Information Flow and Genetics

TWiM #196: I Hear You

Podcast and Annotation Information
  • Annotation by Amelia Kung, Hannah Wang, Tiffany Lee, Mahdote Abebe, and Maggie Schlarman, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #196 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Harshita Sharma: Access Podcast Transcripts
  • Papers Discussed:
    • Ranger CM, Biedermann PHW, Phuntumart V, Beligala GU, Ghosh S, Palmquist DE, Mueller R, Barnett J, Schultz PB, Reding ME, et al. 2018. Symbiont selection via alcohol benefits fungus farming by ambrosia beetles. Proceedings of the National Academy of Sciences. 115(17):4447–4452. doi: 10.1073/pnas.1716852115.
    • Jung J, Yoo JE, Choe YH, Park SC, Lee HJ, Lee HJ, Noh B, Kim SH, Kang GY, Lee KM, Yoon SS, Jang DS, Yoon JH, Hyun YM, Choi JY. 2019. Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear. Cell Host Microbe. 25(4):513-525.e6. doi: 10.1016/j.chom.2019.02.001.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:50 minutes

The Most Interesting Things (according to students)

  • It was very interesting to see how ethanol acts as an attractant for ambrosia beetles and how it is used to optimize the beetles’ ability to cultivate specific fungi. Decaying trees produce alcohol which inhibits mold but ambrosia fungi are not affected by alcohol because they have alcohol dehydrogenase activity.
  • Leaf ants have developed their own way of farming for the food that they eat. Even though they are known for cutting leaves, that is not what they actually consume. Instead, they chew up the leaves into pulp and use it as a growth medium for their actual food, which is a type of fungi. They also carry strains of bacteria that create antibiotics to prevent mold from growing on their pulp without harming the fungi.

“Animal–microbe mutualisms are typically maintained by vertical symbiont transmission or partner choice. A third mechanism, screening of high-quality symbionts, has been predicted in theory, but empirical examples are rare. Here we demonstrate that ambrosia beetles rely on ethanol within host trees for promoting gardens of their fungal symbiont and producing offspring. Ethanol has long been known as the main attractant for many of these fungus-farming beetles as they select host trees in which they excavate tunnels and cultivate fungal gardens. More than 300 attacks by Xylosandrus germanus and other species were triggered by baiting trees with ethanol lures, but none of the foundresses established fungal gardens or produced broods unless tree tissues contained in vivo ethanol resulting from irrigation with ethanol solutions. More X. germanus brood were also produced in a rearing substrate containing ethanol. These benefits are a result of increased food supply via the positive effects of ethanol on food-fungus biomass. Selected Ambrosiella and Raffaelea fungal isolates from ethanol-responsive ambrosia beetles profited directly and indirectly by (i) a higher biomass on medium containing ethanol, (ii) strong alcohol dehydrogenase enzymatic activity, and (iii) a competitive advantage over weedy fungal garden competitors (Aspergillus, Penicillium) that are inhibited by ethanol. As ambrosia fungi both detoxify and produce ethanol, they may maintain the selectivity of their alcohol-rich habitat for their own purpose and that of other ethanol-resistant/producing microbes. This resembles biological screening of beneficial symbionts and a potentially widespread, unstudied benefit of alcohol-producing symbionts (e.g., yeasts) in other microbial symbioses.” (Ranger et al. 2018)

1.2. Main paper; discussion starts at 16:05 minutes

The Most Interesting Things (according to students)

Cochlin protein is a powerful player in aggregating bacteria and inducing an immune response to protect hearing.

“In the inner ear, endolymph fluid surrounds the organ of Corti, which is important for auditory function; notably, even slight environmental changes mediated by trauma or infection can have significant consequences. However, it is unclear how the immune response is modulated in these tissues. Here, we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C, Cochlin, and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection, the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly, hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss.” (Jung et al. 2019)

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Information Flow and Genetics (V&C_IFG)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
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 20 (ASM_20) Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.
  • Fundamental Statement 6 (ASM_6) The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatment, and host immunity.
  • Fundamental Statement Statement 16 (ASM_16) Genetic variation can influence microbial structures and their functions.
  • 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 the pro- and anti-fungal events in the ambrosia beetle-fungus symbiotic relationship.
  • Describe how a new colony is formed that contains both beetles and Ambrosiella.
 S L
  • Predict the effect of a mutation in fungal alcohol dehydrogenase on the ambrosia beetle-fungus symbiotic relationship.
 S H
  • Recall where cochlin cleavage products accumulate during infection.
  • Identify the evidence that cochlin is involved in the defense against inner ear bacterial infections.
 M L
  • Analyze the potential consequences of cochlin mutations on hearing function and susceptibility to infections.
 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

  • Chemoattractant Experiments (7:59–8:54): These types of experiments include those where a chemical compound is applied to something and its attraction is measured. Here, ethanol was applied to tree trunks and beetle attraction was measured to demonstrate that ambrosia beetles prefer trees with ethanol.
  • Alcohol Tolerance Mechanisms (9:07–11:31): Several mechanisms might be involved in alcohol tolerance.  Here, the researchers utilized genome level resources to identify that the species had an alcohol dehydrogenase activity.  This explains why beneficial fungi survive ethanol exposure while contaminants do not.
  • Dose-response Experiments (15:16–16:01): These types of experiments include those where different concentrations are used to see if there is a concentration effect. Researchers varied ethanol concentrations in the soil and measured beetle attraction and egg-laying to quantify the relationship between ethanol levels and beetle behavior.

4.2. Main Paper

  • Knock Out Mouse (25:08–27:35): These are mice that have a specific gene deleted.  These researchers used mice with a deletion in the cochlin gene, a cochlin knock out.
  • Auditory Testing (26:06–28:22): Electrodes are placed in the brain to identify brain activity.  Here it was used to assess how mouse hearing activity differed at different frequencies, when the ear was infected, and in cochlin knock out mice.
  • Explant Culture (29:15- 32:00): This is where tissues are removed from the body and cultured in vitro as tissues.  Here, ear explants were cultured for a number of experiments.
  • Bacterial Growth (32:45–36:25): Bacteria growth and/or inhibition can be observed in a number of ways including optical density and colony forming units (CFU).  Here the researchers compared bacterial numbers in infected cochlin-knockout vs wild-type mice to measure Cochlin’s antibacterial effect by quantifying living bacteria present as CFU.
  • Fluorescent Staining (37:31–39:00): This is a method where a protein of interest is engineered to have green fluorescent protein (GFP) attached.  Here, the researchers used it to identify the movement of neutrophils and monocytes into the tissue.

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

5.1. Snippet Paper

  • Symbiosis (2:03–6:41): Beetles and fungi have a mutual relationship where beetles use fungi for food and fungi use beetles for dispersal. Also, leaf cutting ants rely on bacteria that produce antibiotics against mold that could potentially grow on the pulp they create from leaves, while also avoiding harm to the fungi that grow on the pulp, which is their food.
  • Anaerobic Respiration (11:31–12:24): Ethanol is a natural byproduct of anaerobic respiration.

5.2. Main Paper

  • Protein Domains (23:24–23:34): Cochlin is a protein that has three domains, one with homology to a protease involved in immune responses.
  • Inflammation (37:31–39:34): When bacteria enter the cochlea, an inflammatory response occurs to clear out the infection. This also causes swelling and pain that could damage the membranous labyrinths of the ear. Neutrophils and macrophages are attracted to infected areas using cochlin to prevent bacterial spread.

6. Podcast Questions

  1. Which of the following promote or inhibit fungal survival or growth for the symbiont Ambrosiella or other fungi? (Ambrosiella fungus = A; Other fungi or bacteria = O; Inhibit growth or survival = I; Promote growth or survival = P)
    1. ____ Ethanol production by the tree
    2. ____ Queen packs spores into mycangium
    3. ____ Beetles extract sap and coat tunnels
    4. ____ Fungal alcohol dehydrogenase
  2. Order the events for when a new beetle colony is formed formed with 1 being first.
    1. ____ Fungal garden is trimmed
    2. ____ Ethanol attracts the beetle to a new tree
    3. ____ Queen brings fungal spores
    4. ____ Dying tree produces ethanol
    5. ____ Beetles collect sap and coat the tunnels
  3. What would be the most likely effect if the fungi that are moved to the new beetle colony had a deletion in the gene encoding alcohol dehydrogenase?
    1. The new beetle colony would likely move to another tree.
    2. The new beetle colony would die because it lacks food.
    3. The new beetle colony would adapt to using another fungus.
    4. The new beetle colony would use other antifungal compounds.
  4. Into which chamber of the cochlea is soluble Cochlin released when the inner ear is infected?
    1. organ of Corti
    2. scala vestibulli
    3. scala media
    4. scala tympani
  5. What experimental evidence supports the role of cochlin in bacterial containment?
    1. Cochlin-deficient mice have better hearing, but only after infection.
    2. Wild-type mice with Pseudomonas infections lose hearing faster.
    3. Mice without cochlin show increased bacterial spread in the cochlea.
    4. Cochlin knockout mice are completely immune to ear infections.
  6. What would be the most likely outcome for a person with a mutation in the cochlin gene that encoded a protein that was not able to be cleaved by the proteases?
    1. The person would get severe ear infections.
    2. The person would not get inner ear infections.
    3. The person would get only middle ear infections.
    4. The person would not get any ear infections.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 3AB) and one from the main paper (Figure 1DE).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key aspects of the experimental design including controls, variable types, and methodology.
  • Analyze the data to make conclusions about ethanol effects on Ambrosiella grosmanniae growth in laboratory conditions.
  • Deduce the importance of fungal ethanol effects on beetle survival.
  • Predict how environmental ethanol levels in tree hosts are likely to influence beetle colonization success.
Experimental Background (Ranger et al., Figure 3AB)

Ambrosia beetles (Xylosandrus germanus) reside inside decaying trees where they cultivate the fungus Ambrosiella grosmanniae in fungal gardens.  These fungal gardens serve as the beetle’s primary food source and so are important for their survival and reproduction.  Healthy trees normally emit a small amount of ethanol, but trees that are decaying, dying, or stressed have increased levels due to a lack of aerobic respiration.  In this study, Ranger et al. (2018) identified that ambrosia beetles are attracted to trees with higher levels of ethanol.  Knowing that ethanol can be toxic, they next determined the effects of alcohol concentration on beetle larvae and pupae survival.  Having evaluated the beetle side of the symbiosis, they next wanted to understand how ethanol affects the Ambrosiella fungus. To do this, they cultured fungus on media with different ethanol concentrations: 0%, 1%, 2.5%, and 5%.  They quantified two growth features:  biomass (quantified by dry weight; panel A) and spread (quantified by surface area; panel B).

line graphs and agar plates
Figure 3.  “(A–C) Agar plate bioassays characterized the effects of ethanol incorporated into the medium at concentrations of 0%, 1%, 2.5%, or 5% (vol/vol) on the dry weight (A), surface area (B), and density (C) of A. grosmanniae, the fungal symbiont of the ethanol-responsive ambrosia beetle X. germanus. Data points represent mean growth values as a function of ethanol percentage using a standard weighting factor of 1/variance (n = 7‒10 per ethanol concentration; see Table S1 for regression equations). (A) r2 = 0.99; F4 = 22,685.60; P = 0.00004. (B) r2 = 0.99; F4 = 34,770.7; P = 0.00003. (C) r2 =0.91; F4 = 31.68; P = 0.031. (D) Representative growth of A. grosmanniae on agar medium infused with ethanol.” (Ranger et al. 2018)

 

7.1.2. Questions

  1. What concentrations of ethanol were used for the fungal cultures?
    1. 0%, 0.5%, 1%, 2%
    2. 1%, 2%, 3%, 4%, 5%
    3. 0%, 1%, 2.5%, 5%
    4. 0%, 2%, 4%, 6%
  2. In this experiment ______ is the negative control and _______ is the positive control.
    1. 0% ethanol, no positive control
    2. 0% ethanol, 5% ethanol
    3. No negative control; 5% ethanol
    4. 5% ethanol; 0% ethanol
  3. In this experiment, _____ is the dependent variable and ______ is the independent variable.
    1. Malt agar medium; ethanol level
    2. Ethanol concentration; fungal growth
    3. Fungal growth; beetle larvae survival
    4. Fungal growth; ethanol concentration
  4. What is the relationship of Ambrosiella grosmanniae growth (as quantified by biomass) and ethanol concentration (panel A)?
    1. Biomass increases steadily as the ethanol concentration increases.
    2. Biomass peaks at moderate ethanol concentrations, then declines.
    3. Biomass remains constant regardless of the ethanol concentration.
    4. Biomass decreases steadily as the ethanol concentration increases.
  5. What is the relationship of Ambrosiella grosmanniae growth (as quantified by surface spread) and ethanol concentration (panel B)?
    1. Surface area increases steadily as the ethanol concentration increases.
    2. Surface area peaks at moderate ethanol concentrations, then declines.
    3. Surface area remains stable until a sharp drop at higher ethanol levels.
    4. Surface area remains constant regardless of the ethanol concentration.
  6. Why is the response of Ambrosiella grosmanniae to ethanol important for ambrosia beetles?
    1. It determines the beetle’s ability to invade new habitats
    2. It supports the beetles’ immune system defenses
    3. It influences fungal cultivation which is used for nutrition
    4. It determines the strength of the beetle residence trees
  7. If the researchers were able to engineer healthy trees to produce different ethanol levels (0.75%, 1.5%, 2.25%, 4.25%, and 8%), which of the following trees would likely be unable to have successful beetle colonies?
    1. 0.75%
    2. 1.5%
    3. 2.25%
    4. 4.25%
    5. 8%

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify the results you would expect for hearing, reduced hearing, and not hearing in these experiments.
  • Identify key aspects of the experimental design including controls, variable types, and experimental questions.
  • Analyze the data to make conclusions about the impact of cochlin deficiency and Pseudomonas infection on mouse hearing (loudness and pitch).
Experimental Background (Jung et al., Figure 1DE)

The ability to sense the environment is important for a multicellular organism’s survival.  These organs, which include the eyes, ears, nose, and skin provide information about danger as well as nutrition.  When infection occurs in these areas, the sensory input and structures can be compromised.  In this study, Jung et al. (2019) investigate infection in the inner ear and the role of a specific protein, cochlin, in limiting damage to the delicate structures involved in hearing.  To investigate the role of cochlin in hearing and hearing during infection, the researchers acquired mice that were either wild-type (Coch +/+) or had deletions in the cochlin gene (knock out mice; Coch-/-) and infected them (or not) with Pseudomonas aeruginosa, a common ear infection bacterium.  These mice were implanted with electrical response equipment to detect when the auditory nerves were activated (hearing).  The researchers tested two kinds of hearing: the softest “hearable” click (panel D)  and increasing frequency (panel E) for wild-type non-infected mice (Coch+/+; culture media), wild-type infected mice (Coch-/-; Pseudomonas), knock out uninfected mice (Coch-/–; culture media), and knock out infected mice (Coch-/-; Pseudomonas).  Please note that a decibel is a measure of sound loudness and frequency is a measure of sound pitch.

schematic, microscopy, sound races, bar chars, line graph
Figure 1. “Cochlin in the Cochlea Protects Hearing Function during Bacterial Infection (A) Overview of the cochlea. The mammalian cochlea consists of the scala media that harbors the sensory epithelial structure organ of the Corti (filled with endolymph) and the scala tympani/vestibuli (filled with perilymph). The lateral cochlear wall is surrounded by spiral ligaments consisting of fibrocytes and various types of collagen.(B) Cochlin protein (green) is distinctly expressed in the spiral ligaments in the lateral wall and many supporting cells along the basement membrane of the cochlea and crista ampullaris but is not expressed in cochlin knockout mice (Coch−/−). Myosin7A expression (red) indicates cochlear hair cells. Nuclei are stained in blue (DAPI). Cochlin mRNA is dominantly expressed in the spiral ligament at 8 weeks of age (lower panels). Scale bars, 50 μm. (C–E) Representative traces of auditory brainstem response (ABR) tests (C) performed in both Coch+/+ and Coch−/− mice at 4 weeks of age with and without P. aeruginosa infection. The response to click stimuli (n = 13 mice in each group) (D) and tone-burst stimuli at 4000, 8000, 16,000, and 32,000 Hz (n = 4 mice in each group) (E) was evaluated during the ABR tests. ∗∗p < 0.01, ∗p < 0.05 by two-way ANOVA with Bonferroni correction for multiple comparisons. See also Figure S1.” (Jung et al. 2019)

 

7.2.2. Questions

  1. The auditory responses for the different mouse experimental groups (genotypes and infection states) in response to “clicks” of different loudness (decibels) are shown in panel D.  Would a mouse that hears well show a tall bar or a short bar in this assay? Why?
    1. Low; a hearing response was shown by a low decibel sound
    2. High; a hearing response was shown by a low decibel sound
    3. Low; a hearing response was shown by the greatest frequency
    4. High; a hearing response was shown by the smallest frequency
  2. Controls are an important part of any experiment.  The ability to hear the click for each mouse genotype and infection state was quantified, with data shown as a bar plot in panel D.  This experiment has several layers, but there is one “main” control. What is it and what does it tell us?
    1. Coch+/+ Pseudomonas is a control.  It shows the most sensitive hearing we should expect.
    2. Coch+/+ culture medium is a control.  It shows the most sensitive hearing we can expect.
    3. Coch-/- culture medium is a control.  It shows the least sensitive hearing we could expect.
    4. Coch-/- Pseudomonas is a control.  It shows the least sensitive hearing we would expect.
  3. Match the comparison with the question we can answer using the comparison.
Comparison Group Experimental Question 
_____ uninfected wild-type to infected wild-type a. Are infection and cochlin deficiency effects on hearing additive?
_____ uninfected knockout to infected knockout b. Does cochlin deficiency affect hearing?
_____ uninfected wild-type to uninfected knock out c. Does infection affect hearing differently in cochlin deficiency and wild-type?
_____ infected wild-type to infected knock out d. Does infection affect hearing?
  1. The experiment that shows the results for hearing loud/soft sounds is shown in panel D.  What can you conclude from this experiment?
    1. Cochlin-deficiency alone causes hearing loss in mice.
    2. Cochlin-deficiency alone causes hearing enhancement.
    3. Cochlin-deficiency protects from infection-based hearing loss.
    4. Cochlin-deficiency in mice causes infection-based hearing loss.
    5. Infection causes hearing loss for wild-type and cochlin-deficient.
  2. The experiment that shows the results for hearing sounds with different pitches is shown in panel E. What can you conclude from this experiment?
    1. Cochlin-deficiency protects from all infection-based hearing loss.
    2. Cochlin-deficiency causes infection-based hearing loss for all pitches.
    3. Pseudomonas infection causes high pitch hearing loss in both genotypes.
    4. Cochlin-deficient mice have hearing loss in the high pitch range only.
    5. Cochlin-deficient mice have hearing loss in the high pitch range only.

8. Paper Information and Licensing

8.1. Snippet paper

  • Ranger CM, Biedermann PHW, Phuntumart V, Beligala GU, Ghosh S, Palmquist DE, Mueller R, Barnett J, Schultz PB, Reding ME, et al. 2018. Symbiont selection via alcohol benefits fungus farming by ambrosia beetles. Proceedings of the National Academy of Sciences. 115(17):4447–4452. https://doi.org/10.1073/pnas.1716852115.
  • This article is licensed for Creative Commons use using CC BY-NC-ND 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See https://www.pnas.org/doi/full/10.1073/pnas.1716852115 .

8.2. Main paper

  • Jung J, Yoo JE, Choe YH, Park SC, Lee HJ, Lee HJ, Noh B, Kim SH, Kang GY, Lee KM, Yoon SS, Jang DS, Yoon JH, Hyun YM, Choi JY. 2019. Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear. Cell Host Microbe. 25(4):513-525.e6. doi: 10.1016/j.chom.2019.02.001.
  • This article is licensed under an Elsevier user license, which allows non-commercial re-use with proper attribution so long as the product is Creative Commons licensed as CC BY 4.0, which it is.  There are no adaptations or derivatives of the work permitted.  So, the license is equivalent to the Creative Commons CC BY-NC-ND 4.0 license. See https://www.elsevier.com/about/policies-and-standards/copyright/permissions

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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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