans are having a major impact on the availability of many nutrients important in the development of these traits. For instance, nitrogen and phosphorus availability has increased dramatically because of fertilizer application (5, 6), with drastic consequences for biomass and nutrient content of producers and consumers (7–9). Although the effects of changing macronutrients have been well-studied, the importance of human-induced changes in micronutrients is less established. Are anthropogenic changes in once-limited micronutrients enough to drive differences in trait development, potentially altering selection dynamics?This research focuses on changing availability of an important micronutrient: sodium. Sodium is a key component of animal development, important for the function of neural and muscle tissue (10–12) and affecting the development of traits, such as brain size (13–16). However, sodium availability is limited in most ecosystems (17–19), which is thought to have led to the evolution of sodium cravings (20, 21) and specific foraging behavior to acquire sodium (22–25). Humans are increasing sodium availability, particularly through the application of road salt (26–29) but also, through agricultural activity (30). In the metropolitan area of Minneapolis and St. Paul, Minnesota, ∼300,000 tons of sodium chloride are applied to roads each winter (31). Research on the ecological impact of road salt has mostly focused on the negative effects of chloride entering waterways (32–34). However, road salt application can also increase the availability of dietary sodium for animals. A handful of studies suggest that road salt application may affect sodium foraging in animals from ants to moose (35, 36). We know little about whether local increases in sodium along roadsides have significant effects on development of fitness-related traits for species feeding along roadsides, thus altering evolutionary dynamics in the anthropocene.Butterflies are an excellent study system to test the consequences of changing sodium availability. Sodium availability has been shown to affect the development and activity of flight muscle in male Lepidoptera (37–39). Many adult male Lepidoptera actively forage for sodium through puddling, transferring much of this sodium to females during mating (23, 40–43). In addition, host plants of many butterfly species commonly grow along roadsides and would be affected by roadside runoff. Butterfly larvae also have limited movement (44, 45), such that the spatially restricted effects of salt runoff are biologically relevant. This work starts by documenting the effects of roadside salt runoff on sodium availability in common butterfly host plants. Two rearing experiments—one using roadside-collected and control host plants and the other using a controlled artificial diet manipulation—show the importance of changing sodium availability on trait development. In particular, we focus on two fitness-related traits—muscle and neural tissue—where sodium availability has a shown importance in trait development and function.     

Functional evolution of Erg potassium channel gating reveals an ancient origin for IKr

Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K⁺ channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for I_Kr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K⁺ channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the I_Kr-like gating phenotype with mammalian Erg channels. Thus, the I_Kr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral I_Kr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.Voltage-gated ion channel families are highly conserved across the Eumetazoa (cnidarians and bilaterians) (1, 2). Vertebrates recently expanded the number of ion channel genes within each of the conserved families because of vertebrate-specific gene duplications. Additionally, phylogenetically restricted duplications of ion channel genes appear common throughout the Eumetazoa (1, 3–5). Thus, there is little 1:1 gene orthology between the eumetazoan phyla (1). However, numerous studies show extremely high functional conservation, including family-specific gating properties. For example, Shaker-related voltage-gated K⁺ channels first cloned in Drosophila show a high fidelity of gating phenotype to their mammalian counterparts (6). Subsequent studies have shown this functional conservation extends to cnidarians (4, 7–10), which separated from bilaterians near the base of the eumetazoan tree over 500 Mya (11). One exception to this pattern of high conservation is the Ether-a-go-go related gene (Erg) family (or Kv11) of voltage-gated K⁺ channels. The three mammalian Erg orthologs show striking gating differences compared with Drosophila Erg (seizure, DmErg).The mammalian Erg gating phenotype is typified by human Erg1 (HsErg1), which underlies I_Kr, a K⁺ current that repolarizes the late plateau phase of ventricular action potentials (12, 13). HsErg1 loss-of-function mutations prolong the QT interval in ECG recordings, indicating impaired action potential repolarization (14). Several key gating features adapt Erg1 for ventricular action potential plateau repolarization. First, Erg1 channels inactivate rapidly in response to depolarization (Fig. 1 A–C). Second, recovery from inactivation through the open state is extremely rapid (Fig. 1B), whereas channel deactivation is slow (Fig. 1D); the combination produces a jump in Erg1 current in response to repolarization (15). The net effect is that peak Erg1 current flow is delayed and specifically accelerates cardiac action potential plateau repolarization (15), and the length of the plateau is dependent on Erg1 current density (16). The physiological role of mammalian Erg2 and Erg3 channels has not been extensively characterized, but they share an I_Kr-like gating phenotype (17).Open in a separate windowFig. 1.Comparison of HsErg1 and DmErg gating phenotypes. (A) Families of outward currents recorded from Xenopus oocytes expressing HsErg1 (Left) and DmErg + DAO (Right) in response to depolarizations (Inset). Scale bars indicate time and current amplitude. Currents elicited by a step to +60 mV are highlighted, and arrows indicate (1) rectification of HsErg1 during depolarization by inactivation, (2) rebound in HsErg1 current in response to repolarization due to rapid recovery and slow deactivation, and (3) rapid DmErg deactivation. (B) Comparison of HsErg1 (black) and DmErg (red) currents during a protocol in which channels were first activated by a 1 s step to +60 mV, returned to –100 mV for 10 ms, and then returned to +60 mV. Currents are normalized in peak amplitude for comparison. HsErg1 is inactivated at the end of the first depolarization, recovers to the open state at −100 mV, and inactivates rapidly from a high peak during the second pulse. DmErg1 remains active throughout the first +60 mV pulse, closes at –100 mV, and reactivates during the second +60 mV pulse. (C) Peak HsErg1 current during an initial depolarization (* in B) normalized to peak current after recovery from inactivation (# in B): inactivation reduces the HsErg1 current >20-fold during the first step. Data show mean ± SEM, n = 6 cells. (D) Time constant of deactivation (Tau_DEACT) measured from tail currents recorded at the indicated voltages for HsErg1 (black) and DmErg (red). Data show mean ± SEM, n = 6–7 cells. (E) Normalized GV curves for HsErg1 and DmErg fit with a single Boltzmann distribution (parameters in SI Methods. Scale bar indicates that time and current amplitudes have been normalized.In contrast, DmErg does not inactivate during depolarization (Fig. 1 A and B) and deactivates rapidly upon repolarization (Fig. 1D) (18). The voltage-activation curve (GV) of DmErg is shifted to hyperpolarized potentials, suggesting influence on subthreshold excitability (Fig. 1E). Modeled HsErg1 and DmErg responses to a crude plateau action potential waveform (Fig. 1F and Fig. S1) point to distinct physiological roles. HsErg1 current is attenuated during the plateau by inactivation and rebounds sharply as the plateau decays. These features allow HsErg1 to accelerate late repolarization without blocking the plateau itself (15). Peak DmErg current flows during the plateau, and the current decays rapidly during repolarization. DmErg would therefore directly combat plateau formation. Loss of HsErg1 inactivation in humans indeed leads to a shortened QT interval based on premature action potential repolarization (16). The specific contribution of DmErg to firing patterns in native cells is unknown, but its gating features are consistent with regulation of subthreshold excitability or rapid action potential repolarization. Temperature-sensitive mutations in the seizure locus that encodes DmErg cause bursts of uncoordinated motor output (19) suggestive of changes in subthreshold excitability. The Caenorhabditis elegans Erg ortholog (CeErg, encoded by unc-103) has not been functionally expressed, but genetic analysis demonstrates that it regulates the excitation threshold of vulva muscles in females and protractor muscles in males (20–23).The Erg, Ether-a-go-go (Eag), and Elk gene families comprise the EAG superfamily of voltage-gated K⁺ channels. These gene families are highly conserved in eumetazoan genomes, and Eag channels display a high functional conservation in the bilaterians. Given the distinct gating phenotypes of the Erg genes in Drosophila and mammals, we decided to explore the functional evolution of the Erg gene family to determine the origins of the distinct I_Kr-like and DmErg gating phenotypes in the Erg gene family. We functionally characterized CeErg and Erg paralogs from the starlet sea anemone Nematostella vectensis. We examined CeErg to determine whether the DmErg gating phenotype was present in multiple protostome invertebrate phyla. We reasoned that comparison of bilaterian and Nematostella Erg channels would provide insight into ancestral Erg gating phenotypes present before the cnidarian/bilaterian divergence. Functional and phylogenetic analysis presented here supports an I_Kr-like phenotype as the ancestral gating pattern. An alternate DmErg-like gating phenotype has emerged independently at least twice during metazoan evolution (once in cnidarians and at least once in protostomes) and correlates with loss of the cytoplasmic eag gating domain.     

From the Cover: Evolution of herbivory in Drosophilidae linked to loss of behaviors,antennal responses,odorant receptors,and ancestral diet

Herbivory is a key innovation in insects, yet has only evolved in one-third of living orders. The evolution of herbivory likely involves major behavioral changes mediated by remodeling of canonical chemosensory modules. Herbivorous flies in the genus Scaptomyza (Drosophilidae) are compelling species in which to study the genomic architecture linked to the transition to herbivory because they recently evolved from microbe-feeding ancestors and are closely related to Drosophila melanogaster. We found that Scaptomyza flava, a leaf-mining specialist on plants in the family (Brassicaceae), was not attracted to yeast volatiles in a four-field olfactometer assay, whereas D. melanogaster was strongly attracted to these volatiles. Yeast-associated volatiles, especially short-chain aliphatic esters, elicited strong antennal responses in D. melanogaster, but weak antennal responses in electroantennographic recordings from S. flava. We sequenced the genome of S. flava and characterized this species’ odorant receptor repertoire. Orthologs of odorant receptors, which detect yeast volatiles in D. melanogaster and mediate critical host-choice behavior, were deleted or pseudogenized in the genome of S. flava. These genes were lost step-wise during the evolution of Scaptomyza. Additionally, Scaptomyza has experienced gene duplication and likely positive selection in paralogs of Or67b in D. melanogaster. Olfactory sensory neurons expressing Or67b are sensitive to green-leaf volatiles. Major trophic shifts in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires.Understanding the origins and consequences of trophic shifts, especially the transition to herbivory, has been a central problem in evolutionary biology. The paleontological record suggests that evolutionary transitions to herbivory have been rare in insects (1), and the first transitions to herbivory in vertebrates occurred long after the colonization of land (2). However, species radiations result from herbivorous transitions in insects and vertebrates, suggesting that herbivory is a key innovation (3, 4). Identifying functional genomic changes associated with the evolutionary transition to herbivory could yield insight into the mechanisms that have driven their success. However, the origins of the most diverse clades of herbivorous insects are ancient and date to the Jurassic or earlier (5), limiting meaningful genomic comparisons. In contrast, herbivory has evolved more times in Diptera than in any other order (3). The Drosophilidae is an excellent system to study the evolution of herbivory from a functional genomic perspective because it includes several transitions to herbivory, and the genomic model Drosophila melanogaster (6, 7).The transition to herbivory involves adaptations in physiology (8–10), morphology (11), and behavior (12). The evolution of sensory repertoires could reinforce or even precipitate these adaptations through adaptive loss or relaxation of functional constraint subsequent to a trophic shift (13). Adaptive loss of chemoreceptors has been rarely shown but occurs in nematodes, although their olfactory systems are distinct from insects (14). Families of mammalian olfactory receptor proteins have been remodeled during transitions to flight, aquatic lifestyles, and frugivory (15–18). Similarly, the evolution of diet specialization in Drosophila species correlates with chemoreceptor gene losses (19–21), and hematophagous flies have lost gustatory receptors that detect sweet compounds (22). More profound changes such as the evolution of new protein families are associated with major evolutionary transitions such as the evolution of flight in insects (23). Although gene loss is unlikely to be a driving force of innovation, loss-of-function mutations may be exaptations that allow novel behaviors to evolve by disrupting ancestral attractions. If detection of different chemical cues becomes selected in a novel niche, then loss through relaxed constraint may indicate which chemical cues have changed during a trophic shift.The chemosensory repertoires of many drosophilid species have been functionally annotated. The genus Drosophila includes 23 species with published genome sequences (24–27), and D. melanogaster presents the most fully characterized insect olfactory system (28), allowing potential linkage of receptor remodeling to a mechanistic understanding of behavioral change.Most drosophilids feed on yeast and other microbes growing on decaying plant tissues (29). Adult female D. melanogaster and distantly related species innately prefer yeast chemical cues to those produced by the fruit on which they oviposit (30, 31). D. melanogaster detects volatiles with chemoreceptors of several different protein families, but especially receptors from the odorant receptor (OR) gene family, some of which, such as Or42b, are highly conserved across species (31). Or42b is necessary for attraction and orientation to vinegar and aliphatic esters (32–34). Similar compounds activate Or42b across many Drosophila species (35), suggesting that volatile cues for yeast, and the associated receptors, are conserved across the Drosophilidae.The ancestral feeding niche for the genus Scaptomyza (Drosophilidae) is microbe-feeding, but Scaptomyza use decaying leaves and stems rather than the fermenting fruit used by D. melanogaster and other members of the subgenus Sophophora (29, 36). The close association of Scaptomyza with decaying plant tissues may have precipitated the evolution of herbivory <20 MyBP (Fig. 1; ref. 36). Adult females of the species S. flava feed and oviposit on living leaves of many cruciferous plants (Brassicales) including Arabidopsis thaliana. Females puncture leaves with serrated ovipositors to create feeding and oviposition sites, and larvae mine and pupate within the living leaves (7).Open in a separate windowFig. 1.Time-calibrated Bayesian phylogeny of Drosophila and Scaptomyza species with herbivorous taxa in dark green. Scaptomyza flies are nested phylogenetically within the Drosophila genus. (Inset) Adult female S. flava fly with green-pigmented abdomen after feeding on Arabidopsis thaliana. Arrowhead indicates serrated ovipositor used to create feeding and oviposition holes within leaf tissue. Node labels indicate posterior clade probability (PP). Unlabeled nodes have PP = 1. Error bars are 95% highest posterior density interval. Pie graphs indicate probability of change to herbivory (green) or microbe-feeding (white) traits reconstructed at each node. Herbivorous taxa are in brackets indicated by leaf.Here, we use Scaptomyza as a model to test the hypothesis that functional loss of chemosensory genes has played a role in a major ecological transition to herbivory in insects. We hypothesized that the conserved detection of yeast volatiles would be lost in the herbivorous Scaptomyza lineage. We tested this loss by comparing D. melanogaster and S. flava at behavioral, physiological, and genetic levels. First, we hypothesized that gravid ovipositing S. flava females would not be attracted to yeast volatiles. Second, we hypothesized that the olfactory sensory organs of S. flava would have a decreased ability to detect individual yeast volatiles and volatile mixtures. Third, chemoreceptor genes from the OR gene family implicated in detection of yeast volatiles would be lost in the S. flava genome. Finally, we predict chemoreceptor genes potentially mediating detection of plant volatiles would show evidence of positive selection and possibly, neofunctionalization.     

From the Cover: FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex

In bacteria, the FtsK/Xer/dif (chromosome dimer resolution site) system is essential for faithful vertical genetic transmission, ensuring the resolution of chromosome dimers during their segregation to daughter cells. This system is also targeted by mobile genetic elements that integrate into chromosomal dif sites. A central question is thus how Xer/dif recombination is tuned to both act in chromosome segregation and stably maintain mobile elements. To explore this question, we focused on pathogenic Neisseria species harboring a genomic island in their dif sites. We show that the FtsK DNA translocase acts differentially at the recombination sites flanking the genomic island. It stops at one Xer/dif complex, activating recombination, but it does not stop on the other site, thus dismantling it. FtsK translocation thus permits cis discrimination between an endogenous and an imported Xer/dif recombination complex.In all organisms, the processing of chromosome ends or termini relies on specific activities for replication and segregation. In eukaryotes, telomeres are often targeted by mobile genetic elements, which may even substitute for telomeric functions (1). Circular chromosomes found in prokaryotes have no telomeres but harbor chromosome dimer resolution sites, called dif sites, on which dedicated Xer recombinases (XerC and XerD in most cases) act (2, 3). Besides their role in chromosome maintenance, dif sites are targeted by numerous mobile genetic elements, referred to as integrating mobile element exploiting Xer (IMEX) (4). How IMEXs integrate into dif without inactivating its cellular function and how they are stably maintained in their integrated state has remained unclear despite study over the past decade (4–7). Here we answer these questions by studying the gonococcal genomic island (GGI), an IMEX stably integrated into the dif site of pathogenic Neisseria species that encodes crucial functions for gene exchange and virulence (8, 9).In Escherichia coli, chromosome dimers form by homologous recombination during replication and are resolved by site-specific recombination between sister dif sites catalyzed by the XerC and XerD recombinases (Fig. 1) (3). The 28-bp dif site carries binding sites for each recombinase, separated by a 6-bp central region at the border of which strand exchanges are catalyzed. After assembly of the recombination complex (synapse), one pair of strands is exchanged by the XerD monomers, leading to a branched DNA intermediate (Holliday junction, HJ) subsequently resolved by XerC. Dimer resolution is integrated into the general processing of the terminal region of the chromosome (ter region) during cell division (10). FtsK, a DNA translocase associated with the division apparatus, segregates this region at the onset of cell division (10, 11). The translocation motor, FtsKαβ, is located in the C terminal of FtsK (12). Translocation is oriented toward the dif site located at the center of the ter region via a direct interaction between the extreme C-terminal subdomain of FtsK, FtsKγ, and the KOPS DNA motifs (13). Upon reaching the XerCD/dif complex, FtsK stops translocating and activates recombination via direct interaction with XerD (14, 15) (Fig. 1). The mechanisms of translocation arrest and of recombination activation are poorly understood but they both involve FtsKγ. However, these activities appear to be distinct from each other because FtsKγ can activate recombination in vivo and in vitro when isolated from the FtsKαβ motor or fused to XerC or XerD (16).Open in a separate windowFig. 1.The XerCD/dif recombination. (A) Chromosome dimer formation by homologous recombination (HR) during replication and resolution by site-specific recombination between the two dif sites. The dif site is represented as green and purple boxes for the XerC-binding and the XerD-binding sites, respectively. ori (black circle), some KOPS motifs (arrows), and the ter domain (thick line) are represented. The mechanism of XerCD/dif recombination is represented in the box. XerC (green circles) and XerD (purple circles) bind two distant dif sites to create a synapse. Hexamers of the FtsK C-terminal domain [FtsKC: FtsKαβ: (diamonds) + FtsKγ: (triangle) contacting XerD] translocate toward dif and contact XerD. This activates XerD (Y indicates the active recombinases), which catalyzes the first-strand exchange. This process leads to the formation of an HJ intermediate within which XerC is active and catalyzes the second-strand exchange (3). (B) Integration and excision of the GGI (dotted line) by XerCD catalysis. KOPS, dif_Ng, and dif_GGI sites are represented as in A. An alignment of dif_Ng, dif_GGI and consensus dif sequence (27, 28) is shown on the left. Substituted positions in dif_GGI are represented as lowercase characters and highlighted by stars.In numerous bacteria, the XerCD/dif system is hijacked by IMEXs, which integrate their host genome into dif sites by using XerCD-mediated catalysis (4). In all of the reported cases, integration of IMEXs recreates a bona fide dif site, thereby not interfering with chromosome dimer resolution, which would lead to their counter-selection. The best-described examples are Vibrio cholerae IMEXs, which carry crucial virulence determinants (5–7, 17). These IMEXs have developed different strategies to integrate and to remain stably integrated, although the mechanisms ensuring their stable maintenance are not fully understood. Neisseria species contain an unusually long IMEX called the gonococcal genomic island (GGI) (8). In Neisseria gonorrheae, the GGI is 57 kb long and encodes a type IV secretion system that exports the chromosomal DNA of its host, rendering it available to neighboring cells for gene exchange by genetic transformation (8, 18). The GGI carries a dif site, dif_GGI, consisting of a XerC-binding site, a central region homologous to the Neisseria dif site, dif_Ng, and a divergent XerD-binding site (Fig. 1B). Comparison of N. gonorrheae strains harboring or lacking the GGI, together with functional data, indicates that the GGI integrates by XerCD-dependent recombination (9). The nonreplicative excised circular form of the GGI can be detected and the GGI can also be lost, showing that excision occurs, although at low frequencies (9). Although the GGI was identified over a decade ago, it has remained unclear how DNA flanked by two Xer recombination sites is stably maintained at a chromosomal locus processed by FtsK during each cell cycle. In this study, we have combined in vitro and in vivo approaches to show that dif_GGI is indeed an active Xer recombination site at which the Neisseria Xer recombinases catalyze recombination when activated by FtsKγ. However, we find that recombination between dif_Ng and dif_GGI is inhibited by translocating FtsK. Inhibition is a result of the absence of translocation arrest at XerCD_Ng/dif_GGI complexes that most likely precludes recombination activation, an absence that causes the complex to dismantle. We conclude that, depending on the sequence of the recombination site, Xer recombination complexes have the intrinsic capacity to be activated or inhibited by FtsK.     

From the Cover: Cooperation and individuality among man-eating lions

Cooperation is the cornerstone of lion social behavior. In a notorious case, a coalition of two adult male lions from Tsavo, southern Kenya, cooperatively killed dozens of railway workers in 1898. The “man-eaters of Tsavo” have since become the subject of numerous popular accounts, including three Hollywood films. Yet the full extent of the lions'' man-eating behavior is unknown; estimates range widely from 28 to 135 victims. Here we use stable isotope ratios to quantify increasing dietary specialization on novel prey during a time of food limitation. For one lion, the δ¹³C and δ¹⁵N values of bone collagen and hair keratin (which reflect dietary inputs over years and months, respectively) reveal isotopic changes that are consistent with a progressive dietary specialization on humans. These findings not only support the hypothesis that prey scarcity drives individual dietary specialization, but also demonstrate that sustained dietary individuality can exist within a cooperative framework. The intensity of human predation (up to 30% reliance during the final months of 1898) is also associated with severe craniodental infirmities, which may have further promoted the inclusion of unconventional prey under perturbed environmental conditions.     

From the Cover: Widespread metabolic potential for nitrite and nitrate assimilation among Prochlorococcus ecotypes

The marine cyanobacterium Prochlorococcus is the most abundant photosynthetic organism in oligotrophic regions of the oceans. The inability to assimilate nitrate is considered an important factor underlying the distribution of Prochlorococcus, and thought to explain, in part, low abundance of Prochlorococcus in coastal, temperate, and upwelling zones. Here, we describe the widespread occurrence of a genomic island containing nitrite and nitrate assimilation genes in uncultured Prochlorococcus cells from marine surface waters. These genes are characterized by low GC content, form a separate phylogenetic clade most closely related to marine Synechococcus, and are located in a different genomic region compared with an orthologous cluster found in marine Synechococcus strains. This sequence distinction suggests that these genes were not transferred recently from Synechococcus. We demonstrate that the nitrogen assimilation genes encode functional proteins and are expressed in the ocean. Also, we find that their relative occurrence is higher in the Caribbean Sea and Indian Ocean compared with the Sargasso Sea and Eastern Pacific Ocean, which may be related to the nitrogen availability in each region. Our data suggest that the ability to assimilate nitrite and nitrate is associated with microdiverse lineages within high- and low-light (LL) adapted Prochlorococcus ecotypes. It challenges 2 long-held assumptions that (i) Prochlorococcus cannot assimilate nitrate, and (ii) only LL adapted ecotypes can use nitrite. The potential for previously unrecognized productivity by Prochlorococcus in the presence of oxidized nitrogen species has implications for understanding the biogeography of Prochlorococcus and its role in the oceanic carbon and nitrogen cycles.