Name: Investigating Bacterial-Fungal Interactions using Fungal Highway Columns in Diverse Environments and Substrates (Video) | JoVE Journal
Uploaded: 2025-01-24T12:40:00.000+00:00
Duration: 5 min 22 s
Description: Learn to construct and use fungal highway columns for enriching bacterial-fungal pairs from environmental substrates. This protocol enables researchers to study microbial interactions and ecological dynamics effectively.

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5 minutes

This research was supported by a Science Focus Area Grant from the U.S. Department of Energy (DOE), Biological and Environmental Research (BER), Biological System Science Division (BSSD) under grant number LANLF59T.

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The authors do not have any conflicts of interest to disclose.

When generating the column components, the selection of a 3D printer and printing material can be modified based on availability and desired material properties17,18. The biocompatibility, surface texture, autoclavability, ability to print fine-scale details, and relative transparency were all considered in our group's material selection. Other features, such as porosity, hydrophobicity, printing parameters, etc., should also be considered. Various resins were tested (see Table of Materials) prior to the final selection, and many biocompatible materials will work for the printing of these columns. The material chosen for the construction of the column components will determine which cleaning, post-curing, and sterilization approaches should be used. Not all materials will be autoclavable, and ultraviolet light, bleach, or other sterilization techniques may be required, depending on the material manufacturer's instructions. Some sterilization or cleaning techniques may also damage or not be compatible with the chosen material, so particular attention should be given to this information from the material manufacturer. For 3D printers, some considerations include printing time, material compatibility, build platform size, printing technology, and cost19. The 3D-printed components of the columns can be fragile and may break if handled too forcefully. The threading for the ring and the caps may not always exactly align, therefore we recommend that extra components are printed and sterilized prior to the assembly step, or preliminary printing is done to test how the parameters and material chosen affect the threading. The design specifications for the threading within the caps and the ring may need to be adjusted depending on the chosen 3D printing material. The dimensions, lattice complexity, and other physical features can all be modified in CAD design software prior to printing. As designed, the column itself is 4 cm tall, and the lattice structure within the center of the column has a 2 mm-sized unit cell, a strut diameter of 0.5 mm, and the entire lattice height is 22 mm9. These parameters can be adjusted if a researcher wants, for example, a larger or more complex lattice structure. Overall, the 3D printed manufacturing of these devices enables design flexibility while also ensuring that a single design can be used in a standardized manner across organizations and groups, and even used as classroom teaching tools9.
Several steps in the protocol may require troubleshooting depending on the environment or experimental setup. The fungal highway columns are not very effective in low humidity conditions, as the media plugs quickly desiccate before facilitating fungal growth, which can limit the duration of experiments in these environments (Figure 5A). Techniques that have improved the effectiveness of the columns in low-humidity environments include artificially increasing humidity through the addition of moisture to the substrate and/or sealing the column and substrate in a secondary container with a water source (e.g., a small container of pure water). The hourglass shape and lattice structure were incorporated to prevent bacterial movement alone (without the establishment of a fungal highway) if condensation were to form in high-humidity environments. Fast-growing fungi may overgrow the target and bait media surface area and extend out of the top or bottom of the column (Figure 5C). Decreasing the incubation time of the bait fungus or the duration of the experiment can minimize or eliminate this overgrowth. Additionally, a limitation of these devices is that fast-growing fungi in the substrate of interest may limit colonization of bait and target media by slow-growing fungi, potentially biasing which highway interactions are observed. Some fungi, especially slower-growing fungi, may not colonize the bait media in a manner that allows them to grow through the agar plug and into the lattice structure. If there is sufficient humidity in the environment, thinner agar plugs can be used to encourage growth into the lattice after bait agar plug colonization. Media can be chosen based on whether a researcher wants to select for fungal or bacterial growth, but this can also limit the subculturing to organisms that prefer that media type20. If no growth is seen in the target media, it may be necessary to inoculate the bait medium or substrate with a fungus that is known to create fungal highways. 
Metagenomic or amplicon sequencing can be performed as part of these experiments, and both of these strategies impart their own limitations and strengths21. Metagenomic sequencing is ideal for obtaining additional genomic information about the microbes. However, the recoverable amount of nucleic acids directly from the target media can be very low, which may require the utilization of amplicon sequencing or other amplification methods prior to sequencing. Amplicon sequencing libraries must be prepared separately (16S and ITS), and this method lacks taxonomic resolution and limits any assessments about genome features or functional potential that can be achieved using metagenomic sequencing. Direct sequencing methods from the plugs may be preferred in cases where microbes are not able to be subcultured. It is recommended that plugs be split into multiple sections to enable both culturing and sequencing approaches.
A benefit of these devices is that they can be used both in the laboratory and the field. Special care must be taken to ensure that the columns in the field can remain upright and are protected from animals and environmental perturbations that could disturb their placement. The columns have not yet been tested in a horizontal position, in a position where they are fully covered by a substrate, and they have not been tested in environments that are exposed to substantial rainfall or snow. As stated above, the lattice structure was designed to minimize the probability of bacteria being able to move to the target medium in high-humidity environments. However, it is possible that if the column were exposed to larger volumes of water and this water fully saturated the column, the bacterial movement would be facilitated throughout the column independent of any fungal highways present. For lab-based experiments, the columns can be used within 50 mL conical tubes, small microcosms of substrates, in the soil surrounding potted plants, in boxes, or within other controlled experimental systems. The columns have been successfully utilized in soil, rhizospheres, and dung, and their utility can be expanded to other substrates, including leaf litter, sludge, sand, snow, compost, etc. 
The fungal highway columns enable a number of comparisons to understand this BFI phenotype within diverse sample types. Comparing the community composition between the bait and target media can indicate which bacteria can utilize fungal highways and which fungi can serve as potential highways9. If metagenome sequencing is used, genomic features that distinguish organisms from the bait versus target media can also be examined. It is also possible to compare target media from columns placed in different substrates (e.g., soil versus dung) or placed in the same substrate under different conditions (e.g., temperature or humidity). Overall, the fungal highway columns expand upon the capabilities of previous methods for interrogating this form of BFI and enable extensive examinations into these interactions that shape the spatial dynamics of complex environmental microbiomes.

Bacterial-fungal interactions (BFIs) are extremely important in shaping the structural, spatial, and functional properties of environmental microbiomes. For example, the growth and expansion of filamentous fungi or other fungi-like filamentous microorganisms generates a biological network that can function as a &#39;highway&#39; to facilitate the movement of other microorganisms, such as bacteria. Heterogeneity and inconsistent saturation within environmental substrates can hinder bacterial motility; however, bacteria can use these highways to facilitate access to additional areas of the environment1,2. These interactions are critical to understanding the spatial dynamics of microbial communities. Several techniques and methods have been used to examine fungal highways, however, they are largely limited to laboratory-based investigations3,4.
In one plate-based method, a large section of agar is removed from the middle of the Petri dish, creating a gap between two agar islands. Fungal hyphae can traverse this gap, providing the means for compatible bacteria to cross from one agar island to the other5. Other modified Petri-dish methods include inverted plates where soil is placed in the lid so fungal hyphae can grow vertically and colonize the media without direct contact, providing the means for bacterial transport5,6. A growth media droplet-based method that has recently been developed can be used to evaluate selective hyphal transport of bacteria towards certain nutrient profiles7. Bacterial bridge and trail devices have also been used to investigate the effect of abiotic factors on bacterial movement8. Although several methods and techniques have been utilized to investigate fungal highways, there remains a need for standardized devices that maintain a sterile microenvironment while promoting the establishment of fungal highways from complex environmental substrates such as dung, soil, and rhizospheres.
Our group designed a 3D-printed version of fungal highway columns where fungi can transport bacteria from one end to the other9. These devices are assembled from four printed components: the column itself with an hourglass shape and a complex inner lattice structure, a threaded ring, and two caps (a large cap and a small cap), as well as a piece of sterilized nylon mesh (Figure 1). The assembled column is added directly to the desired environmental substrate. The column then allows microbes to colonize an agar growth medium plug known as the &#39;bait&#39; media plug that is at the bottom of the column and in contact with the environmental substrate through the mesh. This piece of nylon mesh size-excludes other soil dwellers that can transport bacteria, thus limiting the bacterial movement within the columns to fungal highways. Once this bait plug has been colonized, filamentous fungi can extend and grow through the inner lattice within the center of the column that is designed to create an unsaturated system that resembles soil (or other unsaturated media) and minimize potential contamination from the bait medium. The fungi then grow towards and colonize the target medium plug at the top of the column. Columns can either be inoculated with specific fungal isolates to test their ability to transport bacteria, or they can be left uninoculated to identify which fungi from the substrate are capable of transporting bacteria. Organisms that reach the target medium can be further cultured, isolated, and subjected to sequencing analyses (either from pure cultures or from mixed communities using amplicon or metagenomic sequencing approaches). Overall, the columns provide a standardized, reproducible, reusable, and intuitive method for interrogating fungal highways in diverse substrates. These devices can be used for research and as a classroom teaching tool, and herein, we provide instructional steps for using them based on the experiments that have been performed in the past. Although this method facilitates protocol standardization, the design and construction of the devices can be modified for other applications and additional substrates.

<table><tbody><tr><td>50 mL tubes</td><td>Greiner BIO-ONE</td><td>5622-7261</td><td>50 mL tubes for performing column experiments in the lab</td></tr><tr><td>90 mm Petri dishes</td><td>Thermo Scientific Nunc</td><td>08-757-099</td><td>Petri dishes for preparation of agar and for microbial growth</td></tr><tr><td>Asiga Freeform Pico Plus 39 digital light processing (DLP) 3D printer</td><td>Asiga Germany</td><td>Freeform Pico Plus 39</td><td>3D printer used to generate batches of the columns; other 3D printers can be used</td></tr><tr><td>Autoclave</td><td>Fisher Scientific</td><td>LS40F20</td><td>Benchtop autoclave to sterilize the column components</td></tr><tr><td>Beaker</td><td>Fisher Scientific</td><td>FB100600</td><td>600 mL beaker for various uses throughout the protocol</td></tr><tr><td>Dental LT Clear Resin V2</td><td>Formlabs</td><td>RS-F2-DLCL-02</td><td>Alternative resin for 3D printing that was tested</td></tr><tr><td>Dental Surgical Guide Resin</td><td>Formlabs</td><td>RS-F2-SGAM-01</td><td>Was used to generate the columns discussed in manuscript; Other photosensitive resins can be used in place of this material</td></tr><tr><td>DNA Low Bind 1.5 mL tubes</td><td>Eppendorf</td><td>13-698-791</td><td>Tubes used for various preparations including nucleic acid extractions</td></tr><tr><td>DNA/RNA shield preservative</td><td>Zymo Research</td><td>R1100-50</td><td>Preservative used prior to nucleic acid extractions</td></tr><tr><td>EDGE Bioinformatics</td><td>Open source; Developed by the Los Alamos National Laboratory (LANL)</td><td>n/a</td><td>Bioinformatics platform for processing amplicon data</td></tr><tr><td>FastDNA spin kit for soil</td><td>MP Biomedicals LLC</td><td>116560200-CF</td><td>DNA extraction kit option for soil</td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>10-300</td><td>Forceps that can be sterilized</td></tr><tr><td>Formlabs BioMed Clear Resin</td><td>Formlabs</td><td>RS-F2-BMCL-01</td><td>Alternative resin for 3D printing that was tested</td></tr><tr><td>Formlabs Form 3B+ stereolithography (SLA) 3D printer</td><td>Formlabs</td><td>Form 3B+</td><td>Alternative 3D printer</td></tr><tr><td>Formlabs IBT Resin</td><td>Formlabs</td><td>RS-F2-IBCL-01</td><td>Alternative resin for 3D printing that was tested</td></tr><tr><td>Inoculating Loops</td><td>Fisher Scientific</td><td>22-363-598</td><td>Used to isolate/transfer microbes</td></tr><tr><td>Malt Extract Agar (MEA)</td><td>Criterion</td><td>89405-654</td><td>A media type used in columns</td></tr><tr><td>MiSeq sequencer + MiSeq sequencing kit</td><td>Illumina</td><td>SY-410-1003</td><td>Can use other sequencers</td></tr><tr><td>Mortar &amp;amp; Pestle</td><td>Fisher Scientific</td><td>FB961K; FB961A</td><td>Can use any common mortar &amp;amp; pestle that can be sterilized between uses</td></tr><tr><td>NEBNext Ultra II DNA Library Prep Kit for Illumina</td><td>New England Biolabs</td><td>E7805S</td><td>Library prep kit for metagenomic sequencing</td></tr><tr><td>Nextera XT DNA Library Preparation Kit (24 samples)</td><td>Illumina</td><td>FC-131-1024</td><td>Library prep kit for amplicon sequencing</td></tr><tr><td>NMDC EDGE</td><td>Open source: Developed by the National Microbiome Data Collaborative (NMDC)</td><td>n/a</td><td>Bioinformatics platform for processing metagenomic data</td></tr><tr><td>Nylon mesh</td><td>Sefar</td><td>03-25/19</td><td>The mesh used as part of the column construction</td></tr><tr><td>Pipette tips</td><td>Rainin</td><td>30807966</td><td>Can use many different sterilized pipette tips for the protocol steps</td></tr><tr><td>Potato Dextrose Agar</td><td>Cole Parmer</td><td>EW-14200-28</td><td>A media type used in columns</td></tr><tr><td>QIIME2</td><td>Open source</td><td>n/a</td><td>Software for processing amplicon data</td></tr><tr><td>Qubit dsDNA HS assay kit</td><td>Thermo Fisher Scientific</td><td>Q32851</td><td>Used to quantify DNA after extractions</td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33238</td><td>Used to quantify DNA after extractions</td></tr><tr><td>Quick-DNA Fungal/Bacterial Miniprep Kit</td><td>Zymo Research</td><td>D6005</td><td>DNA extraction kit option that works with both bacteria and fungi</td></tr><tr><td>R2A agar</td><td>BD Difco</td><td>218263</td><td>A media type used in columns (bacterial media)</td></tr><tr><td>Rack for 50 mL tubes</td><td>Fisher Scientific</td><td>03-448-11</td><td>Rack to hold 50 mL tubes upright</td></tr><tr><td>Scissors</td><td>Fisher Scientific</td><td>12-000-155</td><td>Fine precision scissors that can be sterilized</td></tr><tr><td>Sodium carboxymethyl cellulose medium</td><td>Aldrich</td><td>419273-100G</td><td>A media type used in columns</td></tr><tr><td>SolidWorks CAD software</td><td>SolidWorks</td><td>n/a</td><td>Software used to design the columns</td></tr><tr><td>Trowel scoop</td><td>Fisher Scientific</td><td>S41701</td><td>To make a depression in the substrate prior to adding the column</td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>Invitrogen: ThermoFisher Scientific</td><td>10977015</td><td>Water for the ultrasonicator water bath</td></tr><tr><td>Ultrasonicator</td><td>Fisher Scientific</td><td>FB-11201</td><td>Ultrasonicator for cleaning the columns</td></tr></tbody></table>

investigating bacterial-fungal interactions using fungal highway columns in diverse environments and substrates

Investigating Bacterial-Fungal Interactions using Fungal Highway Columns in Diverse Environments and Substrates

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and microbial dispersal. Mycelial networks created by filamentous fungi or other filamentous microorganisms (e.g., Oomycetes) act as 'fungal highways' that can be utilized by bacteria for transport throughout heterogeneous environments, greatly facilitating their mobility and granting them access to regions that may be challenging or impossible to reach on their own (e.g., due to air pockets within the soil). Several devices and experimental protocols have been created to study these fungal highways, including fungal highway columns. The fungal highway column designed by our group can be used for a variety of in situ or in vitro applications, as well as with diverse environmental and host-associated sample types. Herein, we describe the methods for performing experiments with these columns, including designing, printing, sterilizing, and preparing the devices. The options for analyzing data obtained from the use of these devices are also discussed here, and troubleshooting advice regarding potential pitfalls associated with experiments using fungal highway columns is offered. These devices can be used to gain a more comprehensive understanding of the diversity, mechanisms, and dynamics of fungal highway BFIs to provide valuable insights into the structural and functional dynamics within complex environments (e.g., soils) and across diverse habitats in which bacteria and fungi co-exist.

The fully assembled fungal highway column is approximately 5 cm in length (Figure 1). The column should not be broken in any area, and the caps and threaded ring should fit together easily and tightly to create microenvironments within the column. The filter mesh can extend beyond the threaded ring (as shown in Figure 1 and Figure 2), or it can be trimmed with sterilized scissors. The agar plugs should fit snugly at each end of the column. When placed into the substrate, the filter mesh should come into contact with the substrate, and the column should not be completely buried.
Columns were previously tested in horse dung9. The columns were also placed into bulk and rhizosphere soil at a research field site, as well as into small volumes of soil in 50 mL tubes in the lab (Figure 3). After the fungal highway columns were removed from the substrate and disassembled, microbial growth was visible on both the bait and target media plugs (examples shown in Figure 4A). Bacteria and fungi were isolated from target and bait media via subculturing techniques (Figure 4B), and the microbes present on the media plugs were taxonomically identified using amplicon sequencing (Figure 4C,D). Figure 4C,D depict the combined results of the amplicon sequencing across multiple experiments, showing which microbes were able to reach the target media plug from columns added to horse dung9. Visualizations of this bacterial and fungal data were generated as outlined in step 17. Results may also be displayed as relative abundances of taxa.
Suboptimal results have been obtained in cases where the columns were added to extremely low humidity environments, and the media plugs were completely desiccated within a matter of days, leading to no recovery of colonized microbes (Figure 5A). We have also seen cases where microbes simply do not grow from the target media plug (Figure 5B), and cases where we do not recover sufficient sequencing data from the target media plug for meaningful analyses. Other cases where fungi overgrow out of the columns have also resulted in the need to re-do the experiments (Figure 5C).
<img alt="Soil moisture equilibrium, tube setup, outdoor experiment, soil moisture retention test." class="xfigimg" src="/files/ftp_upload/66989/66989fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Examples of the columns placed in environmental samples in laboratory and field settings. (A) Column placed inside a 50 mL tube with moistened soil in a laboratory setting. Also shown with a ruler for scale. (B) Column placed into soil in the field. (C) Column placed into the root network of a plant in the field. <a href="https://www.jove.com/files/ftp_upload/66989/66989fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fungal growth and phylogenetic diagrams; petri dish cultures, spore samples, evolutionary analysis." class="xfigimg" src="/files/ftp_upload/66989/66989fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Representative results from successful column experiments. (A) Examples of colonized media plugs extracted from columns. (B) Examples of fungal isolates subcultured from target media. The substrate was soil. Top ITS sequence NCBI BLAST identity from left to right: Rhizopus azygosporous, Aspergillus novofumigatus, Curvularia subpapendorfii, and Phaeomycocentrospora cantuariensis. (C,D) Circular cladograms displaying the phylogenetic diversity of (C) fungal ITS and (D) bacterial 16S sequences recovered from the target media following multiple fungal highway experiments using horse dung. Sections are colored and labeled by phylum, with end nodes representing unique genera. <a href="https://www.jove.com/files/ftp_upload/66989/66989fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Petri dishes with fungal growth, experimental setup for spore development analysis." class="xfigimg" src="/files/ftp_upload/66989/66989fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5. Suboptimal results from column experiments. (A) An example of a desiccated media plug resulting from low humidity environmental conditions. (B) Example of no microbial growth from a column media plug. (C) Example of overgrowth of the fungus through the top (target medium) of the column. <a href="https://www.jove.com/files/ftp_upload/66989/66989fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol provides detailed instructions on how to construct, sterilize, assemble, utilize, and reuse the fungal highway columns to enrich bacterial-fungal pairs interacting through fungal highways from diverse environmental substrates.

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and ...

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Biology

Source: Schlafer, S, and Frost Kristensen, M. Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy. J. Vis. Exp. (2020)This video demonstrates the cultivation of Streptococcus mutans and Candida albicans cross-kingdom biofilms under oral-like conditions to model bacterial–fungal interactions, enabling the study of microbial synergy, glucan-mediated adhesion, and acidogenic virulence in oral pathogenesis.

Establishing Bacterial–Fungal Cross-Kingdom Biofilms for Studying Oral Pathogenesis

Source: Miquel Guennoc, C., et al. New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy. J. Vis. Exp. (2017)This video demonstrates the preparation of a fungal culture on treated cellophane membranes to support colony formation and radial growth. It then shows the cultivation and preparation of soil bacterial cells for interaction assays. Finally, it illustrates how bacterial cells adhere to the fungal colony...

Establishing a Fungal-Bacterial Co-culture for Biofilm Formation

This video describes a method to grow and qualitatively analyze bacterial biofilms on fungal hyphae using microscopy. Bacterial biofilms frequently form on fungal surfaces and involve numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation.

A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

Using Coculture to Detect Chemically Mediated Interspecies Interactions

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Learn to construct and use fungal highway columns for enriching bacterial-fungal pairs from environmental substrates. This protocol enables researchers to study microbial interactions and ecological dynamics effectively.

Investigating Bacterial-Fungal Interactions using Fungal Highway Columns in Diverse Environments and Substrates

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