Structure and Function

TWiM #245: Bacteria That Protect Bees from Fungi

Podcast and Annotation Information

  • Annotation by Blythe Lander, Isabel Lee, Hanin Ghanam, Laura Brambilla, Alex Bray, and Maggie Schlarman
  • Podcast audio by TWiM: Listen to TWiM #245 Podcast
  • Podcast transcript by Otter.ai and edited by Eden Anderson and Isabelle Norris: Access Podcast Transcripts
  • Papers Discussed:
    • Maeda K, Okuda Y, Enomoto G, Watanabe S, Ikeuchi M. 2021. Biosynthesis of a sulfated exopolysaccharide, synechan, and bloom formation in the model cyanobacterium Synechocystis sp. strain PCC 6803. Elife. doi: 10.7554/eLife.66538
    • Miller DL, Smith EA, Newton ILG. 2021. A Bacterial Symbiont Protects Honey Bees from Fungal Disease. mBio. 12(3):e0050321. doi: 10.1128/mBio.00503-21.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:18 minutes

The Most Interesting Things (according to students)

  • There are industrial applications of sulfated polysaccharides in food, medicine, and materials.
  • The way cyanobacteria regulate buoyancy by trapping gases rather than relying solely on traditional gas vesicles was fascinating, as it highlights a novel mechanism for microbial survival and photosynthesis.

“Extracellular Polysaccharides of bacteria contribute to biofilm formation, stress tolerance, and infectivity. Cyanobacteria, the oxygenic photoautotrophic bacteria, uniquely produce sulfated extracellular polysaccharides among bacteria to support phototrophic biofilms. In addition, sulfated polysaccharides of cyanobacteria and other organisms have been focused as beneficial biomaterial. However, very little is known about their biosynthesis machinery and function in cyanobacteria. Here, we found that the model cyanobacterium, Synechocystis sp. strain PCC 6803, formed bloom-like cell aggregates embedded in sulfated extracellular polysaccharides (designated as synechan) and identified whole set of genes responsible for synechan biosynthesis and its transcriptional regulation, thereby suggesting a model for the synechan biosynthesis apparatus. Because similar genes are found in many cyanobacterial genomes with wide variation, our findings may lead to elucidation of various sulfated polysaccharides, their functions, and their potential application in biotechnology.” (Maeda et al. 2021)

1.2. Main paper; discussion starts at 16:45 minutes

The Most Interesting Things (according to students)

  • A discussion that really engaged me was the conversation on the specific life stages of bees that are most affected by fungi. I would be interested to hear more about what fungi target adult bees, specifically, and if there would be a way to mitigate that while bee larvae are still developing.
  • It was interesting to hear them discuss the feasibility of Bombella apis supplementation, since this may not be a catch-all solution, since Bombella apis may just be one symbiotic bacteria that, in combination with other anti-fungal symbionts, produces a net-positive effect on honeybee fungal infection resistance.

“Fungal pathogens, among other stressors, negatively impact the productivity and population size of honey bees, one of our most important pollinators (1, 2), in particular their brood (larvae and pupae) (3, 4). Understanding the factors that influence disease incidence and prevalence in brood may help us improve colony health and productivity. Here, we examined the capacity of a honey bee-associated bacterium, Bombella apis, to suppress the growth of fungal pathogens and ultimately protect bee brood from infection. Our results showed that strains of B. apis inhibit the growth of two insect fungal pathogens, Beauveria bassiana and Aspergillus flavus, in vitro. This phenotype was recapitulated in vivo; bee broods supplemented with B. apis were significantly less likely to be infected by A. flavus. Additionally, the presence of B. apis reduced sporulation of A. flavus in the few bees that were infected. Analyses of biosynthetic gene clusters across B. apis strains suggest antifungal candidates, including a type 1 polyketide, terpene, and aryl polyene. Secreted metabolites from B. apis alone were sufficient to suppress fungal growth, supporting the hypothesis that fungal inhibition is mediated by an antifungal metabolite. Together, these data suggest that B. apis can suppress fungal infections in bee brood via secretion of an antifungal metabolite.” (Miller et al. 2021)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

Snippet Main
Vision and Change Topics
  • Structure and Function (V&C_SF)
  • Information Flow and Genetics  (V&C_IFG)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host
  • Fundamental Statement 29 (ASM_29): The extent of microbial damage can be minimized by host-derived and external factors, including the microbiome, antibiotics, and immunity.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify characteristics of cyanobacteria.
  • Identify the role(s) of polysaccharides in cyanobacteria life.
  • Recall the uses of cyanobacterial sulfated polysaccharides in different industries.
 S L
  • Analyze the role of synechan in enabling cyanobacteria to float to the surface of a body of water.
 S H
  • Recall the techniques used in the main paper to understand the effects of Bombella apis on fungi in honeybees.
  • Identify the data used to show that a bacterial metabolite is likely the anti-fungal component of Bombella apis effectiveness.
 M L
  • Make a recommendation for a next study on honey bee health based on the results of this study.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

  • Visual Assay (8:15–8:30): This is a test used to measure a biological reaction by eye. In this paper’s case, the assay was used to measure the isolation of polysaccharides from the rest of the bacteria and to measure formation of cyanobacteria blooms.
  • Bioinformatics (8:38–8-45):  This is an interdisciplinary field that uses computational tools and techniques to analyze and interpret biological data, particularly in the context of genomics, proteomics, and other large-scale molecular datasets. It combines elements of computer science, biology, statistics, and mathematics to address complex biological questions.

4.2. Main Paper

  • in vitro Inhibition Assay (28:50–29:05): This is an assay to measure how the addition of a particular molecule negatively affects the production or efficiency of another reaction.
  • Infection Assay (28:50–30:56): This is an assay that measures particles capable of replicating in a particular cell type or animal, infecting others along the way. Raised bee larvae, either on a diet with Bombella apis or a diet without Bombella apis, were then infected with fungi. The infection level of the bees was then evaluated in adulthood.
  • Spent Medium Experiment (33:18–35:20): Spent medium is medium that has been used to grow a culture and contains by products and excreted metabolites produced by the cultured organism.  The medium is usually cleared of living organisms by filtering out the organism or by centrifugation.  Fungal pathogens were cultured either in a combination of fresh and Bombella apis spent medium or in just fresh medium. Fungal growth was sufficiently suppressed by the spent medium, indicating the presence of an anti-fungal metabolite.
  • Antibiotics and Secondary Metabolites Analysis Shell (antiSMASH) (35:30–36:03): This is a computational tool used to identify and analyze biosynthetic gene clusters (BGCs) in bacterial and fungal genomes that encode secondary metabolites, including antibiotics. They used this to help search for loci to understand the identity of the metabolite that prevents the fungi from killing bee larvae.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • Photosynthesis (4:18–4:43): Photosynthesis is the process by which plants and some bacteria use photons of light to produce energy for the cell to use. In the context of the podcast, the podcasters explain that cyanobacteria uses light to photosynthesize, so it has to form blooms and float. The podcast then goes on to discuss the mechanisms for flotation.
  • Flotation Model (5:55–8:38):  Cyanobacteria float due to gas vesicles and exopolysaccharides.
  • Biofilm (9:23–10:42): Diverse bacterial species that adhere to one another and grow on a surface in a thin layer. For example, your teeth (especially unbrushed) and mouth are coated with biofilms.
  • Endotoxins and Exotoxins (12:16–14:20): Sulfated polysaccharides are a crucial part of the floatation mechanism of cyanobacteria, but they are also toxins that have great potential for use in industry, and cyanobacteria are the only bacteria that produce sulfated polysaccharides.

5.2. Main Paper

  • Bacterial Symbiosis (18:23–20:40): A close interacting relationship between two different organisms, such as bees and bacteria in the paper.
  • Microbial Defense Mechanisms (20:40–25:00): Defense mechanisms are how beneficial bacteria help protect hosts from infections. The podcasters discuss how Bombella has a gene producing polyketide synthases, which is characteristic of other antifungal-producing microbes. They discuss whether supplementation of Bombella in honeybee diet is a feasible solution to honeybee fungal infection.
  • Animal Microbiome (26:53–28:30): Bombella apis is the most prevalent bacterial species found in the microbiome of honeybee larva, and increased concentration of Bombella in adults correlates with lower fungal infection by Nosema.
  • Selective Medium (31:15–31:25): This is a medium that is used to grow specific microorganisms while inhibiting the growth of others.

6. Podcast Questions

  1. What are some characteristics of cyanobacteria?
    1. They live in low salt environments.
    2. They have type n pili.
    3. They form biofilms.
    4. They produce oxygen.
    5. They have anti-gravity properties.
    6. Are prokaryotes
  2. What role do polysaccharides play in cyanobacteria’s ability to perform photosynthesis in ocean environments?
    1. They are mediators to help balance ATP production using photosynthesis and chemoautotrophy.
    2. They provide structural support so cells have a large surface area for optimal UV absorption.​
    3. They assist in photosynthesis-driven nutrient absorption from seawater using transport pumps.​
    4. They aid buoyancy and aggregation which keep them at the surface where sunlight is available.
  3. What is synechan and what unique structure and behavior does it provide cyanobacteria?
    1. Synechan is a sulfated polysaccharide involved in producing a viscous capsule that captures extracellular gas, allowing cyanobacteria to float and form blooms.
    2. Synechan is a heavily phosphorylated polysaccharide involved in the removal of intracellular hydrogen gas from the cell, allowing them to sink and form blooms.
    3. Synechan is a hydrophobic polysaccharide involved in the capture of carbon dioxide within intracellular vesicles, allowing cyanobacteria to float and form blooms.
    4. Synechan is an sulfated polysaccharide involved in capturing oxygen within intracellular vesicles, allowing cyanobacteria lose density, sink, and form blooms.
  4. How are sulfated polysaccharides used in industry?
    1. They improve the efficiency of water treatment plants
    2. They act as natural preservatives in food processing
    3. They enhance the texture of food products like ice cream
    4. They aid in the production of biodegradable plastics
  5. In vitro inhibition assays showed that Bombella apis has antifungal properties.  To determine the source of this property, the researchers used “spent medium” which is medium that was used to grow Bombella apis bacterium.  This “spent medium” was cleared of bacteria and treated with different destructive treatments (heat, protease) to determine the nature of the anti-fungal component.  The fungus was still inhibited in “spent medium” treated with heat and proteases.  What did this result indicate to the researchers?
    1. The antifungal is sensitive to heat-killing.
    2. The antifungal is a small 20 amino acid peptide.
    3. The antifungal is not a peptide or a protein.
    4. The antifungal is a type III polysaccharide.
  6. Which of the following is a good recommendation for a next step in promoting honeybee health based on this study?
    1. Bombella apis could be tested as a way to infect and kill bee predators to reduce honey bee predation.
    2. Bombella apis could be tested as a probiotic to strengthen honey bee immunity to fungal infection.
    3. Bombella apis could be tested as a chemosensor to direct honeybees to more nutrient-rich nectar
    4. Bombella apis could be tested as a regulator to increase immunity-related enzymes in honeybees.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1) and one from the main paper (Figure 1).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify how you can visually confirm biofilm/aggregate formation.
  • Identify important features of bar charts.
  • Defend the conclusions that bloom formation depends on photosynthesis and active EPS production by identifying relevant data.
  • Use data to evaluate the functional role of slr5054 in exopolysaccharide production and/or bloom formation.
  • Propose a model for EPS involvement in biofilm aggregation and flotation based on these data.

Experimental Background (Maeda et al., Figure 1)

Cyanobacteria are important members of Earth’s ecosystem, producing oxygen photosynthetically and contributing to global nitrogen cycling. They are involved in cyanobacterial blooms (formerly known as algal blooms) in the ocean in which they dynamically switch between floating biofilm structure and planktonic structure.  As a group, these prokaryotes make extracellular sulfated polysaccharides that allow them to form the aggregates.  These polysaccharides have medical, biotechnological, and environmental applications. To learn more about these polysaccharides, Maeda et al. (2021) investigated the type, production, and function of the sulfated polysaccharide, which they call sulfated EPS or synechan, produced by a substrain of Synechocystis sp. PCC 6803 that forms floating aggregates. Their first experiment involved growing the cyanobacterium to characterize biofilm/aggregate formation over time (panels A, B).  Next, they investigated whether aggregate formation was dependent on light and/or photosynthesis by growing the wild-type and non-motile variant cyanobacterium with and without light, as well as with an inhibitor of photosynthesis (panel C). The researchers then used bioinformatics to identify genes that might encode membrane-bound glycosyltransferases, which are important for EPS biosynthesis.  They investigated how deletion of one of the putative membrane-bound glycosyltransferases, Slr5054, whose predicted domain structure is shown in panel D, affected biofilm formation (panel E). Finally, to determine whether the change in biofilm/aggregation might be due to a change in polysaccharide, the researchers examined capsular polysaccharide, extracellular polysaccharide, and free polysaccharide in wild-type and the strain missing Slr5054 (delta Slr5054) (panels F, G).

Bloom schematics and bar charts.
Figure 1. “Bloom formation and exopolysaccharides (EPS) isolation. (A) Time course of bloom formation by wild-type (WT) Synechocystis 6803 during the second step of culture. Extracellular gas bubbles are formed and trapped in viscous EPS (~1 hr). Green vertical columns with bubbles become apparent at 4 hr. Those trapped gas bubbles slowly rise together with the viscous columns. (B) Enlarged images showing gas bubbles trapped in EPS. Vertically aligned bubbles are indicated by red arrows. (C) Lack of bloom formation in the non-motile substrain (NM) or WT with or without light and the photosynthesis inhibitor DCMU at 48 hr of the second step of culture. (D) Domain architecture of Slr5054. GT, glycosyltransferase domain; TM, transmembrane region. (E) Lack of bloom formation in Dslr5054 after standing culture for 48 hr. (F) Isolation of EPS from the first step of culture. Cells and capsular polysaccharides (CPS) were removed from the culture by centrifugation, and EPS in the supernatant was separated from ‘free’ polysaccharide (PS) by membrane filtration followed by a second centrifugation to remove residual cells. CPS was collected from the cell pellet after vortexing and centrifugation. (G) Sugar content of fractions from WT and Dslr5054. Error bars represent SD (CPS, n = 6; others, n = 3; **p<0.005).” (Maeda et al. 2021)

7.1.2. Questions

  1. As the cyanobacteria culture grows (panels A and B), what features do you see that confirm floating aggregates are forming?
    1. Clear masses rise and fall with the 12 hour dark-light cycling of photosynthesis.
    2. Clear masses are found near the bottom of the tube where cells collect due to gravity.
    3. Green masses are rising to the top of the tube and trapped air bubbles are found.
    4. Green masses are distributed evenly in the tube and grow more abundant over time.
  2. The researchers evaluated changes in capsular (CPS), extracellular (EPS), and free polysaccharides in wild-type and the strain missing a gene encoding a putative membrane-bound glycosyltransferase, Slr5054 (Δslr5054) and display the data as a bar chart (panel G).  Which of the three bar charts show(s) the wild-type and variant strain are different, and what feature in the graph(s) indicates this?
    1. All of them show the wild-type and variant are different because the bars are different heights.
    2. CPS and EPS show the wild-type and variant are different because the bars are different heights.
    3. Only CPS is different between wild-type and variant because there is a statistical difference (*).
    4. None of them show a difference between the wild-type and variant for any of the polysaccharides.
  3. The researchers evaluated the role of the putative membrane-bound glycosyltransferase, Slr5054, in production of in different polysaccharides by comparing the wild-type bacterium to a strain missing a gene encoding a putative membrane-bound glycosyltransferase, Slr5054 (Δslr5054). They used a fractionation procedure (panel F) to then quantify capsular (CPS), extracellular (EPS), and free polysaccharides] in each bacterium (panel G).  What do these results indicate about the normal function of they putative glycosyltranferase, Slr5054?
    1. This gene does not in fact encode a membrane-bound glycosyltransferase.
    2. This gene is involved in formation of EPS, but not CPS or free polysaccharides.
    3. This gene encodes a gene necessary for photosynthesis and oxygen generation.
    4. This gene encodes a glycosyltransferase for production of free polysaccharides.
  4. Which data support the conclusion that bloom formation depends on photosynthesis? [Pick all that apply]
    1. Bloom formation was absent when light was absent.
    2. EPS accumulation was higher in the non-motile strain.
    3. DCMU treatment resulted in continued bloom formation.
    4. The slr5054 deletion (Δslr5054) enhanced bloom formation.
    5. Bloom formation was absent with DCMU treatment.
  5. Based on these studies, propose a model for how exopolysaccharides (EPS) are involved in the flotation and biofilm formation of cyanobacteria.
    1. Exopolysaccharides aid in the formation of intracellular gas vesicles, most abundantly in light conditions, and are used to float.
    2. Exopolysaccharides form a viscous extracellular medium that traps gas bubbles drawing the cyanobacteria to the surface.
    3. Exopolysaccharides help individual cyanobacteria adhere to each other and form biofilms only at the aerated surface of water.
    4. Exopolysaccharides are a byproduct of photosynthesis and reduce the density, which helps them to float up the water column.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify important features of the experimental design, box plots, and statistical measures.
  • Propose an improvement to the experimental design.
  • Analyze the data to draw conclusions about fungal growth inhibition.
  • Draw conclusions based on hypothetical alternate results for the experiment.

Experimental Background (Miller et al., Figure 1)

Honey bees are important pollinators, pollinating over one third of all food crops in the United States.  Honey bees also play significant roles in ecosystem sustainability. The health and maintenance of honey bee populations is therefore important for food security and ecosystem health.  The leading cause of disease in insects, like bees, are pathogenic fungal infections.   In this study Miller et al. (2021) investigate the ability of a bee microbiome bacterial species, Bombella apis, to combat fungal infections of Aspergillus flavus and Beauveria bassiana in bee development.  First, the researchers tested the ability of  B. apis to affect fungal growth from spores.  To do this, they inoculated bacterial lawns of four strains of B. apis (A29, B8, C6, SME1), then added spores of the two fungal pathogens and examined the plates for fungal growth (panels a, b).   Because growth on agar plates can sometimes be different in liquid cultures, the researchers next tested the ability of each B. apis strain to affect spore formation when grown in a liquid co-culture with each fungus.  The researchers were able to quantify the fungal  growth, which is displayed for  B. bassiana  in panel c and A. flavus in panel d.

Schematics, plate growth photos, and box plots showing the growth of fungal pathogens in the presence of B. apis.
Figure 1.  “B. apis outcompetes fungal pathogens in vitro. (a) The ability of each fungal isolate to grow in the presence of B. apis was qualitatively assayed by plating 103 spores of each isolate across a lawn of B. apis. (b) Compared to controls of 103 spores plated on fresh media, the presence of B. apis completely inhibited fungal growth. (c) When cocultured in liquid media, the presence of B. apis significantly reduced the number of spores produced by B. bassiana (Kruskal-Wallis; x 2 = 11.7, df = 4, P = 0.01973; pairwise comparisons to control A29, t = 13.114, df = 2.0996, P = 0.019056; B8, t = 11.147, df = 2.9658, P = 0.00652; C6, t = 10.121, df = 2.7744, P = 0.011404; SME1, t = 12.352, df = 2.0277, P = 0.024652). (d) B. apis also significantly reduced the number of spores produced by A. flavus (Kruskal-Wallis; x 2 = 9.9, df = 4, P = 0.04215); however, pairwise comparisons between control and SM from each strain were not significantly different due to the variation in the control samples. To control for nutritional effects, in panels c and d, experimental wells contained the same volume of fresh media as the controls in addition to B. apis culture. Each experimental group consists of three biological replicates. Sporulation was quantified for each well via hemocytometer.” (Miller et al. 2021, no changes).

7.2.2. Questions

  1. Good experimental design includes controls.  What control condition was included in the agar plate fungal growth experiment (panel b)? What was the purpose of using this as a control?
    1. Fungal spores plated on media without any bacteria;  It was used as a comparison to show reduction from maximal expected fungal growth.
    2. Fungal spores plated on a different bacterial species; It was used to compare the growth of the different B. apis plated on top of the fungal lawn.
    3. Bacterial lawn with fungal spores and antifungal drugs; It was used as a comparison to show enhancement above the expected growth of B. apis.
    4. Media containing only B. apis without fungal spores; It was used as a baseline for quantifying symbiotic growth of the fungal spores and B. apis.
  2. Fungal growth was measured in liquid co-culture with different strains of B. apis to generate the data in panels and d.  The data are displayed by box plots. Match the box plot feature with its description. (1 = mean; 2 = median; 3 = outlier; 4 = interquartile range; 5 = individual measures; 6= full data range excluding outliers)
    1. Box =
    2. Horizontal line in box =
    3. Whiskers =
    4. Symbols, such as circles =
  3. Fungal growth was measured in liquid co-culture with different strains of B. apis to generate the data in panels and d.  The data are displayed by box plots and the statistical tests performed, with the results noted in the figure legend. Which of the following pairwise comparisons show evidence they are statistically significantly different? [Pick all that apply]
    1. Control B. bassiana; A29
    2. Control B. bassiana; B8
    3. Control B. bassiana; C6
    4. Control B. bassiana; SME6
    5. Control A. flavus; A29
    6. Control A. flavus; B8
    7. Control A. flavus; C6
    8. Control A. flavus; SME6
  4. How did the presence of different B. apis strains affect fungal spore growth on plates? [Pick all that apply]
    1. Fungal growth was enhanced by all strains.
    2. Fungal growth was dependent on the strain used.
    3. Fungal growth was inhibited by all B. apis strains.
    4. Only the SME1 B. apis strain inhibited fungal growth.
  5. If you performed the same experiment noted in panels a and b with a different fungus that was not sensitive to the B. apis-derived component, what would you expect to see?
    1. No growth on any of the plates except the control (no B. apis).
    2. Growth on all of the plates including the control (no B. apis).
    3. Reduced growth on B. apis plates compared to the control.
    4. Growth on some B. apis plates, but not all B. apis plates.
  6. Which of the following would improve the experimental design?
    1. Use a non-fungicidal bacteria as a control rather than no bacterium.
    2. Use a known fungicidal bacterium as a positive control for inhibition.
    3. Use a known fungicide as a positive control rather than no positive control.
    4. All of the above.
    5. None of the above.

8. Paper Information and Licensing

8.1. Snippet paper

  • Maeda K, Okuda Y, Enomoto G, Watanabe S, Ikeuchi M. 2021. Biosynthesis of a sulfated exopolysaccharide, synechan, and bloom formation in the model cyanobacterium Synechocystis sp. strain PCC 6803. Elife. doi: 10.7554/eLife.66538
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article’s information on its web page.

8.2. Main paper

  • Miller DL, Smith EA, Newton ILG. 2021. A Bacterial Symbiont Protects Honey Bees from Fungal Disease. mBio.12(3):e0050321. doi: 10.1128/mBio.00503-21.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article’s informaiton on its web page.

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Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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