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Results suggest a reduction in immune gene repertoires in termites and possible complementary expression between termite castes. With comparative genomics we will investigate the evolution of gene families related to immunity, try to understand where reductions and expansions take place and relate these changes to shifts in sociality and ecology. The role of TEs in expansions and contractions of immune gene families will be investigated. For these analyses, we propose to generate high quality, highly contiguous genomes of species from different levels of sociality, covering all major termite families. With comparative transcriptomics we will investigate the expression of immune genes in different castes. Via network analyses we will identify pathways indicated in differential immunity between castes and between species of different sociality levels. We will investigate how these pathways have been rewired along the transitions to higher levels of sociality and how, intra-specifically, they change between castes.
Social insects nesting in soil environments are in constant contact with entomopathogens and have evolved disease resistance mechanisms within a colony to prevent the occurrence and spread of infectious diseases. Among these mechanisms: mutual grooming reduces the cuticular load of pathogens, and burial of cadavers and cannibalism can prevent pathogens from replicating within the group. We explored how the rate and type of collective behavioural response is determined by stepwise infection dynamics operating at the level of the individual. Specifically, we infected the eastern subterranean termite Reticulitermes flavipes with different types of infectious particle and infection route of the entomopathogenic fungus Metarhizium anisopliae and recorded behavioural responses of nestmates to individuals at different times during the progression of infections. As expected, termites groomed conidia-exposed individuals significantly more than controls. Interestingly, grooming was significantly elevated after fungal germination than before, suggesting that pathogen growth cues act as strong stimulators of allogrooming. Conidia-exposed termites were cannibalized, but only after they became visibly ill. By contrast, termites did not groom blastospore-injected individuals more than controls at any time-point following infection. Instead, we found that blastospore-injected individuals were continually cannibalized at a low-level following injection with either viable or heat-killed blastospores, with a marked increase in cannibalism after termites injected with viable blastospores became visibly ill and were close to death. Together, these findings point to the importance of host condition as a cue for social hygienic behavior, and that the host itself appears to emit essential sickness cues that act as targets for its own sacrifice. This demonstrates that termites have independently evolved to both identify and destructively respond to sickness.
The termites are a derived eusocial lineage of otherwise non-social cockroaches. Understanding the proximal and ultimate drivers of this major evolutionary transition represents an important goal in biology. One outstanding question concerns the evolution of termite immunity, which is thought to have undergone broad-sweeping adaptations in order to enhance group-level immune protection. To understand the evolutionary origins of termite immunity, we conducted qualitative and quantitative transcriptome analyses along a gradient of sociality. Firstly, we aimed to identify large-scale genetic shifts in immune traits linked to eusociality by comparing immune gene repertoires in solitary and subsocial cockroaches and across a range of eusocial termite lineages. Secondly, we compared the responses of a solitary cockroach, a subsocial wood-roach and different castes of a lower termite species to a non-specific immune challenge, in order to understand how sociality may have influenced the evolution of immune gene regulation. Firstly, we found that termites have a broadly representative repertoire of canonical immune genes as compared to gregarious cockroaches and subsocial wood-roaches. Secondly, with respect to immune challenge, the solitary cockroach and the subsocial wood-roach displayed a similarly comprehensive induced response, while the termite response was considerably dampened by comparison and strongly influenced by caste; with reproductives displaying a generally higher constitutive level of immune-gene expression compared to sterile castes. In summary we did not find termite eusociality to be associated with significant changes in immune gene diversity, but rather to be linked with significant modifications to the regulation of immunity following the origin of division of labour.
Black yeasts form a polyphyletic group of Ascomycota that colonize bare surfaces like rocks, facades of buildings, and solar panels. Their protective adaptations enable an adequate response to fluctuating and diverse temperature, water and UV radiation stresses. Together with bacteria and algae they form sub-aerial biofilms (SAB) this way discoloring and weathering the surfaces they grow on. Strain A95 of Knufia petricola (Eurotiomycetes, Chaetothyriales) displays both typical yeast-like cell growth and constitutive dihydroxynaphthalene (DHN) melanogenesis. Along with the cyanobacterium Nostoc punctiforme as photobiont, it is already used in a model system for studying SAB formation and bio-weathering. Applying the recently developed tools for the generation of deletion mutants will allow to define gene functions and to identify genes critical for abiotic and biotic interactions. We present a chromosome-level genome assembly and annotation for K. petricola A95. The genome was assembled with MaSuRCA using a hybrid assembly approach of Illumina MiSeq and PacBio SMRT sequencing data. The resulting assembly consists of 17 contigs including the complete mitochondrial genome and five complete chromosomes. It shows indication of repeat-induced point mutations (RIP). Supported by RNA sequencing data from eight different growth conditions, 10,994 genes were predicted with the BRAKER2 pipeline. Functional annotation of genes was obtained from general functional annotation databases and the fungal specific database FungiPath. Comparative analyses are in progress to identify genes specific to black yeasts, that may facilitate the survival on exposed surfaces. In sum, the genome sequence of K. petricola is a valuable resource to gain insight into the protein inventory and functional pathways of extremotolerant and symbiosis-capable fungi.
Black microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in hostile desert environments. In these niches, MCF have evolved mineral-weathering and symbiotic capabilities as well as mechanisms to cope with multiple stresses such as solar irradiation, temperature extremes and low water activity. Due to their stress tolerance these ascomycetes are prominent in modern terrestrial ecosystems – like man-made material surfaces from roof to solar panels. MCF interactive capabilities support their facultative symbiotic relationships with cyanobacteria and ensure their rock-weathering geochemical activity.
Using the rock-inhabiting fungus K. petricola A95 (Chaetothyriales), we developed transformation protocols and deleted genes responsible for production of the protective pigments melanins and carotenoids. To confirm that the mutant phenotypes were not due to hidden mutations, melanin synthesis was restored by complementing the mutants with the respective wild type genes. Strains of K. petricola carrying gene variants for fluorescent proteins EGFP and DsRed are available. We successfully labelled the cytoplasm, nuclei, peroxisomes and mitochondria. Targeted and ectopic integrations result in stable transformants suitable for further phenotypical characterization. As K. petricola is a non-pathogenic fungus with all characteristic features of MCF, including meristematic growth, melanized cell-walls, extracellular polymeric substances and extensive pigment production, our results will shed light on protective role of pigments during cell wall maturation and oxidative stress defence in rock-inhabiting MCF. Genes involved in environmental sensing or substrate and phototroph interactions are currently targeted. With the help of a mutant collection and fluorescently labelled K. petricola we will be able to investigate interactions of MCF with environmental stressors, mineral substrates, soil matrices and phototrophic symbionts.
A Genetic Toolbox for Exploring the Life Style of the Rock-inhabiting Black Fungus Knufia petricola
(2019)
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish sub-aerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of black yeasts such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis. For this environmental strain we developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence is paving the way for studying interactions of K. petricola and other black yeasts with environmental stressors, material surfaces, soil matrices and phototrophic symbionts.
In arid sun-exposed environments, rock-inhabiting fungi dominate microbial communities on rock surfaces. Fungi are known to enhance rock weathering, although the exact mechanisms are not completely understood. These natural processes can be simulated by reproducible experimental systems. Particularly attractive are genetically amenable rock-weathering fungi, where certain traits can be knocked-out to identify weathering-relevant functions. Here, we studied the effect of the rock-inhabiting fungus Knufia petricola A95 and its melanin-deficient mutant (ΔKppks) on the weathering of a Fe-bearing olivine. We examined the olivine dissolution kinetics and looked at the olivine-fungus interface. For the former we inoculated olivine powder with the fungus in batch and mixed flow reactors and analysed the medium solution by ICP-OES, for the latter biofilm-covered olivine sections from flow-through reactors were analysed by FIB-TEM.
In general olivine dissolution was lower when olivine was incubated without fungi: the abiotic olivine dissolution rates were lowest and the surface of the abiotically reacted olivine sections showed no etching. Various chemical analyses show Fe (oxyhydr)oxide precipitation on top of the abiotically reacted olivine, indicating its role in inhibiting olivine dissolution. Both the wild type (WT) and ΔKppks solubilised and bound significant amounts of Fe released by olivine dissolution. Fe (oxyhydr)oxide precipitation was thus reduced, explaining the enhanced olivine dissolution in their presence. Particularly efficient at preventing Fe precipitation were attached WT cells: the inhibition of olivine dissolution almost completely disappeared. The attachment capacity of the WT is most likely caused by the production of WT-specific extracellular polymeric substances. Our presented experimental systems allowed the precipitation of Fe (oxyhydr)oxides and included a rock-inhabiting fungus and thus simulated weathering processes relevant to natural ecosystems.
Traceable and reliable chemical element analysis of aerosols by X-ray spectrometry was investigated using aerosol samples from field campaigns which have been measured in the GIXRF-beamline at BESSY.
The reference-free XRF approach allows for a traceable analysis of the mass deposition.
Traceable quantification by means of XRF can be transfered to benchtop instrumentation used in the laboratory Chemical and dimensional analysis of deposited aerosol allows for a comprehensive analysis of aerosols, e.g. for toxicity assessment and determination of the source The folowing elements could be identified and quantified in the field samples: Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Y, W, Pb.
Simple particulate matter sensors are gaining popularity due to their low price, easy handling and good temporal resolution. In this presentation, we report on the performance of a Sharp optical PM sensor GP2Y1010AU0F, which costs less than 15 €.
The sensor is built around an infrared emitting diode (ILED) and a phototransistor detecting the light scattered from the aerosol particle. An electronic circuit shapes the detected light in a pulsed signal. The manufacturer advises sampling the output signal 280 microseconds after the ILED pulse. The measured output voltage is an indicator of dust concentration.
We have built two identical simple PM monitoring devices using Raspberry Pi 3 computer interfacing the PM sensor with Microchip’s MCP3002 ADC via SPI. The ADC is capable of more than 100 ksamples/s at 10-bit resolution. The Rpi3 was pulsing the sensor at 10Hz, digitizing and saving the data and sending the results wirelessly.
Sensor’s output pulse shape was sampled with 10 microsecond time steps and saved, thus making offline analysis possible. A time jitter of output pulses can be observed and suggests a peak fitting as a better approach to the signal readout compared to the single sampling at a fixed time after pulse triggering We compared both methods.
Free-living and (ecto)-mycorrhizal fungi enhance rock weathering. In their roles as mineral weathering agents and mutualistic partners of phototrophs, fungi supply primary producers like plants and phototrophic microorganisms with mineral-derived nutrients. The exact mechanisms behind fungus-induced mineral weathering processes are however not well understood. Progress can be achieved here by reproducible experimental simulations of the natural processes, using well-characterised model organisms and minerals.
In this study, the weathering-affecting, rock-inhabiting fungus, Knufia petricola A95 and the Fe-bearing olivine (Fe0.2Mg1.8SiO4) were selected to investigate fungi-induced effects on mineral dissolution. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, that produced more extracellular polymeric substances (EPS) than the wild type (WT), enabled comparative studies of the role of melanin and EPS in weathering processes.
Three experimental systems, which generate long-term microbiological stability, were developed to study the impact of the WT and ΔKppks on olivine weathering: (1) batch and (2) mixed flow dissolution experiments, and (3) biofilm cultivation experiments. In addition, state-of-the-art analytical techniques were used to monitor changes in the growth medium, as well as of the mineral surface and biofilm-mineral interface.
Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis of the Mg, Si and Fe concentrations in the reacted growth medium was used to quantify olivine dissolution. In abiotic controls, Mg and Si dissolved congruently, while Fe precipitated. The measured olivine dissolution rates at pH 6 were two orders of magnitude lower than previously reported, but similar at acidic pH. X-ray photoelectron spectroscopy (XPS) analyses of the olivine surface confirmed the presence of Fe (oxyhydr)oxide precipitates. Transmission electron microscopy (TEM) imaging of an abiotically reacted polished olivine section from the long-term cultivation experiment showed the presence of an amorphous layer enriched in Fe. All these observations indicate that the precipitation of Fe (oxyhydr)oxides on the olivine surface inhibits olivine dissolution.
Both tested rock-inhabiting fungal strains affect Fe precipitation as well as olivine dissolution. Evaluation of the WT and ΔKppks revealed that the WT formed less biomass but could take up higher amounts of metals (e.g. Fe) and was more efficient in its attachment to olivine. The WT and ΔKppks enhanced olivine dissolution as demonstrated by higher Mg and Si concentration in the reacted growth medium. They furthermore prevented Fe precipitation by binding Fe and retaining it in solution, thereby allowing olivine dissolution to proceed. The WT cells that were attached to the olivine surface were particularly efficient at inhibiting Fe precipitation. By binding Fe directly at the olivine surface, the WT cells removed the inhibition of olivine dissolution almost completely. TEM analysis of polished olivine sections, colonised by a fungal biofilm for seven months, supported this hypothesis. After long-term fungus-olivine interaction, the Fe-enriched, amorphous layer did not develop, and the olivine surface was stronger etched compared to the abiotic control.
To study the effect of mutualism on mineral weathering, K. petricola was grown with the cyanobacterium, Nostoc punctiforme ATCC 29133. Both partners showed an enhanced growth and formed a stratified biofilm which attached more strongly to olivine. Nevertheless, the olivine dissolution rate of the fungus-cyanobacterium consortium was moderate.
Rock weathering simulation systems developed here are promising research instruments. The experimental conditions allow for the alteration of the studied mineral surface, while the clear definition of these conditions delivers a stable growth of microorganisms. The latter makes these systems universally applicable, especially in combination with integrative multidisciplinary analytics. Processes underlying environmental and biological effects on rock weathering, metal corrosion, plastic degradation, or the deterioration of any other substrate can be studied reproducibly and over a long period of time. The chemical and biological complexity of these simulation systems mimics natural rock weathering processes. The mineral dissolution rates generated in this study are therefore relevant to natural ecosystems.