Largely invariant communities of bacterial endophytes in the nonphotosynthetic mycoheterotrophic plant Pterospora andromedea.

PREMISE: Mycoheterotrophic plants rely on fungi to obtain their carbon requirements. Recent experiments demonstrated the presence of endophytic bacteria associated with mycoheterotrophs. Although mycoheterotrophs show high specificity for their fungal partners, it is not known whether they also show high specificity for associated bacteria or whether the bacteria have a definite function in the symbiosis. METHODS: Two 16S rRNA sequencing experiments were designed to explore endophytic microbial community composition and function in root ball fractions of the mycoheterotroph Pterospora andromedea (Ericaceae), and rhizosphere soil and control soil 5 m away from each plant. One experiment compared microbial assemblages in fractions of six plants to those in rhizosphere and control soil samples. Another experiment documented bacterial endophyte diversity in root balls of 97 plants from across North America. RESULTS: Soil samples were similar in bacterial community structure but were significantly more diverse and less consistently structured than were bacterial communities within root balls. The proportion of endophytic bacterial species varied slightly but not their community composition despite differences in P. andromedea lineage, geography, conifer species, and fungi. Predictive metagenomic profiling of the endophytes in P. andromedea-only root ball fractions showed many of the bacterial endophytes likely function in N-metabolism and N-fixation. CONCLUSIONS: Our results document a consistent and largely invariant community of endophytic bacteria in P. andromedea across biotic and abiotic environmental conditions at a continental scale. It is unknown what role these bacteria may play in the quad-partite symbiotic network centered on P. andromedea; however, the predictive metagenomic profiling suggests a possible function in N-metabolism or N-fixation. Discovery of a ubiquitous community of endophytic bacteria with a putative function centered on N-metabolism or N-fixation could have a previously unrecognized impact on understanding of mycoheterotroph ecophysiology.

Survivorship and growth in staghorn coral (Acropora cervicornis) outplanting projects in the Florida Keys National Marine Sanctuary.

Significant population declines in Acropora cervicornis and A. palmata began in the 1970s and now exceed over 90%. The losses were caused by a combination of coral disease and bleaching, with possible contributions from other stressors, including pollution and predation. Reproduction in the wild by fragment regeneration and sexual recruitment is inadequate to offset population declines. Starting in 2007, the Coral Restoration Foundation™ evaluated the feasibility of outplanting A. cervicornis colonies to reefs in the Florida Keys to restore populations at sites where the species was previously abundant. Reported here are the results of 20 coral outplanting projects with each project defined as a cohort of colonies outplanted at the same time and location. Photogrammetric analysis and in situ monitoring (2007 to 2015) measured survivorship, growth, and condition of 2419 colonies. Survivorship was initially high but generally decreased after two years. Survivorship among projects based on colony counts ranged from 4% to 89% for seven cohorts monitored at least five years. Weibull survival models were used to estimate survivorship beyond the duration of the projects and ranged from approximately 0% to over 35% after five years and 0% to 10% after seven years. Growth rate averaged 10 cm/year during the first two years then plateaued in subsequent years. After four years, approximately one-third of surviving colonies were ≥ 50 cm in maximum diameter. Projects used three to sixteen different genotypes and significant differences did not occur in survivorship, condition, or growth. Restoration times for three reefs were calculated based on NOAA Recovery Plan (NRP) metrics (colony abundance and size) and the findings from projects reported here. Results support NRP conclusions that reducing stressors is required before significant population growth and recovery will occur. Until then, outplanting protects against local extinction and helps to maintain genetic diversity in the wild.

An Investigation of Computer-based Brain Training on the Cognitive and EEG Performance of Employees.

Neurocognitive skills (e.g., processing speed, attention and memory) were hypothesized to be critical for workplace performance and by extension for the work-life balance of employees. Twenty-one employee volunteers underwent a neurocognitive training program - which consisted of an initial pre-test assessment, a six week "boost" or intervention period, and then a re-assessment to track the progress of each participant. A median split of the group created two training groups: a long-training group that averaged 30 hours of total training during the boost period; and a short-training group that averaged 7 hours of training. On pre-training measures of neurocognitive performance, group differences in performance did not reach statistical significance. Following training participants experienced a positive impact from the program as measured in three ways: standardized higher behavioral metrics, improved cognitive state metrics using EEG and positive self-reported data. From a quantitative perspective, participants' cognitive efficiency increased by 12% for the high-training group and 5% for the low-training group (cognitive efficiency refers to a behavioral measure which combines accuracy and speed). Qualitatively, study participants reported improvements in their productivity and mental performance post-study.

Study of three interesting Amanita species from Thailand: Morphology, multiple-gene phylogeny and toxin analysis.

Amanita ballerina and A. brunneitoxicaria spp. nov. are introduced from Thailand. Amanita fuligineoides is also reported for the first time from Thailand, increasing the known distribution of this taxon. Together, those findings support our view that many taxa are yet to be discovered in the region. While both morphological characters and a multiple-gene phylogeny clearly place A. brunneitoxicaria and A. fuligineoides in sect. Phalloideae (Fr.) Quél., the placement of A. ballerina is problematic. On the one hand, the morphology of A. ballerina shows clear affinities with stirps Limbatula of sect. Lepidella. On the other hand, in a multiple-gene phylogeny including taxa of all sections in subg. Lepidella, A. ballerina and two other species, including A. zangii, form a well-supported clade sister to the Phalloideae sensu Bas 1969, which include the lethal "death caps" and "destroying angels". Together, the A. ballerina-A. zangii clade and the Phalloideae sensu Bas 1969 also form a well-supported clade. We therefore screened for two of the most notorious toxins by HPLC-MS analysis of methanolic extracts from the basidiomata. Interestingly, neither α-amanitin nor phalloidin was found in A. ballerina, whereas Amanita fuligineoides was confirmed to contain both α-amanitin and phalloidin, and A. brunneitoxicaria contained only α-amanitin. Together with unique morphological characteristics, the position in the phylogeny indicates that A. ballerina is either an important link in the evolution of the deadly Amanita sect. Phalloideae species, or a member of a new section also including A. zangii.

Increased phylogenetic resolution within the ecologically important Rhizopogon subgenus Amylopogon using 10 anonymous nuclear loci.

Rhizopogon species are ecologically significant ectomycorrhizal fungi in conifer ecosystems. The importance of this system merits the development and utilization of a more robust set of molecular markers specifically designed to evaluate their evolutionary ecology. Anonymous nuclear loci (ANL) were developed for R. subgenus Amylopogon. Members of this subgenus occur throughout the United States and are exclusive fungal symbionts associated with Pterospora andromedea, a threatened mycoheterotrophic plant endemic to disjunct eastern and western regions of North America. Candidate ANL were developed from 454 shotgun pyrosequencing and assessed for positive amplification across targeted species, sequencing success, and recovery of phylogenetically informative sites. Ten ANL were successfully developed and were subsequently used to sequence representative taxa, herbaria holotype and paratype specimens in R. subgenus Amylopogon. Phylogenetic reconstructions were performed on individual and concatenated data sets by Bayesian inference and maximum likelihood methods. Phylogenetic analyses of these 10 ANL were compared with a phylogeny traditionally constructed using the universal fungal barcode nuc rDNA ITS1-5.8S-ITS2 region (ITS). The resulting ANL phylogeny was consistent with most of the species designations delineated by ITS. However, the ANL phylogeny provided much greater phylogenetic resolution, yielding new evidence for cryptic species within previously defined species of R. subgenus Amylopogon. Additionally, the rooted ANL phylogeny provided an alternate topology to the ITS phylogeny, which inferred a novel set of evolutionary relationships not identified in prior phylogenetic studies.

The M1 Muscarinic Receptor Antagonist VU0255035 Delays the Development of Status Epilepticus after Organophosphate Exposure and Prevents Hyperexcitability in the Basolateral Amygdala.

Exposure to organophosphorus toxins induces seizures that progress to status epilepticus (SE), which can cause brain damage or death. Seizures are generated by hyperstimulation of muscarinic receptors, subsequent to inhibition of acetylcholinesterase; this is followed by glutamatergic hyperactivity, which sustains and reinforces seizure activity. It has been unclear which muscarinic receptor subtypes are involved in seizure initiation and the development of SE in the early phases after exposure. Here, we show that pretreatment of rats with the selective M1 receptor antagonist, VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)-benzo[c][1,2,5]thiadiazole-4 sulfonamide], significantly suppressed seizure severity and prevented the development of SE for about 40 minutes after exposure to paraoxon or soman, suggesting an important role of the M1 receptor in the early phases of seizure generation. In addition, in in vitro brain slices of the basolateral amygdala (a brain region that plays a key role in seizure initiation after nerve agent exposure), VU0255035 blocked the effects produced by bath application of paraoxon-namely, a brief barrage of spontaneous inhibitory postsynaptic currents, followed by a significant increase in the ratio of the total charge transferred by spontaneous excitatory postsynaptic currents over that of the inhibitory postsynaptic currents. Furthermore, paraoxon enhanced the hyperpolarization-activated cation current Ih in basolateral amygdala principal cells, which could be one of the mechanisms underlying the increased glutamatergic activity, an effect that was also blocked in the presence of VU0255035. Thus, selective M1 antagonists may be an efficacious pretreatment in contexts in which there is risk for exposure to organophosphates, as these antagonists will delay the development of SE long enough for medical assistance to arrive.

Long-term neuropathological and behavioral impairments after exposure to nerve agents.

One of the deleterious effects of acute nerve agent exposure is the induction of status epilepticus (SE). If SE is not controlled effectively, it causes extensive brain damage. Here, we review the neuropathology observed after nerve agent-induced SE, as well as the ensuing pathophysiological, neurological, and behavioral alterations, with an emphasis on their time course and longevity. Limbic structures are particularly vulnerable to damage by nerve agent exposure. The basolateral amygdala (BLA), which appears to be a key site for seizure initiation upon exposure, suffers severe neuronal loss; however, GABAergic BLA interneurons display a delayed death, perhaps providing a window of opportunity for rescuing intervention. The end result is a long-term reduction of GABAergic activity in the BLA, with a concomitant increase in spontaneous excitatory activity; such pathophysiological alterations are not observed in the CA1 hippocampal area, despite the extensive neuronal loss. Hyperexcitability in the BLA may be at least in part responsible for the development of recurrent seizures and increased anxiety, while hippocampal damage may underlie the long-term memory impairments. Effective control of SE after nerve agent exposure, such that brain damage is also minimized, is paramount for preventing lasting neurological and behavioral deficits.

A rat model of nerve agent exposure applicable to the pediatric population: The anticonvulsant efficacies of atropine and GluK1 antagonists.

Inhibition of acetylcholinesterase (AChE) after nerve agent exposure induces status epilepticus (SE), which causes brain damage or death. The development of countermeasures appropriate for the pediatric population requires testing of anticonvulsant treatments in immature animals. In the present study, exposure of 21-day-old (P21) rats to different doses of soman, followed by probit analysis, produced an LD50 of 62µg/kg. The onset of behaviorally-observed SE was accompanied by a dramatic decrease in brain AChE activity; rats who did not develop SE had significantly less reduction of AChE activity in the basolateral amygdala than rats who developed SE. Atropine sulfate (ATS) at 2mg/kg, administered 20 min after soman exposure (1.2×LD50), terminated seizures. ATS at 0.5mg/kg, given along with an oxime within 1 min after exposure, allowed testing of anticonvulsants at delayed time-points. The AMPA/GluK1 receptor antagonist LY293558, or the specific GluK1 antagonist UBP302, administered 1h post-exposure, terminated SE. There were no degenerating neurons in soman-exposed P21 rats, but both the amygdala and the hippocampus were smaller than in control rats at 30 and 90days post-exposure; this pathology was not present in rats treated with LY293558. Behavioral deficits present at 30 days post-exposure, were also prevented by LY293558 treatment. Thus, in immature animals, a single injection of atropine is sufficient to halt nerve agent-induced seizures, if administered timely. Testing anticonvulsants at delayed time-points requires early administration of ATS at a low dose, sufficient to counteract only peripheral toxicity. LY293558 administered 1h post-exposure, prevents brain pathology and behavioral deficits.

Characterization of microsatellite markers for pinedrops, Pterospora andromedea (Ericaceae), from Illumina MiSeq sequencing.

PREMISE OF THE STUDY: Pterospora andromedea (Ericaceae) is a mycoheterotrophic plant endemic to North America with a disjunct distribution. Eastern populations are in decline compared to western populations. Microsatellite loci will allow comparison of genetic diversity in endangered to nonthreatened populations. • METHODS AND RESULTS: Illumina MiSeq sequencing resulted in development of 12 polymorphic microsatellite loci from 63 perfect microsatellite loci tested. One polymorphic locus was obtained from a traditional enrichment method. These 13 loci were screened across two western and two eastern populations. For western and eastern populations, respectively, number of alleles ranged from one to 10 and one to four, and observed heterozygosity ranged from 0.000 to 0.389 and 0.000 to 0.143. • CONCLUSIONS: These are the first microsatellite loci developed for Pterospora. They will be useful in conservation efforts of the eastern populations and for examination of population genetic parameters at different geographic scales and comparison with mycorrhizal fungal hosts.

Microsatellite primers for the fungi Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) from 454 shotgun pyrosequencing.

PREMISE OF THE STUDY: Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) are ectomycorrhizal fungi symbiotic with pines and the mycoheterotrophic plant Pterospora andromedea (Ericaceae). Microsatellite loci will allow population genetic study of fungal hosts to P. andromedea. • METHODS AND RESULTS: Shotgun pyrosequencing of R. kretzerae DNA resulted in primer development of 23 perfect microsatellite loci and screened across two populations each for R. kretzerae and R. salebrosus. Twelve loci were polymorphic in R. kretzerae populations, and 11 loci cross-amplified in R. salebrosus populations. For R. kretzerae and R. salebrosus, number of alleles was one to eight and one to nine, respectively, and observed heterozygosity ranged from 0.00-0.57 and 0.00-0.70, respectively. • CONCLUSIONS: These are the first microsatellite loci developed for any species within Rhizopogon subgenus Amylopogon. These microsatellite loci will be used in conservation genetic studies of rare to endangered eastern populations and to compare plant and fungal population genetic structure at different hierarchical levels.

The limitations of diazepam as a treatment for nerve agent-induced seizures and neuropathology in rats: comparison with UBP302.

Exposure to nerve agents induces prolonged status epilepticus (SE), causing brain damage or death. Diazepam (DZP) is the current US Food and Drug Administration-approved drug for the cessation of nerve agent-induced SE. Here, we compared the efficacy of DZP with that of UBP302 [(S)-3-(2-carboxybenzyl)willardiine; an antagonist of the kainate receptors that contain the GluK1 subunit] against seizures, neuropathology, and behavioral deficits induced by soman in rats. DZP, administered 1 hour or 2 hours postexposure, terminated the SE, but seizures returned; thus, the total duration of SE within 24 hours after soman exposure was similar to (DZP at 1 hour) or longer than (DZP at 2 hours) that in the soman-exposed rats that did not receive the anticonvulsant. Compared with DZP, UBP302 stopped SE with a slower time course, but dramatically reduced the total duration of SE within 24 hours. Neuropathology and behavior were assessed in the groups that received anticonvulsant treatment 1 hour after exposure. UBP302, but not DZP, reduced neuronal degeneration in a number of brain regions, as well as neuronal loss in the basolateral amygdala and the CA1 hippocampal area, and prevented interneuronal loss in the basolateral amygdala. Anxiety-like behavior was assessed in the open field and by the acoustic startle response 30 days after soman exposure. The results showed that anxiety-like behavior was increased in the DZP-treated group and in the group that did not receive anticonvulsant treatment, but not in the UBP302-treated group. The results argue against the use of DZP for the treatment of nerve agent-induced seizures and brain damage and suggest that targeting GluK1-containing receptors is a more effective approach.

ASIC1a activation enhances inhibition in the basolateral amygdala and reduces anxiety.

The discovery that even small changes in extracellular acidity can alter the excitability of neuronal networks via activation of acid-sensing ion channels (ASICs) could have therapeutic application in a host of neurological and psychiatric illnesses. Recent evidence suggests that activation of ASIC1a, a subtype of ASICs that is widely distributed in the brain, is necessary for the expression of fear and anxiety. Antagonists of ASIC1a, therefore, have been proposed as a potential treatment for anxiety. The basolateral amygdala (BLA) is central to fear generation, and anxiety disorders are characterized by BLA hyperexcitability. To better understand the role of ASIC1a in anxiety, we attempted to provide a direct assessment of whether ASIC1a activation increases BLA excitability. In rat BLA slices, activation of ASIC1a by low pH or ammonium elicited inward currents in both interneurons and principal neurons, and increased spontaneous IPSCs recorded from principal cells significantly more than spontaneous EPSCs. Epileptiform activity induced by high potassium and low magnesium was suppressed by ammonium. Antagonism of ASIC1a decreased spontaneous IPSCs more than EPSCs, and increased the excitability of the BLA network, as reflected by the pronounced increase of evoked field potentials, suggesting that ASIC1a channels are active in the basal state. In vivo activation or blockade of ASIC1a in the BLA suppressed or increased, respectively, anxiety-like behavior. Thus, in the rat BLA, ASIC1a has an inhibitory and anxiolytic function. The discovery of positive ASIC1a modulators may hold promise for the treatment of anxiety disorders.

NEURO.TV: Neuroscience Education on the Internet

NEURO.tv is a new educational project that seeks to bring advanced concepts in neuroscience to the general public. We film one-hour discussions with leading neuroscientists, philosophers, and psychologists who have had significant impact on our current understanding of brain function, and we publish these discussions on YouTube, iTunes, and other social media outlets. Here, we explain the motivations behind this new program.

Genetic divergence among disjunct populations of three Russula spp. from Africa and Madagascar.

This study was a preliminary analysis of the genetic structure of the ectomycorrhizal species Russula discopus, R. pseudocarmecina and R. ochraceorivulosa with disjunct distributions in continental Africa and Madagascar. Phylogenetic analyses of the nuclear ITS1/5.8S/ITS2 region and the mitochondrial atp6 gene were performed with specimens from both locations for each species along with a suitable outgroup for each of the three taxa. Additional analyses of the ITS1/5.8S/ITS2 region using the African and Malagasy specimens and additional taxa in the genus Russula also were performed. R. pseudocarmecina and R. discopus both exhibited genetic structure as shown by a relatively high percentage difference in ITS and atp6 sequences, high bootstrap support for African or Malagasy groups and the presence of indels in the ITS sequence that are unique to either Africa or Madagascar. African and Malagasy groups of each species were more closely related to each other than to other taxa in Russula. Genetic structure also existed in populations of R. ochraceorivulosa, but bootstrap support was weaker than in the other two species. In addition, there was less sequence divergence in R. ochraceorivulosa and this species was the only one for which the same atp6 haplotype was found in both Africa and Madagascar. Reciprocal monophyly for all three species was consistent with the hypothesis that the same vicariance event may be responsible for the genetic structure observed here, although shorter branch lengths, lower bootstrap support and the presence of the same atp6 haplotype in Africa and Madagascar for R. ochraceorivulosa suggested slower evolutionary rates or geographical isolation after the other two taxa. In addition to the geological events that separated Africa and Madagascar, environmental changes during the Miocene or later might have had an effect on the distribution of these species.

New Elaphomyces species (Elaphomycetaceae, Eurotiales, Ascomycota) from Guyana.

Elaphomyces compleximurus sp. nov. and E. digitatus sp. nov. are described from the Pakaraima Mountains of Guyana. Macromorphological, micromorphological, habitat and DNA sequence data are provided for each new species. This is the first report of Elaphomyces ascomata associated with ectomycorrhizal members of the Fabaceae and also for the genus from the lowland South American tropics.

Probability sampling of stony coral populations in the Florida Keys.

Principles of probability survey design were applied to guide large-scale sampling of populations of stony corals and associated benthic taxa in the Florida Keys coral reef ecosystem. The survey employed a two-stage stratified random sampling design that partitioned the 251-km(2) domain by reef habitat types, geographic regions, and management zones. Estimates of the coefficient of variation (ratio of standard error to the mean) for stony coral population density and abundance ranged from 7% to 12% for four of six principal species. These levels of survey precision are among the highest reported for comparable surveys of marine species. Relatively precise estimates were also obtained for octocoral density, sponge frequency of occurrence, and benthic cover of algae and invertebrates. Probabilistic survey design techniques provided a robust framework for estimating population-level metrics and optimizing sampling efficiency.

Climate change, coral reef ecosystems, and management options for marine protected areas.

Marine protected areas (MPAs) provide place-based management of marine ecosystems through various degrees and types of protective actions. Habitats such as coral reefs are especially susceptible to degradation resulting from climate change, as evidenced by mass bleaching events over the past two decades. Marine ecosystems are being altered by direct effects of climate change including ocean warming, ocean acidification, rising sea level, changing circulation patterns, increasing severity of storms, and changing freshwater influxes. As impacts of climate change strengthen they may exacerbate effects of existing stressors and require new or modified management approaches; MPA networks are generally accepted as an improvement over individual MPAs to address multiple threats to the marine environment. While MPA networks are considered a potentially effective management approach for conserving marine biodiversity, they should be established in conjunction with other management strategies, such as fisheries regulations and reductions of nutrients and other forms of land-based pollution. Information about interactions between climate change and more "traditional" stressors is limited. MPA managers are faced with high levels of uncertainty about likely outcomes of management actions because climate change impacts have strong interactions with existing stressors, such as land-based sources of pollution, overfishing and destructive fishing practices, invasive species, and diseases. Management options include ameliorating existing stressors, protecting potentially resilient areas, developing networks of MPAs, and integrating climate change into MPA planning, management, and evaluation.

Worldwide phylogeny of Lactarius section Deliciosi inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences.

A phylogenetic analysis of Lactarius sect. Deliciosi was performed based on collections of all known species. Several samples of each species were included, originating from a wide geographic range. The two DNA regions we used (ITS and a part of the gene encoding glyceraldehyde-3-phosphate dehydrogenase) showed an incongruent phylogenetic signal. Much attention was paid to carefully observed macro- and micromorphological characters to draw taxonomic conclusions. We currently accept 38 taxa (31 species and seven varieties) in Lactarius sect. Deliciosi worldwide; four species are new to science. More sampling is needed to resolve the status of the North American varieties. Our knowledge of the Asian species in this section remains fragmentary. The monophyly of the section and its position within Lactarius subgenus Piperites, as proposed in recent morphology-based classification schemes, is confirmed. The intrasectional relationships however do not coincide with the color of the latex (as previously supposed). Intercontinental conspecificity is low in general. The name L. deliciosus is wrongfully applied in North and Central America and only two species seem to occur in both Asia and Europe.

Cantharellus pleurotoides, a new and unusual basidiomycete from Guyana.

Cantharellus pleurotoides sp. nov. (Cantharellaceae, Cantharellales, Basidiomycota) is described from the Pakaraima Mountains of Guyana, occurring in rainforests dominated by ectomycorrhizal Dicymbe spp. (Caesalpiniaceae). This fungus is singular among Cantharellus species described worldwide in possessing a pleurotoid basidioma. Macromorphological, micromorphological, and habitat data are provided for the new species.

Gymnomyces xerophilus sp. nov. (sequestrate Russulaceae), an ectomycorrhizal associate of Quercus in California.

Gymnomyces xerophilus sp. nov., a sequestrate species in the Russulaceae, is characterized and described morphologically as a new species from Quercus-dominated woodlands in California. ITS sequences recovered from healthy, ectomycorrhizal roots of Quercus douglasii and Q. wislizeni matched those of G. xerophilus basidiomata, confirming the ectomycorrhizal status of this fungus. Phylogenetic analysis of the ITS region places G. xerophilus in a clade with both agaricoid (Russula in the section Polychromae) and sequestrate (Gymnomyces, Cystangium) relatives. We include a dichotomous key to the species of Gymnomyces associated with Quercus.