Spore germination: Two ion channels are better than one.

Germination is the process by which spores emerge from dormancy. Although spores can remain dormant for decades, the study of germination is an active field of research. In this issue of Genes & Development, Gao and colleagues (pp. 31-45) address a perplexing question: How can a dormant spore initiate germination in response to environmental cues? Three distinct complexes are involved: GerA, a germinant-gated ion channel; 5AF/FigP, a second ion channel required for amplification; and SpoVA, a channel for dipicolinic acid (DPA). DPA release is followed by rehydration of the spore core, thus allowing the resumption of metabolic activity.

Microtubules and actin filaments direct nuclear movement during the polarisation of Marchantia spore cells.

The multicellular haploid stage of land plants develops from a single haploid cell produced by meiosis - the spore. Starting from a non-polar state, these spores develop polarity, divide asymmetrically and establish the first axis of symmetry. Here, we show that the nucleus migrates from the cell centroid to the basal pole during polarisation of the Marchantia polymorpha spore cell. A microtubule organising centre on the leading edge of the nucleus initiates a microtubule array between the nuclear surface and the cortex at the basal pole. Simultaneously, cortical microtubules disappear from the apical hemisphere but persist in the basal hemisphere. This is accompanied by the formation a dense network of fine actin filaments between the nucleus and the basal pole cortex. Experimental depolymerisation of either microtubules or actin filaments disrupts cellular asymmetry. These data demonstrate that the cytoskeleton reorganises during spore polarisation and controls the directed migration of the nucleus to the basal pole. The presence of the nucleus at the basal pole provides the cellular asymmetry for the asymmetric cell division that establishes the apical-basal axis of the plant.

Temperate rainforests near the South Pole during peak Cretaceous warmth.

The mid-Cretaceous period was one of the warmest intervals of the past 140 million years1-5, driven by atmospheric carbon dioxide levels of around 1,000 parts per million by volume6. In the near absence of proximal geological records from south of the Antarctic Circle, it is disputed whether polar ice could exist under such environmental conditions. Here we use a sedimentary sequence recovered from the West Antarctic shelf-the southernmost Cretaceous record reported so far-and show that a temperate lowland rainforest environment existed at a palaeolatitude of about 82° S during the Turonian-Santonian age (92 to 83 million years ago). This record contains an intact 3-metre-long network of in situ fossil roots embedded in a mudstone matrix containing diverse pollen and spores. A climate model simulation shows that the reconstructed temperate climate at this high latitude requires a combination of both atmospheric carbon dioxide concentrations of 1,120-1,680 parts per million by volume and a vegetated land surface without major Antarctic glaciation, highlighting the important cooling effect exerted by ice albedo under high levels of atmospheric carbon dioxide.

Stepwise recombination suppression around the mating-type locus in an ascomycete fungus with self-fertile spores.

Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i.e. the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma. However, it is unclear whether this intriguing association holds in other species. We show here that Schizothecium tetrasporum, a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina. The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.47 Mb around the mating-type locus. We detected three evolutionary strata, indicating a stepwise extension of recombination suppression. The three strata displayed no rearrangement or transposable element accumulation but gene losses and gene disruptions were present, and precisely at the strata margins. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association between pseudo-homothallism and recombination suppression across lineages and the presence of gene disruption at the strata limits are consistent with a recently proposed mechanism of sheltering deleterious alleles to explain stepwise recombination suppression.

Proteome plasticity during Physcomitrium patens spore germination - from the desiccated phase to heterotrophic growth and reconstitution of photoautotrophy.

The establishment of moss spores is considered a milestone in plant evolution. They harbor protein networks underpinning desiccation tolerance and accumulation of storage compounds that can be found already in algae and that are also utilized in seeds and pollen. Furthermore, germinating spores must produce proteins that drive the transition through heterotrophic growth to the autotrophic plant. To get insight into the plasticity of this proteome, we investigated it at five timepoints of moss (Physcomitrium patens) spore germination and in protonemata and gametophores. The comparison to previously published Arabidopsis proteome data of seedling establishment showed that not only the proteomes of spores and seeds are functionally related, but also the proteomes of germinating spores and young seedlings. We observed similarities with regard to desiccation tolerance, lipid droplet proteome composition, control of dormancy, and ß-oxidation and the glyoxylate cycle. However, there were also striking differences. For example, spores lacked any obvious storage proteins. Furthermore, we did not detect homologs to the main triacylglycerol lipase in Arabidopsis seeds, SUGAR DEPENDENT1. Instead, we discovered a triacylglycerol lipase of the oil body lipase family and a lipoxygenase as being the overall most abundant proteins in spores. This finding indicates an alternative pathway for triacylglycerol degradation via oxylipin intermediates in the moss. The comparison of spores to Nicotiana tabacum pollen indicated similarities for example in regards to resistance to desiccation and hypoxia, but the overall developmental pattern did not align as in the case of seedling establishment and spore germination.

Nanozyme-Enhanced Probiotic Spores Regulate the Intestinal Microenvironment for Targeted Acute Gastroenteritis Therapy.

Antibiotic therapeutics to combat intestinal pathogen infections often exacerbate microbiota dysbiosis and impair mucosal barrier functions. Probiotics are promising strategies, because they inhibit pathogen colonization and improve intestinal microbiota imbalance. Nevertheless, their limited targeting ability and susceptibility to oxidative stress have hindered their therapeutic potential. To tackle these challenges, Ces3 is synthesized by in situ growth of CeO2 nanozymes with positive charges on probiotic spores, facilitating electrostatic interactions with negatively charged pathogens and possessing a high reactive oxygen species (ROS) scavenging activity. Importantly, Ces3 can resist the harsh environment of the gastrointestinal tract. In mice with S. Typhimurium-infected acute gastroenteritis, Ces3 shows potent anti-S. Typhimurium activity, thereby alleviating the dissemination of S. Typhimurium into other organs. Additionally, owing to its O2 deprivation capacity, Ces3 promotes the proliferation of anaerobic probiotics, reshaping a healthy intestinal microbiota. This work demonstrates the promise of combining antibacterial, anti-inflammatory, and O2 content regulation properties for acute gastroenteritis therapy.

Identification of a putative cell wall-hydrolyzing amidase involved in sporangiospore maturation in Actinoplanes missouriensis.

Actinoplanes missouriensis is a filamentous bacterium that differentiates into terminal sporangia, each containing a few hundred spores. Previously, we reported that a cell wall-hydrolyzing N-acetylglucosaminidase, GsmA, is required for the maturation process of sporangiospores in A. missouriensis; sporangia of the gsmA null mutant (ΔgsmA) strain released chains of 2-20 spores under sporangium dehiscence-inducing conditions. In this study, we identified and characterized a putative cell wall hydrolase (AsmA) that is also involved in sporangiospore maturation. AsmA was predicted to have a signal peptide for the general secretion pathway and an N-acetylmuramoyl-l-alanine amidase domain. The transcript level of asmA increased during the early stages of sporangium formation. The asmA null mutant (ΔasmA) strain showed phenotypes similar to those of the wild-type strain, but sporangia of the ΔgsmAΔasmA double mutant released longer spore chains than those from the ΔgsmA sporangia. Furthermore, a weak interaction between AsmA and GsmA was detected in a bacterial two-hybrid assay using Escherichia coli as the host. Based on these results, we propose that AsmA is an enzyme that hydrolyzes peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in cooperation with GsmA in A. missouriensis.IMPORTANCEActinoplanes missouriensis produces sporangiospores as dormant cells. The spores inside the sporangia are assumed to be formed from prespores generated by the compartmentalization of intrasporangium hyphae via septation. Previously, we identified GsmA as a cell wall hydrolase responsible for the separation of adjacent spores inside sporangia. However, we predicted that an additional cell wall hydrolase(s) is inevitably involved in the maturation process of sporangiospores because the sporangia of the gsmA null mutant strain released not only tandemly connected spore chains (2-20 spores) but also single spores. In this study, we successfully identified a putative cell wall hydrolase (AsmA) that is involved in sporangiospore maturation in A. missouriensis.

An improved method to study Phytophthora cinnamomi Rands zoospores interactions with host.

Phytophthora cinnamomi Rands is a highly prevalent phytopathogen worldwide, ranking among the top ten in terms of distribution. It inflicts crown rot, canker, and root rot on numerous plant species, significantly impacting the biodiversity of both flora and fauna within affected environments. With a host range spanning over 5,000 species, including important plants like Quercus suber, Quercus ilex, Castanea sativa, and commercially significant crops such as avocado (Persea americana), maize (Zea mays), and tomato (Solanum lycopersicum), Phytophthora cinnamomi poses a substantial threat to agriculture and ecosystems. The efficient dissemination of the oomycete relies on its short-lived asexually motile zoospores, which depend on water currents to infect host roots. However, managing these zoospores in the laboratory has long been challenging due to the complexity of the life cycle. Current protocols involve intricate procedures, including alternating cycles of growth, drought, and flooding. Unfortunately, these artificial conditions often result in a rapid decline in virulence, necessitating additional steps to maintain infectivity during cultivation. In our research, we sought to address this challenge by investigating zoospore survival under various conditions. Our goal was to develop a stable stock of zoospores that is both easily deployable and highly infective. Through direct freezing in liquid nitrogen, we have successfully preserved their virulence. This breakthrough eliminates the need for repeated culture transfers, simplifying the process of plant inoculation. Moreover, it enables more comprehensive studies of Phytophthora cinnamomi and its interactions with host plants.

Stepwise changes in flavonoids in spores/pollen contributed to terrestrial adaptation of plants.

Protecting haploid pollen and spores against UV-B light and high temperature, 2 major stresses inherent to the terrestrial environment, is critical for plant reproduction and dispersal. Here, we show flavonoids play an indispensable role in this process. First, we identified the flavanone naringenin, which serves to defend against UV-B damage, in the sporopollenin wall of all vascular plants tested. Second, we found that flavonols are present in the spore/pollen protoplasm of all euphyllophyte plants tested and that these flavonols scavenge reactive oxygen species to protect against environmental stresses, particularly heat. Genetic and biochemical analyses showed that these flavonoids are sequentially synthesized in both the tapetum and microspores during pollen ontogeny in Arabidopsis (Arabidopsis thaliana). We show that stepwise increases in the complexity of flavonoids in spores/pollen during plant evolution mirror their progressive adaptation to terrestrial environments. The close relationship between flavonoid complexity and phylogeny and its strong association with pollen survival phenotypes suggest that flavonoids played a central role in the progression of plants from aquatic environments into progressively dry land habitats.

Systematics and evolutionary dynamics of insect-fern interactions in the specialized fern-spore feeding Cuprininae (Lepidoptera, Stathmopodidae).

Fern-spore-feeding (FSF) is rare and found in only four families of Lepidoptera. Stathmopodidae is the most speciose family that contains FSF species, and its subfamily Cuprininae exclusively specializes on FSF. However, three species of Stathmopodinae also specialize on FSF. To better understand the evolutionary history of FSF and, more generally, the significance of specialization on a peculiar host, a phylogenetic and taxonomic revision for this group is necessary. We reconstructed the most comprehensive molecular phylogeny, including one mitochondrial and four nuclear genes, of Stathmopodidae to date, including 137 samples representing 62 species, with a particular focus on the FSF subfamily, Cuprininae, including 33 species (41% of named species) from 6 of the 7 Cuprininae genera. Species from two other subfamilies, Stathmopodinae and Atkinsoniinae, were also included. We found that FSF evolved only once in Stathmopodidae and that the previous hypothesis of multiple origins of FSF was misled by inadequate taxonomy. Moreover, we showed that (1) speciation/extinction rates do not differ significantly between FSF and non-FSF groups and that (2) oligophage is the ancestral character state in Cuprininae. We further revealed that a faster rate of accumulating specialists over time, and thus a higher number of specialists, was achieved by a higher transition rate from oligophagages to specialists compared to the transition rate in the opposite direction. We finish by describing three new genera, Trigonodagen. nov., Petalagen. nov., and Pediformisgen. nov., and revalidating five genera: Cuprina, Calicotis, Thylacosceles, Actinoscelis, Thylacosceloides in Cuprininae, and we provide an updated taxonomic key to genera and a revised global checklist of Cuprininae.

Ubiquitin-dependent Argonauteprotein MEL1 degradation is essential for rice sporogenesis and phasiRNA target regulation.

MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.

An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

The effect of sedimentation on spore settlement and recruitment of the endemic Arctic kelp, Laminaria solidungula (Phaeophyceae).

Environmental changes associated with rapid climate change in the Arctic, such as the increased rates of sedimentation from climatic or anthropogenic sources, can enhance the impact of abiotic stressors on coastal ecosystems. High sedimentation rates can be detrimental to nearshore kelp abundance and distribution, possibly due to increased mortality at the spore settlement stage. Spore settlement and viability of the Arctic kelp Laminaria solidungula were examined through a series of lab-based sedimentation experiments. Spores were exposed to increasing sediment loads in three experimental designs simulating different sedimentation scenarios: sediment deposition above settled spores, settlement of spores on sediment-covered substrate, and simultaneous suspension of spores and sediments during settlement. Spore settlement was recorded upon completion of each experiment, and gametophyte abundance was assessed following a growth period with sediments removed to examine short-term spore viability via a gametophyte-to-settled-spore ratio. In all three types of sediment exposure, the addition of sediments caused a 30%-40% reduction in spore settlement relative to a no-sediment control. Spore settlement decreased significantly between the low and high sediment treatments when spores were settled onto sediment-covered substrates. In all experiments, increasing amounts of sediment had no significant effect on spore viability, indicating that spores that had settled under different short-term sediment conditions were viable. Our results indicate that depending on spore-sediment interaction type, higher rates of sedimentation resulting from increased sediment loading could affect L. solidungula spore settlement success with potential impacts on the long-term persistence of a diverse and productive benthic habitat.

Biocontrol Mechanisms of Three Plant Essential Oils Against Phytophthora infestans Causing Potato Late Blight.

Late blight, caused by the notorious pathogen Phytophthora infestans, poses a significant threat to potato (Solanum tuberosum) crops worldwide, impacting their quality as well as yield. Here, we aimed to investigate the potential use of cinnamaldehyde, carvacrol, and eugenol as control agents against P. infestans and to elucidate their underlying mechanisms of action. To determine the pathogen-inhibiting concentrations of these three plant essential oils (PEOs), a comprehensive evaluation of their effects using gradient dilution, mycelial growth rate, and spore germination methods was carried out. Cinnamaldehyde, carvacrol, and eugenol were capable of significantly inhibiting P. infestans by hindering its mycelial radial growth, zoospore release, and sporangium germination; the median effective inhibitory concentration of the three PEOs was 23.87, 8.66, and 89.65 µl/liter, respectively. Scanning electron microscopy revealed that PEOs caused the irreversible deformation of P. infestans, resulting in hyphal shrinkage, distortion, and breakage. Moreover, propidium iodide staining and extracellular conductivity measurements demonstrated that all three PEOs significantly impaired the integrity and permeability of the pathogen's cell membrane in a time- and dose-dependent manner. In vivo experiments confirmed the dose-dependent efficacy of PEOs in reducing the lesion diameter of potato late blight. Altogether, these findings provide valuable insight into the antifungal mechanisms of PEOs vis-à-vis late blight-causing P. infestans. By utilizing the inherent capabilities of these natural compounds, we could effectively limit the harmful impacts of late blight on potato crops, thereby enhancing agricultural practices and ensuring the resilience of global potato food production.

A comparison of the biofouling potential of field-collected and laboratory-cultured Ulva.

The marine algae Ulva spp. are commonly used as model biofouling organisms. As biofouling studies are primarily conducted using field-collected specimens, factors including species identity, seasonal availability, and physiological status can hinder the replicability of the results. To address these limitations, a protocol was developed for the on-demand laboratory culture and release of Ulva zoospores. The biofouling potential of laboratory-cultured and field-collected Ulva blades was compared using a waterjet. No significant differences were found between field and laboratory-cultured samples in either spore adhesion (before waterjet) or the proportion of spores retained after waterjet exposure. However, there was significant variability within each session type in pre- and post-waterjet exposures, indicating that spore adhesion and retention levels vary significantly among trial runs. In addition, all our laboratory cultures were Ulva Clade C (LPP complex). In contrast, our field samples contained a mix of Ulva Clade C, U. compressa clade I, and U. flexuosa Clade D. This protocol for on-demand production of Ulva spores can improve biofouling research approaches, enables comparison of results across laboratories and regions, and accelerate the development of anti-biofouling strategies.

Unreduced spore formation in a spontaneous chimeric pinnule in an artificially produced haploid Anisocampium niponicum (Athyriaceae, Polypodiales).

Haploid sporophytes of Anisocampium niponicum with 2n = 40, were produced artificially by induced apogamy in vitro. They were subsequently transplanted into pots and two of them have been cultivated for the investigation of sporogenesis and/or production of chimera for more than 20 years. Haploid A. niponicum is sterile, but an abnormal chimeric pinnule that developed spontaneously in a single frond produced sporangia with spores. Each sporangium bore approximately 32 spores that were almost uniform in size. Sowing of these spores resulted in 50 gametophytes. Of 20 gametophytes cultured individually, five produced sporophytes apogamously after eight months. Both the gametophytes and subsequent apogamous sporophytes showed a chromosome number of 2n = 40. Our study demonstrates that a haploid sporophyte offspring can be produced from a haploid mother sporophyte via haploid spores. Since asexual reproduction is a prominent evolutionary process in ferns, the reproduction of a haploid A. niponicum sporophyte by unreduced spore formation might help to elucidate how apogamous ferns occur and evolve.

Insights into the differential molecular response of non-germinated and germinated spores of Ameson portunus in vitro by comparative transcriptome analysis.

Ameson portunus, the recently discovered causative agent of "toothpaste disease" of pond-cultured swimming crabs in China has caused enormous economic losses in aquaculture. Understanding the process of spore germination is helpful to elucidate the molecular mechanism of its invasion of host cells. Here, we obtained mature and germinating spores by isolation and purification and in vitro stimulation, respectively. Then, non-germinated and germinated spores were subjected to the comparative transcriptomic analysis to disclose differential molecular responses of these two stages. The highest germination rate, i.e., 71.45 %, was achieved in 0.01 mol/L KOH germination solution. There were 9,609 significantly differentially expressed genes (DEGs), with 685 up-regulated and 8,924 down-regulated DEGs. The up-regulated genes were significantly enriched in ribosome pathway, and the down-regulated genes were significantly enriched in various metabolic pathways, including carbohydrate metabolism, amino acid metabolism and other metabolism. The results suggested that spores require various carbohydrates and amino acids as energy to support their life activities during germination and synthesize large amounts of ribosomal proteins to provide sites for DNA replication, transcription, translation and protein synthesis of the spores of A. portunus within the host cells. Functional genes related to spore germination, such as protein phosphatase CheZ and aquaporin, were also analyzed. The analysis of transcriptome data and identification of functional genes will help to understand the process of spore germination and invasion.

Germination and outgrowth of Bacillus mycoides KBAB4 spores are impacted by environmental pH, quantitatively analyzed at single cell level with sporetracker.

Quantifying spore germination and outgrowth heterogeneity is challenging. Single cell level analysis should provide supplementary knowledge regarding the impact of unfavorable conditions on germination and outgrowth dynamics. This work aimed to quantify the impact of pH on spore germination and outgrowth, investigating the behavior of individual spore crops, produced under optimal and suboptimal conditions. Bacillus mycoides (formerly B. weihenstephanensis) KBAB4 spores, produced at pH 7.4 and at pH 5.5 were incubated at different pH values, from pH 5.2 to 7.4. The spores were monitored by microscopy live imaging, in controlled conditions, at 30 °C. The images were analyzed using SporeTracker, to determine the state of single cells. The impact of pH on germination and outgrowth times and rates was estimated and the correlation between these parameters was quantified. The correlation between germination and outgrowth times was significantly higher at low pH. These results suggest that an environmental pressure highlights the heterogeneity of spore germination and outgrowth within a spore population. Results were consistent with previous observations at population level, now confirmed and extended to single cell level. Therefore, single cell level analyses can be used to quantify the heterogeneity of spore populations, which is of interest in order to control the development of spore-forming bacteria, responsible for food safety issues.

Myxospore density of Kudoa inornata varies significantly within symmetrical white muscle tissue replicates of its fish host, the spotted seatrout, Cynoscion nebulosus.

The spotted seatrout, Cynoscion nebulosus, is a popular game fish in the southeastern USA. It is estimated that nearly 90% of the adult population in South Carolina estuaries are infected in their skeletal muscle by the myxosporean, Kudoa inornata. However, little is known about this parasite's biology, including the distribution and densities of myxospores within tissues of infected fish, which we expect affect the physiology of their hosts. In order to correlate densities with physiological parameters in future studies, we quantified the myxospores density in muscle and characterized the variation among individual fish. Naïve juvenile seatrout was experimentally infected via presumed K. inornata actinospores exposure to raw seawater. A plug of muscle was extracted from two bilaterally symmetrical regions in the epaxial fillet from fresh and frozen carcasses. Variation in density data was calculated both within and among individuals. Within individuals, density counts were compared between left- and right-side biopsies. There was no significant difference between fresh and frozen plugs, and variation among individuals accounted for the greatest proportion of variation at 68.8%, while variation within individuals was substantial at 25.6%. Simulation and correlation tests confirmed that bilaterally symmetrical replicates varied significantly within individuals. When sampled from areas surrounding the initial biopsies, myxospore density estimates were more similar than between sides. Our findings have important implications for sampling design, particularly for studies investigating physiological parameters at the cellular or molecular level in association with parasite infection.

Glycine fermentation by C. difficile promotes virulence and spore formation, and is induced by host cathelicidin.

Clostridioides difficile is a leading cause of antibiotic-associated diarrheal disease. C. difficile colonization, growth, and toxin production in the intestine is strongly associated with its ability to use amino acids to generate energy, but little is known about the impact of specific amino acids on C. difficile pathogenesis. The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to C. difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase (GR) genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. Our data show that the grd pathway promotes growth, toxin production, and sporulation. Glycine fermentation also had a significant impact on toxin production and pathogenesis of C. difficile in the hamster model of disease. Furthermore, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.