Evolution

TWiM #168: The Lesser of Two Weevils

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 6:10 minutes

The Most Interesting Things (according to students)

Dairy farmers have a more diverse nasal microbiome than non-farmers and those who live in urban environments.

“Allergic and autoimmune diseases had been attributed to lack of exposure to biodiversity, an important factor in regulating immune homeostasis in a healthy host. We posit that the microbiome of healthy dairy farmers (DF) will be richer than non-farmers (NF) living in urban settings due to exposure to a greater biodiversity in the dairy environment. However, no studies have investigated the relationships between microbiota of dairy farmers (DF) compared with urban non-farmers (NF). We compared the nasal and oral microbiota of dairy farmers (N_DF, O_DF, respectively) with nasal and oral microbiota of NF in the same geographical area. The N_DF showed high microbial diversity with hundreds of unique genera that reflected environmental/occupational exposures. The nasal and oral microbiomes clustered separately from each other using Principal Coordinate Analysis, and with DF harboring two-fold and 1.5-fold greater exclusive genera in their nose and mouth respectively, than did non-farmers. Additionally, the N_DF group had a lower burden of Staphylococcus spp. suggesting a correlation between higher microbial diversity and competition for colonization by staphylococci. The N_DF samples were negative for the mecA gene, a marker of methicillin-resistance in staphylococci. The lower burden of staphylococci was found to be independent of the abundance of Corynebacterium spp. Exposure to greater biodiversity could enhance microbial competition, thereby reducing colonization with opportunistic pathogens. Future studies will analyze whether exposure to livestock microbiomes offers protection from acute and chronic diseases.” (Shukla et al. 2017)

1.2. Main paper; discussion starts at 34:40 minutes

The Most Interesting Things (according to students)

A bacteria that associates with weevils suppresses virulence factors after its population increases in the host.

“Sodalis praecaptivus is a close relative and putative environmental progenitor of the widely distributed, insect-associated, Sodalis-allied symbionts. Here we show that mutant strains of S. praecaptivus that lack genetic components of a quorum-sensing (QS) apparatus have a rapid and potent killing phenotype following microinjection into an insect host. Transcriptomic and genetic analyses indicate that insect killing occurs as a consequence of virulence factors, including insecticidal toxins and enzymes that degrade the insect integument, which are normally repressed by QS at high infection densities. This method of regulation suggests that virulence factors are only utilized in early infection to initiate the insect-bacterial association. Once bacteria reach sufficient density in host tissues, the QS circuit represses expression of these harmful genes, facilitating a long-lasting and benign association. We discuss the implications of the functionality of this QS system in the context of establishment and evolution of mutualistic relationships involving these bacteria.” (Enomoto et al. 2017)

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

Snippet Main
Vision and Change Topics
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Metabolic Pathways (V&C_MP)
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
ASM Fundamental Statements
  • 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 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 24 (ASM_24): Microbes and their communities are essential for supporting all life as we know it.
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 17 (ASM_17): Although the flow of information from DNA to RNA to protein is universal in all cells, aspects of the processes of replication, transcription, and translation differ between Bacteria, Archaea, and Eukarya.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recall how the hygiene hypothesis relates to allergies.
  • Identify the main take-home conclusions of the study.
 S L
  • Defend the choice of sampling farmers and non-farmers from the same region.
  • Propose an experiment to test one of the hypotheses from the discussion.
 S H
  • Define quorum sensing.
  • Compare features of vertical and horizontal endosymbiont transmission.
 M L
  • Predict what would be different if S. praecaptivus became a persistent endosymbiont.
 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

  • Metagenomic 16s rDNA Sequencing (12:55–13:55): This technique is a next generation sequencing method where environmental DNA is isolated, used in polymerase chain reactions to amplify a genomic region that is sequenced to determine the species of origin when compared to a database of known species.  Here, the V4 of the 16S rDNA gene was sequenced to identify species in nasal and oral microbiomes.

4.2. Main Paper

  • Bactoblot (40:37–41:23): Thin layer chromatography is used to fractionate cell lysates and then overlaid with a layer of bacteria with a reporter gene to identify the presence of a molecule.  Here, it was used to show S. praecaptivus produces quorum sensing molecule, homoserine lactone.
  • Transcriptomics (44:11–45:10): This is a next generation sequencing technique to identify and quantify the expressed genes, usually in a comparison situation.  Here it was used to determine which genes were expressed and whether the level was different in bacteria in the presence or absence of the quorum molecule, homoserine lactone.

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

5.1. Snippet Paper

  • Hygiene Hypothesis (7:15–8:40): This hypothesis, also called the Friends Hypothesis, is that a lack of exposure to microbes/parasites early in life increases susceptibility to allergies and autoimmune disease.
  • Human Microbiomes (8:40; 9:45–11:00; 18:05–18:30): Dairy farmers have a more diverse nasal microbiota than non-farmers (about 2x more); (23:20) Pseudomonas spp. was more common in non-farmers than farmers.
  • Competition (11:00): A more diverse nasal/oral microbiota associated with fewer amounts of Staphylococci, so there may be competition.
  • Species Richness (14:40): “Chao 1 richness factor” is statistical measure of species richness.
  • Core Microbiome (16:45–18:05; 20:10–23:30): The idea of a group of bacteria as common among all specific environments, such as the nasal cavity.  These would be those common between both farmers and non-farmers.

5.2. Main Paper

  • Endosymbionts:(35:42–36:50; 53:00 –53:30): Only certain taxa of bacteria are predisposed to become endosymbionts; S. praecaptivus are not endosymbionts yet (transient association), and have not lost essential genes.
  • Quorum Sensing (39:20–40:00; 42:15–44:08): Population density dependent gene expression signaled through the local concentration of small metabolites or peptides.
  • Quorum Sensing-based Gene Regulation (40:11–41:23; 44:11–45:57): Sodalis praecaptivus changes its gene expression after the concentration of n-3-oxo-hexanoyl homoserine lactone hits a threshold.
  • Quorum Sensing and Virulence (45:00–45:57; 55:50–57:15): Quorum sensing inactivated virulence genes like chitinases; S. praecaptivus initially expresses virulence factors and causes disease, but once they reach quorum, responsive elements activate disabling virulence and stunting growth.
  • Suppressor Genetic Screen (46:30–48:42): CMP relieves growth suppression caused by HSL.
  • Virulence Regulation (55:00–55:43): Knockouts of two of the four upregulated genes (cmpJ and cmpA) are not able to effectively establish themselves in weevils.

6. Podcast Questions

  1. What is the hygiene hypothesis?
    1. Childhood exposure to microbes increases susceptibility to allergies and autoimmune diseases.
    2. Childhood exposure to microbes increases susceptibility to infectious disease in adulthood.
    3. Lack of childhood exposure to microbes increases susceptibility to infectious disease in adulthood.
    4. Lack of childhood exposure to microbes increases susceptibility to allergies and autoimmune diseases.
  2. Which statements best describe the conclusions of the dairy farmer microbiome study?
    1. Dairy farmers had higher nasal microbiome species diversity than non-dairy farmers.
    2. Non-dairy farmers had higher nasal microbiome species diversity than dairy farmers.
    3. Dairy farmers had higher oral microbiome species diversity than non-dairy farmers.
    4. Non-dairy farmers had higher oral microbiome species diversity than dairy farmers.
  3. Why was the choice of sampling farmers and non-farmers from the same region a good decision?
    1. Farm pathogens may differ based on the specific crops grown and farming techniques used.
    2. The amount of sunlight and rainfall received will influence harvest time and farming practices.
    3. Environmental microbes, and thus microbiomes, may differ from one location to another.
    4. Microbes from different regions are more likely to come from recent, localized infections.
  4. The podcasters discussed several remaining questions regarding the dairy farmer microbiome study including that other microbiome studies suggest that increased diversity is associated with better health.  How could the researchers extend this study to include that aspect?
    1. Analyze the seasonal changes in microbiome diversity and compare to the species diversity.
    2. Quantify the allergies and autoimmune diseases in all groups and compare to species diversity.
    3. Record vitamin supplement usage across all of the groups and compare to species diversity.
    4. Measure the average dairy field exposure in all groups and compare to the species diversity.
  5. Quorum sensing is _______.
    1. A bacterial communication system that enables bacteria to sense population size and regulate gene expression when a threshold population level is achieved.
    2. A bacterial defense mechanism that enables bacteria to produce toxins and attack host cells when nutrients are depleted in the local environment.
    3. A bacterial transport process that enables bacteria to move through tissues and invade organs; bacteria follow a distinct host-derived chemoattractant molecule.
    4. A bacterial replication method that enables bacteria to divide rapidly and increase population size when conditions are favorable, and then disperse as nutrients deplete.
  6. Match the endosymbiont transmission feature with its type. [1 = vertical transmission; 2 = horizontal transmission]
    1. _____ Acquired from environment
    2. _____ Do not express virulence genes
    3. _____ Express virulence genes
    4. _____ Inherited from mother
    5. _____ Not a reduced genome
    6. _____ Reduced genome
  7. What features would likely be different if a strain of S. praecaptivus became a persistent endosymbiont in the weevil?
    1. Lipid composition would be distorted
    2. Virulence gene expression would decrease
    3. Genome size and complexity would increase
    4. The outer membrane would disappear

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 5A) and one from the main paper (Figure 3AB).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features of Venn diagrams.
  • Analyze the data to make conclusions about core and unique microbial phyla for and across environments
  • Analyze the data to make conclusions about microbial phyla abundance differences for and across environments
  • Defend key experimental design choices.

Experimental Background (Shukla et al., Figure 5A)

The incidence of allergies and autoimmune diseases in developed countries has increased since the mid-1960s. In 1989 David Strachan proposed the hygiene hypothesis to explain these data (Strachan 1989).  He suggested that a lack of exposure to microbes in childhood led to dysfunction in the immune system leading to higher incidences of allergies and autoimmune disease in adults.  Here, Shukla et al. (2017) investigate ideas related to this hypothesis, namely how different the microbiome biodiversity might be in people with different environmental exposures.  They compared the nasal and oral microbiome for a sample of dairy farmers and non-farmers in the same local area of Wisconsin, United States.  Their data regarding the unique and common microbial phyla are presented as a Venn diagram (panel A).

A complex Venn diagram with labels and corresponding number of phyla within each group.
Figure 5. “A. Venn diagram of number of common and exclusive phyla represented in N_DF, N_NF, O_DF, and O_NF groups (N_DF = Nasal dairy farmer; N_NF = Nasal non-farmer; O_DF = Oral dairy farmer; O_NF = Oral non-farmer)….” (Shukla et al. 2017, cropped image and text to panel A only)
Data presented in the Venn diagram:
  • N_DF: 431
  • N_DF and O_NF: 43
  • N_DF and O_DF: 168
  • N_DF, O_NF, and O_DF: 44
  • O_DF: 18
  • O_DF and N_NF: 1
  • O_DF, N_NF, and N_DF: 122
  • N_NF: 38
  • N_NF and N_DF: 104
  • N_NF and O_NF: 7
  • N_NF, N_DF, and O_NF: 48
  • O_NF: 12
  • O_NF and O_DF: 3
  • O_NF, O_DF, and N_NF: 3
  • N_DF, O_NF, O_DF, and N_NF: 229

7.1.2. Questions

  1. Match the researchers’ notation to the group being assayed.
Group Identifier Description
a.____ N_DF 1. Nasal microbiome for non-farmer
b.____ N_NF 2. Oral microbiome for non-farmer
c.____ O_DF 3. Nasal microbiome for dairy-farmer
d.____ O_NF 4. Oral microbiome for dairy-farmer
  1. Which Venn diagram color shows the nasal microbiome data for non-farmers?
    1. Coral
    2. Sky blue
    3. Purple
    4. Bright green
  2. How many phyla are common to both groups (farmers and non-farmers) as well as microbiome environments (nasal and oral)?
    1. 3
    2. 7
    3. 48
    4. 229
  3. If there were a common/core set of microbes for a particular environment, how many phyla are in this “core” group for nasal microbiomes (based on these data)?
    1. 279
    2. 287
    3. 503
    4. 563
  4. If you compare the nasal microbiomes of farmers and non-farmers (panel A), how many unique phyla are present in dairy farmers?
    1. 168
    2. 431
    3. 503
    4. 686
  5. Which microbiome shows the greatest diversity of phyla (panel A)?  What is your evidence?
    1. The nasal microbiome of dairy farmers; the coral oval has the highest number.
    2. The nasal microbiome of non- farmers; the sky blue oval is nearest the top.
    3. The oral microbiome of dairy farmers; the purple oval has the lowest number.
    4. The oral microbiome of non- farmers; the green oval is nearest the right side.
  6. Which statements best describe the conclusions of this study? [pick all that apply]
    1. Dairy farmers had higher nasal microbiome diversity than non-dairy farmers.
    2. Non-dairy farmers had higher nasal microbiome diversity than dairy farmers.
    3. Dairy farmers had higher oral microbiome diversity than non-dairy farmers.
    4. Non-dairy farmers had higher oral microbiome diversity than dairy farmers.
    5. Dairy farmers had higher microbiome diversity than non-dairy farmers.
    6. Non-dairy farmers had higher microbiome diversity than dairy farmers.
    7. The nasal microbiome was more diverse than the oral microbiome.
    8. The oral microbiome was more diverse than the nasal microbiome.
    9. There were no large differences between the microbiomes.
  7. Why was the choice of sampling farmers and non-farmers from the same region a good decision?
    1. Farm pathogens may differ based on the specific crops grown and farming techniques used.
    2. The amount of sunlight and rainfall received will influence harvest time and farming practices.
    3. Environmental microbes, and thus microbiomes, may differ from one location to another.
    4. Microbes from different regions are more likely to come from recent, localized infections.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in disk diffusion type assays.
  • Identify key experimental design features such as controls and variable types.
  • Analyze the data to make conclusions about which genes promote and suppress quorum pathway-related growth.
  • Analyze the data to make conclusions about which genes, when mutant, suppress quorum pathway-related growth defects.

Experimental Background (Onomoto et al., Figure 3AB)

Endosymbionts can be mutualistic bacteria that are inherited from the mother organism (vertical transmission) or be acquired from the environment (horizontal transmission).  One of the endosymbionts that displays horizontal transmission is Sodalis praecaptivus which forms a transient relationship with the grain weevil, Sitophilus zeamais.  Near relatives of this species are obligate endosymbionts, so Onomoto et al. (2017) were interested in investigating how S. praecaptivus functions differently than its obligate endosymbiont relative.   In the earlier experiments of this study, the researchers found that S. praecaptivus produces the same quorum sensing molecule as its relative S. glossinidius, N-(3-oxohexanoyl homoserine lactone (OHHL), and also has orthologs for quorum response regulator genes, ypeR and yenR. When they compared the genes expressed in a wild-type bacterium and one lacking the native ability to produce OHHL (ΔypeI)  in a medium containing high exogenous OHHL levels to mimic high population, they found 30 genes were expressed differently with most being down-regulated.  They also noticed the mutant strain (ΔypeI) grew differently than the wild-type strain. To explore OHHL-related differences in growth, the growth of several strains was assayed using variations of the disk diffusion assay (panels A and B).  Here, the wild-type strain was compared to strains with deletions in the quorum response regulator genes (ΔypeR and ΔyenR) when inoculated near or far from an OHHL-impregnated paper strip (panel A; left agar plate) or inoculated near or far from a methanol-impregnated paper strip (panel A; right agar plate).  The upper eight inoculation spots (including those in the red rectangle) were inoculated with 10 fold more bacteria than the bottom eight inoculation spots (including those in the blue/purple rectangles).  To test whether this effect observed with purified OHHL could be replicated using biological conditions, they next switched out the impregnated disks for bacteria that overexpress the OHHL biosynthetic gene from a plasmid (PypeI; panel B; left agar plate) or lack the ability to express OHHL (ΔypeI; panel B; right agar plate).

Agar plates A and B, next to a disk diffusion assay (C), and a line graph of the results (D).
Figure 3. “QS Induces Growth Suppression in S. praecaptivus. (A and B) Each bacterial strain (labeled according to genotype) was spotted in two positions on the plate. (A) shows spots placed either distal (left) or proximal (right) to a strip of sterile paper that was impregnated with exogenous OHHL in methanol (left plate) or methanol alone (right plate). (B) shows spots placed either distal (left) or proximal (right) to a streak of the S. praecaptivus ΔyenR strain maintaining plasmid pCM66 overexpressing the ypeI gene (left plate) or a streak of the S. praecaptivus ΔypeI strain maintaining plasmid pCM66 lacking ypeI (right plate). The spots highlighted in the red boxes have a 10-fold higher concentration of cells than their counterparts highlighted in blue. (C) S. praecaptivus strains (labeled according to genotype) were spotted in two positions on a plate, either proximal (left) or distal (right) to a strip of sterile paper impregnated with exogenous OHHL in methanol (labeled “HSL”). Note that deletion of cpmA alone relieves QS-mediated growth suppression. Deletion of either cpmJ or cstA (another gene whose transcription is increased under quorum) has no effect on growth rate.(D) Assays of growth performed in liquid media containing 1 mg/mL OHHL or no OHHL as indicated by the “−OHHL” suffix. Data were obtained from three biological replicates, and error bars show standard errors. Note that growth is enhanced by deletion of yenR or cpmAJ.” (Onomoto et al. 2017)

7.2.2. Questions

  1. Growth of different bacterial strains is shown in panel A.  In this experiment, the negative control is _____ and the positive control is ______ .
    1. OHHL-impregnated disk plate; methanol-impregnated disk plate
    2. methanol-impregnated disk plate; no positive control
    3. Methanol-impregnated disk plate; OHHL-impregnated disk plate
    4. No negative control; OHHL-impregnated disk plate
  2. What is the difference in the experimental set up that makes the eight inoculation spots at the top of each plate different from the eight inoculation spots on the bottom of each plate?
    1. The bottom spots are inoculated with different strains of bacteria than the top spots.
    2. The bottom spots are a 10 fold concentration of the bacteria used for the top spots.
    3. The bottom spots are exposed to a higher OHHL concentration than the top spots.
    4. The bottom spots are a 10 fold dilution of the bacteria used for the top spots.
  3. Which bacterial strains show growth suppression in the presence of OHHL (panel A)?  What is your evidence? [select all that apply]
    1. ΔyenR; growth is less with OHHL than without OHHL (left plate vs. right plate)
    2. ΔypeR; growth is less with OHHL than without OHHL (right side of each plate)
    3. Wild-type; growth is less with OHHL than without OHHL (right side of each plate)
    4. None; all strains grow equally well with or without OHHL (left plate vs. right plate)
  4. A mutation in which gene enhances the growth of the bacterium, and is it dependent or independent of exposure to the quorum sensor molecule (panel A)?
    1. ΔyenR; OHHL-dependent
    2. ΔyenR; OHHL-independent
    3. ΔypeR; OHHL-dependent
    4. ΔypeR; OHHL-independent
  5. Growth of different bacterial strains is shown in panel B.  In this experiment, the negative control is _____ and the positive control is ______ .
    1. ΔypeI bacteria plate; PypeI bacteria plate
    2. PypeI bacteria plate; ΔypeI bacteria plate
    3. ΔypeI bacteria plate; no positive control
    4. No negative control; PypeI bacteria plate
  6. Do the results using the biological equivalent (panel B) confirm or refute the results with the purified quorum sensor molecule (panel A)?  What is your evidence?
    1. Refute; the growth for the purified OHHL shows more growth (panel A; left) than the plasmid-based OHHL (panel B; left) for all strains.
    2. Confirm; the growth for the purified OHHL shows equal growth (panel A; left) to the plasmid-based OHHL (panel B; left) for all strains.
    3. Refute; the growth for the purified OHHL shows less growth (panel A; left) than the plasmid-based OHHL (panel B; left), but only for some strains.
    4. Confirm; the growth for the purified OHHL shows more growth (panel A; right) than the plasmid-based OHHL (panel A; left) for all strains.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

License

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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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