RNA stable-isotope probing

At the heart of microbial ecology lies a true scientific dichotomy. On the one hand, we know microbes are responsible for processes on which all other life on Earth is dependent; their removal would mean the cessation of all known life. However, in opposition, the majority of extant microbial species in natural environments have never been cultured or studied in a laboratory as living organisms. Owing to these factors, the question of "who does what?" has been a major barrier to understanding how microbially mediated ecosystem level events occur. Recently, the use of stable isotopes (13C) to trace carbon from specific substrates into microbes that assimilate carbon from that substrate has significantly advanced our understanding of the relationship between environmental processes and microbial phylogeny.     

The sampling scheme matters: Pan troglodytes troglodytes and P. t. schweinfurthii are characterized by clinal genetic variation rather than a strong subspecies break

Populations of an organism living in marked geographical or evolutionary isolation from other populations of the same species are often termed subspecies and expected to show some degree of genetic distinctiveness. The common chimpanzee (Pan troglodytes) is currently described as four geographically delimited subspecies: the western (P. t. verus), the nigerian‐cameroonian (P. t. ellioti), the central (P. t. troglodytes) and the eastern (P. t. schweinfurthii) chimpanzees. Although these taxa would be expected to be reciprocally monophyletic, studies have not always consistently resolved the central and eastern chimpanzee taxa. Most studies, however, used data from individuals of unknown or approximate geographic provenance. Thus, genetic data from samples of known origin may shed light on the evolutionary relationship of these subspecies. We generated microsatellite genotypes from noninvasively collected fecal samples of 185 central chimpanzees that were sampled across large parts of their range and analyzed them together with 283 published eastern chimpanzee genotypes from known localities. We observed a clear signal of isolation by distance across both subspecies. Further, we found that a large proportion of comparisons between groups taken from the same subspecies showed higher genetic differentiation than the least differentiated between‐subspecies comparison. This proportion decreased substantially when we simulated a more clumped sampling scheme by including fewer groups. Our results support the general concept that the distribution of the sampled individuals can dramatically affect the inference of genetic population structure. With regard to chimpanzees, our results emphasize the close relationship of equatorial chimpanzees from central and eastern equatorial Africa and the difficult nature of subspecies definitions. Am J Phys Anthropol 156:181–191, 2015. © 2014 Wiley Periodicals, Inc.     

Electron acceptor-dependent identification of key anaerobic toluene degraders at a tar-oil-contaminated aquifer by Pyro-SIP

The diversity of archaeal communities growing in four hot springs (65-90 °C, pH 6.5) was assessed with 16S rRNA gene primers specific for the domain Archaea. Overall, mainly uncultured members of the Desulfurococcales, the Thermoproteales and the Korarchaeota, were identified. Based on this diversity, a set of chaperonin heat-shock protein (Hsp60) gene sequences from different archaeal species were aligned to design two degenerate primer sets for the amplification of the chaperonin gene: Ths and Kor (which can also detect the korarchaeotal chaperonin gene from one of the samples). A phylogenetic tree was constructed using the chaperonin sequences retrieved and other sequences from cultured representatives. The Alpha and Beta paralogs of the chaperonin gene were observed within the main clades and orthologs among them. Cultivated representatives from these clades were assigned to either paralog in the chaperonin tree. Uncultured representatives observed in the 16S rRNA gene analysis were found to be related to the Desulfurococcales. The topologies of the 16S rRNA gene and chaperonin phylogenetic trees were compared, and similar phylogenetic relationships were observed. Our results suggest that the chaperonin Hsp60 gene may be used as a phylogenetic marker for the clades found in this extreme environment.     

Categorization of Extremely Brief Auditory Stimuli: Domain-Specific or Domain-General Processes?

The present study investigated the minimum amount of auditory stimulation that allows differentiation of spoken voices, instrumental music, and environmental sounds. Three new findings were reported. 1) All stimuli were categorized above chance level with 50 ms-segments. 2) When a peak-level normalization was applied, music and voices started to be accurately categorized with 20 ms-segments. When the root-mean-square (RMS) energy of the stimuli was equalized, voice stimuli were better recognized than music and environmental sounds. 3) Further psychoacoustical analyses suggest that the categorization of extremely brief auditory stimuli depends on the variability of their spectral envelope in the used set. These last two findings challenge the interpretation of the voice superiority effect reported in previously published studies and propose a more parsimonious interpretation in terms of an emerging property of auditory categorization processes.     

Inducible gene manipulations in brain serotonergic neurons of transgenic rats

The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system.     

Subliminal semantic priming in speech

Numerous studies have reported subliminal repetition and semantic priming in the visual modality. We transferred this paradigm to the auditory modality. Prime awareness was manipulated by a reduction of sound intensity level. Uncategorized prime words (according to a post-test) were followed by semantically related, unrelated, or repeated target words (presented without intensity reduction) and participants performed a lexical decision task (LDT). Participants with slower reaction times in the LDT showed semantic priming (faster reaction times for semantically related compared to unrelated targets) and negative repetition priming (slower reaction times for repeated compared to semantically related targets). This is the first report of semantic priming in the auditory modality without conscious categorization of the prime.     

An ethological perspective on defecation as an integral part of anti-predatory behaviour in the grey partridge (<Emphasis Type="Italic">Perdix perdix</Emphasis> L.) at night

Night-time flight behaviour of the grey partridge (Perdix perdix) was studied in three agricultural landscapes in Germany during the winter of 2005/2006. Thermography was used to detect roosting coveys and record flight behaviour. The main focus of the study was an analysis of defecation behaviour during the escape flight. A total of 640 partridges in 102 coveys were detected and approached during 85 scanning hours. Flight initiation distance averaged 22.8 m. In at least 75% of the coveys, defecation occurred upon being flushed. A covey of 15 partridges can produce 60 droppings that appear as a shower on the thermal record distributed over an estimated area of 200 m2. It can be hypothesised that defecation in the context of escape behaviour has various selective advantages in addition to the reduction of body weight. Predators may be irritated or deterred by the excretion of faeces of the partridges taking off. As defecation also appears in flushed partridges not in an immediate life-threatening situation, this behaviour seems to be uncoupled from the parasympathetic reaction of fear. Permanent predator pressure may have selected for a strategic integration of defecation in the escape behaviour of partridges at night.     

Development of stress response in nestling pied flycatchers

Birds respond to unpredictable events by secreting corticosterone, which induces various responses to cope with stressful situations. However, the evidence is still elusive whether altricial nestlings perceive and respond to external stressors. We investigated the development of adrenocortical stress response to handling-related stressor in nestlings of a small passerine bird, the pied flycatcher (Ficedula hypoleuca). Nestlings were held in isolation from their parents during the experiment to ensure that they indeed respond to handling, not to parental alarm calls. We found that both 9- and 13-day-old nestlings were able to elicit hormonal stress response. Although baseline as well as stress-induced corticosterone levels rose slightly with age, the magnitude of difference between the control and stress-induced levels remained similar in both age groups. However, comparison with adults showed that the stress response of nestlings prior to fledging was still incomplete and significantly lower than in adults. Overall, our results indicate that altricial nestlings do respond to acute stressors, but on the contrary to previous predictions the development of corticosterone stress response during growth period is not gradual and varies remarkably between different passerine species.     

Testing the limits of 454 pyrotag sequencing: reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes

The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ~4% read abundance across biological, and ~2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific 'in vivo' fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.     

Characterization of azaspiracids in plankton size-fractions and isolation of an azaspiracid-producing dinoflagellate from the North Sea

Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfish poisoning in humans, particularly in northern Europe. In an attempt to establish the biogeographical distribution of AZA toxins, their association with plankton size-fractions, and to confirm the identity of the causative species responsible for human poisoning, a month-long oceanographic study was undertaken in coastal North Sea waters. The occurrence and abundance of AZA analogues was measured by on board triple quadrupole mass spectrometry coupled to liquid chromatography (LC-MS/MS). In size-fractionated plankton samples collected by net tows (20 μm mesh-size), by pumping from discrete depths and from Niskin entrapment bottle casts to fixed depths, AZA-1 was consistently the major azaspiracid component. In eastern Scottish coastal waters, the highest amounts of AZA-1 in net tow samples were in the 50–200 μm fractions, with lesser amounts detected in the >200 μm and 50–20 μm fractions. At these stations, the 50–200 μm fractions were rich in the ciliate Favella ehrenbergii. Cells of F. ehrenbergii isolated by microcapillary indeed contained AZA-1, but isolated cells grown and fed the non-toxic dinoflagellate Scrippsiella trochoidea for one week failed to contain any detectable AZA-1—evidence that F. ehrenbergii is merely a vector for AZA. Detailed analysis of plankton from Niskin bottle samples from around the North Sea typically showed highest amounts of AZA in the 3–20 μm fraction. From this fraction, a large number of crude cultures were established and subsequently screened for the presence of AZAs. A small photosynthetic thecate dinoflagellate, provisionally designated as strain 3D9, was isolated by microcapillary and brought into pure culture. This dinoflagellate strain was found to produce AZA-1, AZA-2 and an isomer of AZA-2. Sequence comparisons by molecular genetic techniques also indicated that this genotype was present in field samples rich in AZA. This discovery of a novel causative dinoflagellate for AZA toxicity essentially explains the lack of correlation of AZA with the abundance and distribution of the previously postulated culprit species Protoperidinium crassipes. We instead propose that such large phagotrophic dinoflagellates can act as an AZA vector following grazing upon a proximal source, such as the dinoflagellate 3D9 strain.