Search

Bacterial secondary metabolites exhibit diverse remarkable bioactivities and are thus the subject of study for different applications. Recently, the individual effectiveness of tripyrrolic prodiginines and rhamnolipids against the plant-parasitic nematode Heterodera schachtii, which causes tremendous losses in crop plants, was described. Notably, rhamnolipid production in engineered Pseudomonas putida strains has already reached industrial implementation. However, the non-natural hydroxyl-decorated prodiginines, which are of particular interest in this study due to a previously described particularly good plant compatibility and low toxicity, are not as readily accessible. In the present study, a new effective hybrid synthetic route was established. This included the engineering of a novel P. putida strain to provide enhanced levels of a bipyrrole precursor and an optimization of mutasynthesis, i.e., the conversion of chemically synthesized and supplemented monopyrroles to tripyrrolic compounds. Subsequent semisynthesis provided the hydroxylated prodiginine. The prodiginines caused reduced infectiousness of H. schachtii for Arabidopsis thaliana plants resulting from impaired motility and stylet thrusting, providing the first insights on the mode of action in this context. Furthermore, the combined application with rhamnolipids was assessed for the first time and found to be more effective against nematode parasitism than the individual compounds. To obtain, for instance, 50% nematode control, it was sufficient to apply 7.8 μM hydroxylated prodiginine together with 0.7 μg/ml (~ 1.1 μM) di-rhamnolipids, which corresponded to ca. ¼ of the individual EC50 values. In summary, a hybrid synthetic route toward a hydroxylated prodiginine was established and its effects and combinatorial activity with rhamnolipids on plant-parasitic nematode H. schachtii are presented, demonstrating potential application as antinematodal agents. Graphical Abstract

The aim of this study was to investigate the genetic structure and diversity of populations of the yellow-bellied toad in Lower Saxony, Germany. These populations provide a good example of a fragmented distribution of amphibian pioneer species on the outer rim of their occurrence. In total, 150 individuals from 11 sites were genotyped using ten highly polymorphic microsatellites. The genetic diversity, as measured by allelic richness and heterozygosity, was lower than that of other European amphibian populations; the individuals could be assigned to five genetic clusters. The average genetic differentiation between populations was very high (mean FST = 0.27) and no migration was detected between the two major populations, which were about 50 km apart. Nonetheless, natural migration as well as translocations between some subpopulations could be detected. In one population, which was introduced in the 1970s, we were able to verify the potential source population of the introduced toads. The results are discussed in light of conservation management strategies applied to this highly endangered species.

A desmotivação em aprender Física e a dificuldade dos alunos em atribuir significado a conteúdos apresentados em sala de aula são alguns dos problemas enfrentados pelo Ensino de Física nas escolas públicas do Brasil. Na busca por alternativas que melhorem o ensino, sem onerar ainda mais o currículo escolar, a literatura apresenta espaços não formais como um caminho em potencial. Neles, os estudantes enfrentariam problemas autênticos e, na busca por soluções, poderiam vivenciar a aprendizagem como participação social. Nessa linha de investigação, realizamos um estudo de caso, na acepção de Yin, em um espaço não formal, o Centro de Tecnologia Acadêmica Júnior (CTA Jr.), situado no Colégio de Aplicação da Universidade Federal do Rio Grande do Sul. Buscamos identificar os impactos que o CTA Jr. causou tanto no percurso formativo quanto na identidade de quatro estudantes que, após encerrarem o ensino básico, regressaram ao Centro exercendo um novo papel. A partir da análise de conteúdo das entrevistas concedidas pelos regressantes, à luz da Teoria Social da Aprendizagem (TSA) de Étienne Wenger, baseada em cinco dimensões: Prática, Identidade, Significado, Comunidade e Aprendizagem, foi possível: i) avaliar as contribuições da prática do Centro para o percurso formativo dos seus participantes; e ii) identificar valores criados/cultivados no CTA Jr. e nas identidades dos regressantes. Como resultado do estudo, verificamos que, ao participarem do CTA Jr., o percurso formativo dos regressantes foi diferenciado tanto em termos da segurança na escolha profissional quanto no desempenho em disciplinas do currículo formal. Também identificamos o cultivo de valores como colaboração, companheirismo, comunicação, responsabilidade e solidariedade, na identidade dos regressantes, tornando-os melhores cidadãos. Ainda, verificamos que a estrutura social existente no CTA Jr. apresenta características comuns a Comunidades de Prática (CoP). Assim sendo, acreditamos que a utilização de espaços não formais que promovam aprendizagem social enfatizando o aspecto CoP pode contribuir para uma aprendizagem com significado. Para isso, é necessário que a comunidade escolar perceba os ensinos formal e não formal como aliados na busca por uma educação cidadã. Some of the problems faced in physics education in the public school in Brazil are the students' lack of motivation to learn and the difficulties they face in assigning meaning to what is presented to them in class. Seeking for alternatives to improve teaching without overload the scholar curriculum even more, the literature presents non-formal learning spaces as a potential path. In these spaces, students would face authentic problems and experience learning as social participation. In this research line, we carried out a case study, in the sense of Yin, in a non-formal space, the Centro de Tecnologia Acadêmica Júnior (CTA Jr.), located at the Colégio de Aplicação of the Federal University of Rio Grande do Sul. We sought to identify the impacts that the CTA Jr. caused in the formative path and identity of four students who, after finishing high school, returned to the Technology Center in a new role. Based on the content analysis of the returnees interviews, in the light of Étienne Wenger's Social Theory of Learning – based on five dimensions: Practice, Identity, Meaning, Community and Learning – it was possible to: i) evaluate the contributions of the Technology Center's practice to the training path of its participants; and ii) identify values reated/cultivated in CTA Jr. and in the identities of the returnees. The results indicate that, by participating in the CTA Jr., the returnees’ formative path was differentiated both in terms of security in professional choice and in terms of performance in subjects of the scholar curriculum. We also identified the cultivation of values such as collaboration, companionship, communication, responsibility and solidarity, in the identity of returnees. Still, we verify that the existing social structure at CTA Jr. has characteristics common to Communities of Practice (CoP). Therefore, we consider that the use of non-formal spaces that promote social learning emphasizing the CoP aspect can contribute to a meaningful learning. For this, it is necessary that the school community perceives the formal and non-formal teaching as allies in the search for a citizen education.

Most terrestrial animals move with a specific number of propulsive legs, which differs between clades. The reasons for these differences are often unknown and rarely queried, despite the underlying mechanisms being indispensable for understanding the evolution of multilegged locomotor systems in the animal kingdom and the development of swiftly moving robots. Moreover, when speeding up, a range of species change their number of propulsive legs. The reasons for this behaviour have proven equally elusive. In animals and robots, the number of propulsive legs also has a decisive impact on the movement dynamics of the centre of mass. Here, I use the leg force interference model to elucidate these issues by introducing gradually declining ground reaction forces in locomotor apparatuses with varying numbers of leg pairs in a first numeric approach dealing with these measures’ impact on locomotion dynamics. The effects caused by the examined changes in ground reaction forces and timing thereof follow a continuum. However, the transition from quadrupedal to a bipedal locomotor system deviates from those between multilegged systems with different numbers of leg pairs. Only in quadrupeds do reduced ground reaction forces beneath one leg pair result in increased reliability of vertical body oscillations and therefore increased energy efficiency and dynamic stability of locomotion.

Most terrestrial animals move with a specific number of propulsive legs, which differs between clades. The reasons for these differences are often unknown and rarely queried, despite the underlying mechanisms being indispensable for understanding the evolution of multilegged locomotor systems in the animal kingdom and the development of swiftly moving robots. Moreover, when speeding up, a range of species change their number of propulsive legs. The reasons for this behaviour have proven equally elusive. In animals and robots, the number of propulsive legs also has a decisive impact on the movement dynamics of the centre of mass. Here, I use the leg force interference model to elucidate these issues by introducing gradually declining ground reaction forces in locomotor apparatuses with varying numbers of leg pairs in a first numeric approach dealing with these measures’ impact on locomotion dynamics. The effects caused by the examined changes in ground reaction forces and timing thereof follow a continuum. However, the transition from quadrupedal to a bipedal locomotor system deviates from those between multilegged systems with different numbers of leg pairs. Only in quadrupeds do reduced ground reaction forces beneath one leg pair result in increased reliability of vertical body oscillations and therefore increased energy efficiency and dynamic stability of locomotion.Significance statementThe model grants access to the effects of gradual ground force reduction on total ground reaction forces, body dynamics and gait transitions.

Τhe morphology, physiology and immunology, of solid tumors exhibit spatial heterogeneity which complicates our understanding of cancer progression and therapy response. Understanding spatial heterogeneity necessitates high resolution in vivo imaging of anatomical and pathophysiological tumor information. We introduce Rhodobacter as bacterial reporter for multispectral optoacoustic (photoacoustic) tomography (MSOT). We show that endogenous bacteriochlorophyll a in Rhodobacter gives rise to strong optoacoustic signals >800 nm away from interfering endogenous absorbers. Importantly, our results suggest that changes in the spectral signature of Rhodobacter which depend on macrophage activity inside the tumor can be used to reveal heterogeneity of the tumor microenvironment. Employing non-invasive high resolution MSOT in longitudinal studies we show spatiotemporal changes of Rhodobacter spectral profiles in mice bearing 4T1 and CT26.WT tumor models. Accessibility of Rhodobacter to genetic modification and thus to sensory and therapeutic functions suggests potential for a theranostic platform organism., Current optoacoustic probes for cancer imaging have limitations including background noise, long-term toxicity and scarce imaging depth in living tissue. Here the authors use Rhodobacter, purple bacteria rich in bacteriochlorophyll a, as an optoacoustic reporter to image tumor-associated macrophages in mice in vivo.

BackgroundCockroaches are usually typical omnivorous detritivores and their cephalic morphology is considered to be ancestral in various aspects. Thus, several studies addressed the morphology and function of the blattodean head, and the cockroach usually serves as a model for standard mouthparts in text books. However, so far only two of the three major lineages of Blattodea have been studied and no detailed information for the head of any Corydioidea was available. The present study closes this gap by providing a detailed morphological description of the head ofErgaula capucina, studying some important functional parameters of the mandible and discussing it in a phylogenetic framework.MethodsThe cephalic morphology ofErgaulastudied in detail using a broad set of different techniques including digital microscopy, µ-computed tomography, and 3-dimensional reconstructions. Concerning the functional morphology of the mandible, we compared the volume and effective cross sections of the eight compartments of the primary mandibular adductor muscle forErgaula,Blattella germanica, andSalganea rossiand measured the mechanical advantage,i.e., the force transmission ratio for all teeth of the mandible ofErgaula.ResultsThe head capsule ofErgaulais characterized by a strong sexual dimorphism and typical orthopteran mouthparts. It resembles the head capsule of other roaches in several respects and confirms oesotendons, the reduction of the mesal occelus, and bipartite M. verticopharyngealis and M. hypopharyngosalivaris as blattodean apomorphies. But it also shows some unique adaptations. It is the first described cockroach that lacks the dorsal tentorial arms which has various consequences for the cephalic musculature. On the maxillary lacinia,Ergaulais the first described blattodean to show strong and blunt setae instead of a lacinula, which might be homologues to the dentisetae of dragonflies and mayflies. Like other corydiid roaches that inhabit xeric areas,Ergaulahas an atmospheric water-vapor absorption mechanism that includes a gland and a ductus on the epipharnyx and bladders on the hypopharynx. The mandibular adductor is in cockroaches asymmetric, a pattern not found in termites, mantids, or other closely related insects.

The maximum running speed of legged animals is one evident factor for evolutionary selection—for predators and prey. Therefore, it has been studied across the entire size range of animals, from the smallest mites to the largest elephants, and even beyond to extinct dinosaurs. A recent analysis of the relation between animal mass (size) and maximum running speed showed that there seems to be an optimal range of body masses in which the highest terrestrial running speeds occur. However, the conclusion drawn from that analysis—namely, that maximum speed is limited by the fatigue of white muscle fibres in the acceleration of the body mass to some theoretically possible maximum speed—was based on coarse reasoning on metabolic grounds, which neglected important biomechanical factors and basic muscle-metabolic parameters. Here, we propose a generic biomechanical model to investigate the allometry of the maximum speed of legged running. The model incorporates biomechanically important concepts: the ground reaction force being counteracted by air drag, the leg with its gearing of both a muscle into a leg length change and the muscle into the ground reaction force, as well as the maximum muscle contraction velocity, which includes muscle-tendon dynamics, and the muscle inertia—with all of them scaling with body mass. Put together, these concepts’ characteristics and their interactions provide a mechanistic explanation for the allometry of maximum legged running speed. This accompanies the offering of an explanation for the empirically found, overall maximum in speed: In animals bigger than a cheetah or pronghorn, the time that any leg-extending muscle needs to settle, starting from being isometric at about midstance, at the concentric contraction speed required for running at highest speeds becomes too long to be attainable within the time period of a leg moving from midstance to lift-off. Based on our biomechanical model we, thus, suggest considering the overall speed maximum to indicate muscle inertia being functionally significant in animal locomotion. Furthermore, the model renders possible insights into biological design principles such as differences in the leg concept between cats and spiders, and the relevance of multi-leg (mammals: four, insects: six, spiders: eight) body designs and emerging gaits. Moreover, we expose a completely new consideration regarding the muscles’ metabolic energy consumption, both during acceleration to maximum speed and in steady-state locomotion.

The measurement of cuticular strain during locomotion using foil strain gauges provides information both on the loads of the exoskeleton bears and the adaptive value of the specific location of natural strain detectors (slit sense organs). Here, we critically review available literature. In tethered animals,by applying loads to the metatarsus tip,strain and mechanical sensitivity (S = strain/load) induced at various sites in the tibia were determined. The loci of the lyriform organs close to the tibia–metatarsus joint did not stand out by high strain. The strains induced at various sites during free locomotion can be interpreted based onSand, beyond the joint region, on beam theory. Spiders avoided laterad loading of the tibia–metatarsus joint during slow locomotion. Balancing body weight, joint flexors caused compressive strain at the posterior and dorsal tibia. While climbing upside down strain measurements indicate strong flexor activity. In future studies, a precise calculation and quantitative determination of strain at the sites of the lyriform organs will profit from more detailed data on the overall strain distribution, morphology, and material properties. The values and caveats of the strain gauge technology, the only one applicable to freely moving spiders, are discussed.

Arthropods are the most diverse clade on earth with regard to both species number and variability of body plans. Their general body plan is characterised by variable numbers of legs, and many-legged locomotion is an essential aspect of many aquatic and terrestrial arthropod species. Moreover, arthropods belong to the first groups of animals to colonise subaerial habitats, and they did so repeatedly and independently in a couple of clades. Those arthropod clades that colonised land habitats were equipped with highly variable body plans and locomotor apparatuses. Proceeding from their respective specific anatomies, they were challenged with strongly changing environmental conditions as well as altered physical and physiological constraints. This review explores the transitions from aquatic to terrestrial habitats across the different arthropod body plans and explains the major mechanisms and principles that constrain design and function of a range of locomotor apparatuses. Important aspects of movement physiology addressed here include the effects of different numbers of legs, different body sizes, miniaturisation and simplification of body plans and different ratios of inertial and damping forces. The article's focus is on continuous legged locomotion, but related ecological and behavioural aspects are also taken into account.

Nuclear power generation became popular in the 1950s in industrialised countries as an alternative to fossil energy sources to provide large amounts of low cost, low carbon energy. Currently 6% of the world’s energy supply is produced in 451 nuclear reactors across 30 countries. However, nuclear power generation has a serious disadvantage and hidden cost: the accumulation and disposal of spent fuel or high level nuclear waste (HLW) - notably highly radioactive nuclear fission products and the absence of suitable long-term storage solutions, threatening livestock and the environment. Sustainable disposal of HLW holds many challenges: fluid and heat transfer may induce strongly coupled undesirable thermal, hydrological, mechanical and chemical processes.A crystalline rock repository construction license has been accomplished by Finland in 2015 for the first long-term HLW repository worldwide. In Germany, a consortium of federal offices is exploring the opportunity of establishing a long-term underground repository in crystalline rock for HLW as an alternative to potential repositories in salt rock and mudrock.The aim of this research is to de-risk hypothetical storage solutions for long-term HLW repositories in Germany in crystalline rock. As no geological site must be alluded to for legal reasons during the repository site investigation process at the time being, flow is modelled for a generic fractured rock site based on academic studies of crystalline rock. An inverse problem approach is applied to investigate hydraulic site requirements for the long-term storage of HLW and provide footing for the analysis of coupled thermal, hydrological, mechanical and chemical processes. This work demonstrates progress towards finding a long-term storage solution for HLW in Germany through evaluating hydrological processes in a generic crystalline rock site. Through Oda analysis and simulating steady-state flow and particle tracking in a synthetic discrete fracture network (DFN), degrees of fracture connectivity and hydraulic conductivity of fractures have been identified for the hydraulic (boundary) conditions in a repository in crystalline rock.

Summary Bacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5‐based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX‐based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine‐1‐carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putida KT2440 as well as the use of β‐galactosidase‐encoding lacZ as a reporter of production levels., A detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’ is provided: We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in Pseudomonas putida. Cloning and activation of the phenazine‐1‐carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putida KT2440 and the use of β‐galactosidase‐encoding lacZ as a reporter of production levels is demonstrated as an application.

European population history has been shaped by migrations of people, and their subsequent admixture. Recently, ancient DNA has brought new insights into European migration events linked to the advent of agriculture, and possibly to the spread of Indo-European languages. However, little is known about the ancient population history of north-eastern Europe, in particular about populations speaking Uralic languages, such as Finns and Saami. Here we analyse ancient genomic data from 11 individuals from Finland and north-western Russia. We show that the genetic makeup of northern Europe was shaped by migrations from Siberia that began at least 3500 years ago. This Siberian ancestry was subsequently admixed into many modern populations in the region, particularly into populations speaking Uralic languages today. Additionally, we show that ancestors of modern Saami inhabited a larger territory during the Iron Age, which adds to the historical and linguistic information about the population history of Finland., Populations from North-eastern Europe, in particular those speaking Uralic languages, carry additional ancestry in similarity with modern East Asian populations. Here, the authors analyse ancient genomic data from 11 individuals from Finland and Northwest Russia, and identify genomic signals of migrations from Siberia that began at least 3500 years ago.

Target-capture approach has improved over the past years, proving to be very efficient tool for selectively sequencing genetic regions of interest. These methods have also allowed the use of noninvasive samples such as faeces (characterized by their low quantity and quality of endogenous DNA) to be used in conservation genomic, evolution and population genetic studies. Here we aim to test different protocols and strategies for exome capture using the Roche SeqCap EZ Developer kit (57.5 Mb). First, we captured a complex pool of DNA libraries. Second, we assessed the influence of using more than one faecal sample, extract and/or library from the same individual, to evaluate its effect on the molecular complexity of the experiment. We validated our experiments with 18 chimpanzee faecal samples collected from two field sites as a part of the Pan African Programme: The Cultured Chimpanzee. Those two field sites are in Kibale National Park, Uganda (N = 9) and Loango National Park, Gabon (N = 9). We demonstrate that at least 16 libraries can be pooled, target enriched through hybridization, and sequenced allowing for the genotyping of 951,949 exome markers for population genetic analyses. Further, we observe that molecule richness, and thus, data acquisition, increase when using multiple libraries from the same extract or multiple extracts from the same sample. Finally, repeated captures significantly decrease the proportion of off-target reads from 34.15% after one capture round to 7.83% after two capture rounds, supporting our conclusion that two rounds of target enrichment are advisable when using complex faecal samples. JH-R is supported by the Ministerio de Economía y Competitividad, Spain (FPI grant BES-2013-064333). This work was supported by the Ministerio de Economía y Competitividad, Spain, and Fondo Europeo de Desarollo Regional (FEDER) (SAF2012-35025 and SAF2015-68472-C2-2-R to FC). The collection of faecal samples was supported by the Max Planck Society Innovation Fund and the Heinz L. Krekeler Foundation's generous funding for the Pan African Programme: The Cultured Chimpanzee. We thank the Agence Nationale des Parcs Nationaux and the Centre National de la Recherche Scientifique (CENAREST) in Gabon and the Uganda National Council for Science and Technology (UNCST) and Ugandan Wildlife Authority (UWA) for their support and permission to collect and export samples from their respective nations. AN is funded by MINECO BFU2015-68649-P (FEDER). TM-B is supported by MINECO BFU2014-55090-P (FEDER), U01 MH106874 grant, Howard Hughes International Early Career, Fundació Zoo de Barcelona and Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya.

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.

The current society requires solutions for many problems in safety, economy, and health. The social concerns on the high rate of repetitive strain injury, work-related osteomuscular disturbances, and domestic issues involving the elderly and handicapped are some examples. Therefore, studies on complex machines with structures similar to humans, known as humanoids robots, as well as emerging optimization metaheuristics have been increasing. The combination of these technologies may result in robust, safe, reliable, and flexible machines that can substitute humans in multiple tasks. In order to contribute to this topic, the static modeling of a humanoid robot and the optimization of its static force capability through a modified self-adaptive differential evolution (MSaDE) approach is proposed and evaluated in this study. Unlike the original SaDE, MSaDE employs a new combination of strategies and an adaptive scaling factor mechanism. In order to verify the effectiveness of the proposed MSaDE, a series of controlled experiments are performed. Moreover, some statistical tests are applied, an analysis of the results is carried out, and a comparative study of the MSaDE performance with other metaheuristics is presented. The results show that the proposed MSaDE is robust, and its performance is better than other powerful algorithms in the literature when applied to a humanoid robot model for the pushing and pulling tasks.

Taking into account the effect of climatic changes over the last few millions of years on fauna both worldwide and in eastern Africa, hardly anyone would challenge the ecosystem shift addressed recently by Faith et al. (1). In eastern Africa, diminishing precipitation triggered the expansion of C4 grassland; reduced net production and the appearance of large grazers increased the relative impact of megaherbivore consumption, eventually facilitating and stabilizing the development of more open landscapes. Whereas some of the emerging grazing genera had already preadapted their masticatory apparatus, others, like proboscides, did not evolve highly hypsodont grazing teeth until the late Pleistocene period (2). The switch to grass-dominated diets … [↵][1]1Email: tom.weihmann{at}uni-koeln.de. [1]: #xref-corresp-1-1

Cockroaches are usually typical omnivorous detritivores and their cephalic morphology is considered to be ancestral in various aspects. Thus, several studies addressed the morphology and function of the blattodean head, and the cockroach usually serves as a model for standard mouthparts in text books. However, so far only two of the three major lineages of Blattodea have been studied and no detailed information for the head of any Corydioidea was available. The present study closes this gap by providing a detailed morphological description of the head ofThe cephalic morphology ofThe head capsule of

This paper presents the design and kinematic analysis of a 6-DOF foldable/deployable Delta-like parallel manipulator, with a spherical wrist, for a lightweight Intervention Autonomous Underwater Vehicle (I-AUV). To the best of the authors knowledge, this is the first paper to propose the application of a parallel manipulator for underwater vehicles. Most of all underwater manipulators used nowadays are serial. However, parallel manipulators present some interesting features when compared to the serial ones. Lightweight underwater vehicle-manipulator system (UVMS) suffers from disturbances due to several underwater effects, among them, the drag caused by the manipulator motion. When applied to terrestrial and aerial mobile manipulation, parallel manipulators can quickly compensate some disturbances thanks to their geometry (thin links, drives located at the base) and fast motion. Based on these applications and considering that the drag effect can be minimised with a parallel manipulator, this paper proposes a Delta with an optimised geometry, which can be folded for transportation and navigation and deployed for intervention.

Scientific reports 9(1), 7028 (2019). doi:10.1038/s41598-019-43405-1, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

To avoid pathogen-associated molecular pattern recognition, the hemibiotrophic maize pathogen Colletotrichum graminicola secretes proteins mediating the establishment of biotrophy. Targeted deletion of 26 individual candidate genes and seven gene clusters comprising 32 genes of C. graminicola identified a pathogenicity cluster (CLU5) of five co-linear genes, all of which, with the exception of CLU5b, encode secreted proteins. Targeted deletion of all genes of CLU5 revealed that CLU5a and CLU5d are required for full appressorial penetration competence, with virulence deficiencies independent of the host genotype and organ inoculated. Cytorrhysis experiments and microscopy showed that Delta clu5a mutants form pressurized appressoria, but they are hampered in forming penetration pores and fail to differentiate a penetration peg. Whereas Delta clu5d mutants elicited WT-like papillae, albeit at increased frequencies, papillae induced by Delta clu5a mutants were much smaller than those elicited by the WT. Synteny of CLU5 is not only conserved in Colletotrichum spp. but also in additional species of Sordariomycetes including insect pathogens and saprophytes suggesting importance of CLU5 for fungal biology. Since CLU5a and CLU5d also occur in non-pathogenic fungi and since they are expressed prior to plant invasion and even in vegetative hyphae, the encoded proteins probably do not act primarily as effectors.

The morphology, musculature, and function of the feeding apparatus of cockroaches is described in detail and compared with other insects with biting and chewing mouthparts. The mouthparts of cockroaches represent, in most cases, the ancestral condition for winged and neopteran insects. Their head capsule is flattened in a posterior-anterior direction and very similar among the studied species. The right mandible is very constant in shape, while the number of distal incisivi in left mandibles varies among species. With the exception of Tivia sp. (Corydiidae), primary mandibular adductor of the mandible has eight distinct compartments in all studied roaches, for which functional cross section and volume are provided. In all these specimens, the left adductor is smaller than the right one. Bite forces and muscle properties are discussed for Periplaneta americana. The maxilla, labium, and hypopharynx are also highly similar among cockroaches and close to the pterygotan ground plan. The same also applies to the associated musculature for which we also provide functional and kinematic considerations. Cockroaches salivate food outside the mouth cavity before cutting it with the mandibles. The maxillae transport food into the cibarium where the hypopharynx is involved in transporting it between the grinding mandibular molae. The crushed food is sucked into the pharynx via dilation. During the feeding process, most mouthparts exhibit highly concerted activities. This process generally follows the ground pattern for insects with biting and chewing mouthparts, although some salivation processes may differ.

We investigate gcf-Petri nets, a generalization of communication-free Petri nets allowing arbitrary edge multiplicities, and characterized by the sole restriction that each transition has at most one incoming edge. We use canonical firing sequences with nice properties for gcf-PNs to show that the RecLFS, (zero-)reachability, covering, and boundedness problems of gcf-PNs are in PSPACE. By showing, how PSPACE-Turing machines can be simulated by gss-PNs, a subclass of gcf-PNs where additionally all transitions have at most one outgoing edge, we ultimately prove the PSPACE-completess of these problems for gss/gcf-PNs. Last, we show PSPACE-hardness as well as a doubly exponential space bound for the containment and equivalence problems of gss/gcf-PNs.

Balancing selection is an important evolutionary force that maintains genetic and phenotypic diversity in populations. Most studies in humans have focused on long-standing balancing selection, which persists over long periods of time and is generally shared across populations. But balanced polymorphisms can also promote fast adaptation, especially when the environment changes. To better understand the role of previously balanced alleles in novel adaptations, we analyzed in detail four loci as case examples of this mechanism. These loci show hallmark signatures of long-term balancing selection in African populations, but not in Eurasian populations. The disparity between populations is due to changes in allele frequencies, with intermediate frequency alleles in Africans (likely due to balancing selection) segregating instead at low- or high-derived allele frequency in Eurasia. We explicitly tested the support for different evolutionary models with an approximate Bayesian computation approach and show that the patterns in PKDREJ, SDR39U1, and ZNF473 are best explained by recent changes in selective pressure in certain populations. Specifically, we infer that alleles previously under long-term balancing selection, or alleles linked to them, were recently targeted by positive selection in Eurasian populations. Balancing selection thus likely served as a source of functional alleles that mediated subsequent adaptations to novel environments.

Autonomous underwater vehicles (AUVs) operate with limited external support and underwater missions usually have long or continuous duration, taking place in dynamic environments, which makes the creation of resilient, fault-tolerant AUVs a necessity. This paper briefly reviews some fault-tolerant techniques and presents the design of a fault-tolerant hardware architecture for AUVs and its application on a bench test prototype. The main fault tolerant techniques applied to the proposed architecture are hardware redundancy, which apply hybrid strategy, and software redundancy, which apply detection, contention, recovery and multiple version.

When leg number increases, COM oscillations decline more quickly with increasing deviations from alternating leg coordination., The examination of gaits and gait changes has been the focus of movement physiology and legged robot engineering since the first emergence of the fields. While most examinations have focused on bipedal and quadrupedal designs, many robotic implementations rely on the higher static stability of three or more pairs of legs. Thus far, however, the effect of number of pairs of legs on locomotion dynamics has not been examined. Accordingly, the present approach aims to extend available theory to polypedal designs and examines how the number of active walking legs affects body dynamics when combined with changing duty factors and phase relations. The model shows that ground force interference of higher numbers of active pairs of walking legs can prevent effective use of bouncing gaits, such as trot, and their associated advantages, such as energy efficiency, because significantly higher degrees of leg synchronization are required. It also shows that small changes in the leg coordination pattern have a much higher impact on the center-of-mass dynamics in locomotor systems with many legs than in those with fewer legs. In this way, the model reveals coordinative constraints for specific gaits facilitating the assessment of animal locomotion and economization of robotic locomotion.

European history has been shaped by migrations of people, and their subsequent admixture. Recently, evidence from ancient DNA has brought new insights into migration events that could be linked to the advent of agriculture, and possibly to the spread of Indo-European languages. However, little is known so far about the ancient population history of north-eastern Europe, in particular about populations speaking Uralic languages, such as Finns and Saami. Here we analyse ancient genomic data from 11 individuals from Finland and Northwest Russia. We show that the specific genetic makeup of northern Europe traces back to migrations from Siberia that began at least 3,500 years ago. This ancestry was subsequently admixed into many modern populations in the region, in particular populations speaking Uralic languages today. In addition, we show that ancestors of modern Saami inhabited a larger territory during the Iron Age than today, which adds to historical and linguistic evidence for the population history of Finland.

The examination of gaits and gait-changes have been the focus of movement physiology and legged robot engineering since the first emergence of the fields. While most examinations focussed on bipedal and quadrupedal designs many robotic implementations rely on the higher static stability of three or more pairs of legs. Nevertheless, examinations of gait-changes in the biological models, i.e. poly-pedal arthropods such as insects or arachnids, are rare. Except for the well-known change from slow feedback controlled walking to a fast, feedforward controlled running gait, no changes are known or are deemed to be of low significance.However, recent studies in fast moving spiders, mites and cockroaches have revealed an additional gait change also for the transition from intermediate to high running speeds. This change is similar to gait transitions as found in quadrupedal vertebrates.Accordingly, the present approach aims to extend available theory to poly-pedal designs and examines how the number of active walking legs affects body dynamics when combined with changing duty factors and phase relations. The model shows that higher numbers of active leg pairs can prevent effective use of bouncing gaits such as trot and their associated advantages because significantly higher degrees of leg synchronisation are required. It also shows that small changes in the leg coordination pattern have a much higher impact onto the COM dynamics than in locomotor systems with fewer legs. In this way, the model reveals coordinative constraints for specific gaits facilitating the assessment of animal locomotion and economization of robotic locomotion.Significance StatementThe present model approach enables to assess the impact of different numbers of walking legs onto movement dynamics and gait choice in terrestrial legged locomotion. The model’s results are indicatory for research in legged locomotion regardless whether biological examples or legged walking machines are considered. The approach is suitable for all numbers of pairs of walking legs larger than two and is focussed on symmetrical gaits as found in straight and continuous locomotion. The model fills a gaping gap as the impact of phase shifts among the legs in the coordinated sets of legs typical for poly-pedal animals and robots on overall body dynamics are not considered sufficiently in existing dynamic model approaches.

Modern computer-aided techniques foster the availability and quality of 3D visualization and reconstruction of extinct and extant species. Moreover, animated sequences of locomotion and other movements find their way into motion pictures and documentary films, but also gain attraction in science. While movement analysis is well advanced in vertebrates, particularly in mammals and birds, analyses in arthropods, with their much higher variability regarding general anatomy and size, are still in their infancies and restricted to a few laboratory species. These restrictions and deficient understanding of terrestrial arthropod locomotion in general impedes sensible reconstruction of movements in those species that are not directly observable (e.g., extinct and cryptic species). Since shortcomings like over-simplified approaches to simulate arthropod locomotion became obvious recently, in this review we provide insight into physical, morphological, physiological, behavioral, and ecological constraints, which are essential for sensible reconstructions of terrestrial arthropod locomotion. Such concerted consideration along with sensible evaluations of stability and efficiency requirements can pave the way to realistic assessment of leg coordination and body dynamics.

The purpose of offshore support vessels (OSVs) is to support the oil industry in many sea activities, such as supply, anchor handling, towing, and construction. For a proper operation, a vessel requires different installed capabilities on board, and the availability and capacity of these capabilities are directly connected to the operational level of the vessel. In this work, a parametric model of an OSV is developed, taking into account the vessel capabilities and its connection to the main operations that these vessels can perform. Designs are ranked according to their operability score and capital cost. The model consists of parametric equations based on regression analysis from similar vessels and a preliminary configuration-based approach for specific modules, such as cranes, extra accommodation, and larger propulsion. The model takes into account different contexts in which the vessel will operate (e.g., North Sea, Arctic, and Brazil) for the scenario generation.

We investigate several computational problems of communication-free Petri nets, and develop very efficient mostly linear time algorithms for different variations of the boundedness and liveness problems of cf-PNs. For several more complex notions of boundedness, as well as for the covering problem, we show NP-completeness. In the last part, we use our results for cf-PNs to give linear time algorithms for related problems of context-free commutative grammars, and, in turn, use known results for such grammars to give a coNEXPTIME-upper bound for the equivalence problem of cf-PNs.

Background Many legged animals change gaits when increasing speed. In insects, only one gait change has been documented so far, from slow walking to fast running, which is characterised by an alternating tripod. Studies on some fast-running insects suggested a further gait change at higher running speeds. Apart from speed, insect gaits and leg co-ordination have been shown to be influenced by substrate properties, but the detailed effects of speed and substrate on gait changes are still unclear. Here we investigate high-speed locomotion and gait changes of the cockroach Nauphoeta cinerea, on two substrates of different slipperiness. Results Analyses of leg co-ordination and body oscillations for straight and steady escape runs revealed that at high speeds, blaberid cockroaches changed from an alternating tripod to a rather metachronal gait, which to our knowledge, has not been described before for terrestrial arthropods. Despite low duty factors, this new gait is characterised by low vertical amplitudes of the centre of mass (COM), low vertical accelerations and presumably reduced total vertical peak forces. However, lateral amplitudes and accelerations were higher in the faster gait with reduced leg synchronisation than in the tripod gait with distinct leg synchronisation. Conclusions Temporally distributed leg force application as resulting from metachronal leg coordination at high running speeds may be particularly useful in animals with limited capabilities for elastic energy storage within the legs, as energy efficiency can be increased without the need for elasticity in the legs. It may also facilitate locomotion on slippery surfaces, which usually reduce leg force transmission to the ground. Moreover, increased temporal overlap of the stance phases of the legs likely improves locomotion control, which might result in a higher dynamic stability. Electronic supplementary material The online version of this article (10.1186/s12983-017-0232-y) contains supplementary material, which is available to authorized users.

RESUMO Neste trabalho, apresentamos como os conceitos de liberdade e abertura do conhecimento estao sendo integrados e postos em pratica nos principios e projetos do Centro de Tecnologia Academica do Instituto de Fisica e no Colegio de Aplicacao, ambos da UFRGS. Apontamos o desenvolvimento colaborativo de hardware aberto e livre como o desafio atual para a integracao e consolidacao da cultura livre. Descrevemos como os metodos e principios do software livre, educacao aberta e da ciencia aberta podem ser integradas para a formacao de equipes autogeridas de desenvolvimento de projetos de hardware abertos e livres (HAL), que servem como plataformas para ensino de ciencias e engenharias, embasadas no conceito de hiperobjetos. Palavras-chave: Conhecimento Livre; Ciencia Cidada; Recursos Educacionais Abertos; Software Livre. ABSTRACT In this article we discuss how the concepts of freedom and openness of knowledge are being integrated and adopted in the principles and projects of the Academic Technology Center of the Institute of Physics and of the College of Application, both in the Federal University of Rio Grande do Sul - UFRGS. We point to the collaborative development of Open and Free Hardware as the current challenge for the integration and consolidation of a culture of freedom. We describe how the methods and principles of free software, open education and open science can be integrated into the formation of self-managed Open and Free Hardware project development teams while also serving as educational platforms for teaching sciences and engineering, based on the concept of the hiperobject. Keywords: Free Knowledge; Citizen Science; Open Educational Resources; Free Software.

In den Geowissenschaften gehört es zum Standard, internationale Netzwerke aufzubauen, um Zugang zu bestimmten Regionen zu erhalten oder auf lokales Wissen zuzugreifen. Die DFG-Arbeitsgruppe „Geowissenschaftlicher Nachwuchs“ hat Netzwerkbildung und Internationalisierung im Rahmen eines Rundgespräches analysiert. Ein berufliches Netzwerk ist Grundbest andteil wissenschaftlichen Arbeitens. Es erleichtert Kenntnisse über den state of the art und erlaubt, Ergebnisse zu erzielen, die Einzelkämpfer nicht erreichen. Drittmittelgeber fordern häufig die Etablierung oder Nutzung internationaler Kooperationen. Aktiver wissenschaftlicher Austausch, Einladungen zu Vorträgen oder Fachbeiträgen können als Form des Netzwerkens die eigene Sichtbarkeit erhöhen und Chancen auf dem Bewerbungsmarkt verbessern.

COM kinematics over second legs’ strides for alternating tripodal runs on non-slippery substrate. A stride consists of a contact phase and the subsequent swing phase. The black solid line shows the median course of a value and the grey shaded area the interquartile range. First column: fore-aft direction (X); Second column: lateral direction (Y); Third column: vertical direction (Z). First row: distance in m; Second row: velocity in ms−1; Third row: acceleration in ms−2. Figure S2. COM kinematics over second legs’ strides for metachronal runs on non-slippery substrate. A stride consists of a contact phase and the subsequent swing phase. The black solid line shows the median course of a value and the grey shaded area the interquartile range. First column: fore-aft direction (X); Second column: lateral direction (Y); Third column: vertical direction (Z). First row: distance in m; Second row: velocity in ms−1; Third row: acceleration in ms−2. Figure S3. COM kinematics over second legs’ strides for alternating tripodal runs on slippery substrate. A stride consists of a contact phase and the subsequent swing phase. The black solid line shows the median course of a value and the grey shaded area the interquartile range. First column: fore-aft direction (X); Second column: lateral direction (Y); Third column: vertical direction (Z). First row: distance in m; Second row: velocity in ms−1; Third row: acceleration in ms−2. Figure S4. COM kinematics over second legs’ strides for metachronal runs on slippery substrate. A stride consists of a contact phase and the subsequent swing phase. The black solid line shows the median course of a value and the grey shaded area the interquartile range. First column: fore-aft direction (X); Second column: lateral direction (Y); Third column: vertical direction (Z). First row: distance in m; Second row: velocity in ms−1; Third row: acceleration in ms−2. Figure S5. The courses of pitch, yaw and ∠TA over the second legs’ strides. The black solid line shows the median course of an angle and the grey shaded area the interquartile range. Upper row: Alternating tripodal runs on non-slippery substrate; Second row: Metachronal runs on non-slippery substrate; Third row: Alternating tripodal runs on slippery substrate; Fourth row: Metachronal runs on slippery substrate. Figure S6. Stride frequencies, duty factors, swing rates and contact rates plotted against running speed for all walking legs and slippery conditions. First row: Stride frequency (f T ); Second row: duty factors (β); Third row: swing rates (t S −1 ); Fourth row: contact rates (t C −1 ). Red circles are measured values for runs with metachronal leg coordination while black crosses depict values from alternating tripodal runs. Figure S7. Phase shifts between ipsilateral legs and between the contralateral rear legs during alternating tripodal (white) and metachronal (red) runs on non-slippery (left) and slippery substrate. A, E) Phase values for the touch-downs of the fore legs in the stride period of the rear legs. B, F) Phase values for the touch-downs of the middle legs in the stride period of the rear legs. C, G) Phase values for the touch-downs of the fore legs in the stride period of the middle legs. D, H) Phase values for the touch-downs of the contralateral rear legs. Figure S8. Stride durations, swing durations and contact durations plotted against running speed for all walking legs. Stride duration: row one and four; contact duration: row two and five; swing duration: row three and six. The rows one to three refer to non-slippery conditions whereas the rows four to six refer to slippery conditions. Red circles are measured values for runs with metachronal leg coordination while black crosses depict values from alternating tripodal runs. Black (alternating runs) and red (metachronal) lines in the upper three rows were calculated on the basis of the linear least squares regressions for t S −1 and t C −1 (see Fig. 4). (PDF 2640 kb)

We unify and generalize several inequalities for the number w"k of walks of length k in graphs, and for the entry sum of matrix powers. First, we present a weighted sandwich theorem for Hermitian matrices which generalizes a matrix theorem by Marcus and Newman and which further generalizes our former unification of inequalities for the number of walks in undirected graphs by Lagarias et al. and by Dress and Gutman. The new inequality uses an arbitrary nonnegative weighting of the indices (vertices) which allows to apply the theorem to index (vertex) subsets (i.e., inequalities considering the number w"k(S,S) of walks of length k that start at a vertex of a given vertex subset S and that end within the same subset). We also deduce a stronger variation of the sandwich theorem for the case of positive-semidefinite Hermitian matrices which generalizes another inequality of Marcus and Newman. Further, we show a similar theorem for nonnegative symmetric matrices which is another unification and generalization of inequalities for walk numbers by Erdos and Simonovits, by Dress and Gutman, and by Ilic and Stevanovic. In the last part, we generalize lower bounds for the spectral radius of adjacency matrices and upper bounds for the energy of graphs.

We investigated intraspecific aggression in experimental nests (expN1, expN2) of the giant honey bee Apis dorsata in Chitwan (Nepal), focusing on interactions between surface bees and two other groups of bees approaching the nest: (1) homing “nestmate” foragers landing on the bee curtain remained unmolested by guards; and (2) supposed “non-nestmate” bees, which were identified by their erratic flight patterns in front of the nest, such as hovering or sideways scanning and splaying their legs from their body, and were promptly attacked by the surface bees after landing. These supposed non-nestmate bees only occurred immediately before and after migration swarms, which had arrived in close vicinity (and were most likely scouting for a nesting site). In total, 231 of the “nestmate” foragers (fb) and 102 approaches of such purported “non-nestmate” scouts (sc) were analysed (total observation time expN1: 5.43 min) regarding the evocation of shimmering waves (sh). During their landing the “nestmate” foragers provoked less shimmering waves (relnsh[fb] = 23/231 = 0.0996, relnsh[sc] = 75/102 = 0.7353; p 18 min) quite similarly to the defensive response against predatory wasps. Hence, the surface members of settled colonies respond differently to individual giant honey bees approaching the nest, depending on whether erratic flight patterns are displayed or not.

The open nesting behaviour of giant honeybees (Apis dorsata) accounts for the evolution of a series of defence strategies to protect the colonies from predation. In particular, the concerted action of shimmering behaviour is known to effectively confuse and repel predators. In shimmering, bees on the nest surface flip their abdomens in a highly coordinated manner to generate Mexican wave-like patterns. The paper documents a further-going capacity of this kind of collective defence: the visual patterns of shimmering waves align regarding their directional characteristics with the projected flight manoeuvres of the wasps when preying in front of the bees’ nest. The honeybees take here advantage of a threefold asymmetry intrinsic to the prey–predator interaction: (a) the visual patterns of shimmering turn faster than the wasps on their flight path, (b) they “follow” the wasps more persistently (up to 100 ms) than the wasps “follow” the shimmering patterns (up to 40 ms) and (c) the shimmering patterns align with the wasps’ flight in all directions at the same strength, whereas the wasps have some preference for horizontal correspondence. The findings give evidence that shimmering honeybees utilize directional alignment to enforce their repelling power against preying wasps. This phenomenon can be identified as predator driving which is generally associated with mobbing behaviour (particularly known in selfish herds of vertebrate species), which is, until now, not reported in insects. Electronic supplementary material The online version of this article (doi:10.1007/s00114-014-1220-0) contains supplementary material, which is available to authorized users.

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.

Differentiation of effective and ineffective papillae formed by maize in response to attempted penetration by the C. graminicola wild type reference. Bright field microscopy, serial focussing allowed to follow fungal morphogenesis. Magnification is 600x. Bars represent 50 μm. (PPTX 28444 kb)

Specificity of qPCR. Primers employed were (A) Cg_ITS2-F1.1/Cg_ITS2-R1 and (B) M13new-For/M13new-Rev. Conventional PCR (35 cycles) using as template 10 ng of gDNA of C. graminicola (lane 1) and Zea mays (lane 2), 50 pg of pUC18 (lane3), 10 ng of each gDNA of C. graminicola and Z. mays (lane 4), 10 ng of each gDNA of C. graminicola and Z. mays plus 50 pg of pUC18 (lane 5), and water as a non-template control (lane 6). A low molecular weight ladder was included in lane 7 on a 1.5 % (w/v) Sodium borate-Agarose gel. (PPTX 314 kb)

Construction of a C. graminicola strain constitutively expressing eGFP. Graphical representation of the genomic region from base pair 275,656 to base pair 295830, containing annotated genes GLRG_03629 to GLRG_03636 on supercontig 11 where the eGFP expression cassette was inserted. Annotations are those provided by the Broad Institute (Broad) or based on NCBI protein Blast best hits (BlastP) for genes annotated as hypothetical by the Broad Institute. Transcript abundances in appressoria (AP), during the biotrophic phase of fungal development (BP) and during the necrotrophic phase (NP) were taken from [4]. The insert combines an eGFP expression unit consisting of the P. tritici-repentis ToxB promoter (PtoxB), the eGFP coding sequence and the Agrobacterium tumefaciens nos terminator (Tnos) with a geneticin/G418/neomycin resistance unit consisting of the promoter and terminator of Aspergillus nidulans trpC flanking the transposon Tn5 nptII gene. Flanking sequences homologous to the intergenic region between GLRG_03632 and GLRG_03633 allow targeted integration of the construct. (PPTX 77Â kb)

Primers used. Primers used for the construction of the eGFP expression cassette and the qPCR assay for fungal biomass. Uppercase: target sequence-specific. Bold: BsaI recognition sequence. Underlined: BsaI cleavage site, yielding a 5'-overhang. Italics: XhoI cleavage site for generation of pBR322-Bsa from pBR322. *: Two site-directed mutagenic bases (lowercase) in the target-specific sequence for removal of plasmid-borne BsaI recognition sequence. (DOCX 16Â kb)

In Giant Honey Bees, abdomen flipping happens in a variety of contexts. It can be either synchronous or cascaded, such as in the collective defense traits of shimmering and rearing-up, or it can happen as single-agent behavior. Abdomen flipping is also involved in flickering behavior, which occurs regularly under quiescent colony state displaying singular or collective traits, with stochastic, and (semi-) synchronized properties. It presumably acts via visual, mechanoceptive, and pheromonal pathways and its goals are still unknown. This study questions whether flickering is preliminary to shimmering which is subject of the fs (flickering-shimmering)-transition hypothesis? We tested the respective prediction that trigger sites (ts) at the nest surface (where shimmering waves had been generated) show higher flickering activity than the alternative non-trigger sites (nts). We measured the flickering activity of ts- and nts-surface bees from two experimental nests, before and after the colony had been aroused by a dummy wasp. Arousal increased rate and intensity of the flickering activity of both ts- and nts cohorts (P < 0.05), whereby the flickering intensity of ts-bees were higher than that of nts-bees (P < 0.05). Under arousal, the colonies also increased the number of flickering-active ts- and nts-cohorts (P < 0.05). This provides evidence that cohorts which are specialist at launching shimmering waves are found across the quiescent nest zone. It also proves that arousal may reinforce the responsiveness of quiescent curtain bees for participating in shimmering, practically by recruiting additional trigger site bees for expanding repetition of rate and intensity of shimmering waves. This finding confirms the fs-transition hypothesis and constitutes evidence that flickering is part of a basal colony-intrinsic information system. Furthermore, the findings disprove that the muscle activity associated with flickering would heat up the surface bees. Hence, surface bees are not actively contributing to thermoregulation.