Microbial Ecology

TWiM #258: A Tick’s Meal

Podcast and Annotation Information
  • Annotation by Matthew Longoria, Isabella Matta, Monique Ortega, Janani Rathinam, Nicole L. Podnecky, Rebecca Seipelt-Thiemann, and Maia Larios-Sanz.
  • Podcast audio by TWiM: Listen to TWiM #258 Podcast
  • Podcast transcript Otter.ai and edited by Matthew Longoria, Isabella Matta, Monique Ortega, Janani Rathinam, Maia Larios-Sanz, and Isabelle Norris: Access Podcast Transcripts
  • Papers Discussed:
    • Zhong Z, Zhong T, Peng Y, Zhou X, Wang Z, Tang H, and Wang J. 2021. Symbiont-regulated serotonin biosynthesis modulates tick feeding activity. Cell Host Microbe. 29:1545-1557. doi: 10.1016/j.chom.2021.08.011
    • Spagnolo F, Trujillo M, and Dennehy JJ. 2021. Why do antibiotics exist? mBio.12(6): e01966-21. doi: 10.1128/mbio.01966-21

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:19 minutes

The Most Interesting Things (according to students) 

Ticks are involved in a symbiotic relationship with Coxiella bacteria, which provide basic nutrients for their host but also basically tell the ticks when to feed through metabolic pathways.

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

1.2. Main paper; discussion starts at 20:04 minutes

The Most Interesting Things (according to students)

Antibiotics have been here for millions of years but antibiotic resistance is largely a human-augmented problem due to overuse in high concentrations – something bacteria that naturally produce these antibiotic compounds do not do.

“In the struggle with antibiotic resistance, we are losing. There is now a serious threat of moving into a postantibiotic world. High levels of resistance, in terms of both frequency and strength, have evolved against all clinically approved antibiotics worldwide. The usable life span of new clinically approved antibiotics is typically less than a decade before resistance reaches frequencies so high as to require only guarded usage. However, microbes have produced antibiotics for millennia without resistance becoming an existential issue. If resistance is the inevitable consequence of antibiotic usage, as has been the human experience, why has it not become an issue for microbes as well, especially since resistance genes are as prevalent in nature as the genes responsible for antibiotic production? Here, we ask how antibiotics can exist given the almost ubiquitous presence of resistance genes in the very microbes that have produced and used antibiotics since before humans walked the planet. We find that the context of both production and usage of antibiotics by microbes may be key to understanding how resistance is managed over time, with antibiotic synthesis and resistance existing in a paired relationship, much like a cipher and key, that impacts microbial community assembly. Finally, we put forward the cohesive, ecologically based “secret society” hypothesis to explain the longevity of antibiotics in nature.” (Spagnolo et al. 2021, no changes)

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

Snippet Main
Vision and Change Topics
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Metabolic Pathways (V&C_MP)
  • Evolution (V&C_E)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • 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 2 (ASM_2): The diversity of microbes has arisen because of processes that include horizontal gene transfer, mutation, reassortment, recombination, and natural selection in varying ecological niches favor the growth and survival of certain variants.
  • Fundamental Statement 3 (ASM_3): The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recall features of tick feeding behaviors.
  • Recall the conclusions of the experiments, particularly those involving tetracycline, serotonin, and chorismate.
  • Identify how the tick host and the Coxiella endosymbiont benefit from their interaction.
 S L
  • Predict the effect of altering other aspects of the endosymbiont metabolism on the tick’s feeding behavior.
 S H
  • Recall the length of time an antibiotic is in clinical use before resistance is observed.
  • Identify the hypothesis for why antibiotic resistance observed in human pathogens differs from antibiotic resistance observed in natural environments.
 M L
  • Suggest factors that should be controlled in a mouse infection model for determining optimal antibiotic dose/time combinations.
 M H

1Papers: Snippet (S) or Main (M)
2Learning 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

  • Tick Feeding Assays (5:50–6:15):  These are assays to determine tick feeding behaviors.  Here, antibiotics were used to deplete Coxiella populations and other microbiota to study the effect of infection with specific bacteria on tick feeding.
  • Transcriptome Analysis (7:00–7:53):  This is a next generation sequencing technology that is used to identify and quantify all genes expressed in specific conditions.  Here, RNA sequencing was used to identify tick genes whose expression was affected by presence/absence of Coxiella.
  • Gene Knockdown (9:43–11:32): This is a technique to remove gene expression without deleting the gene from the genome.  Here, a double stranded RNA was used to reduce the protein level for tryptophan hydroxylase, which would then inhibit synthesis of serotonin.

4.2. Main Paper

Note: The main paper is a review exploring consideration of ecology principles to combating antibiotic resistance, and thus no techniques are extensively discussed.

  • Pharmacokinetics and Pharmacodynamics (41:20–42:45): These are measures or features of a drug’s metabolism in the body.  Here, the podcasters discuss the curves produced to evaluate antibiotic concentration in a patient over time – a method to optimize dosage.

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

5.1. Snippet Paper

  • Life-cycle of a Tick & Vectors of Disease (2:09–2:53):  Ticks develop from eggs, going through larvae and nymph stages, and days to weeks feeding on their hosts, during which they can transfer microbes to the host, particularly Lyme disease.
  • Endosymbionts (3:40–5:40): Gut microbiota provide nutrients (e.g., amino acids, vitamins, cofactors) to their hosts but can also have important influence in host health and behavior. In this paper, special focus is on Coxiella, which produces chorismate – a precursor of amino acids and also serotonin, which controls tick feeding behavior.
  • Amino Acid Metabolism & Shikimate Pathway (7:45–9:15): Coxiella species use shikimate pathway to synthesize chorismate, a precursor for serotonin and aromatic amino acids.
  • Coevolution (16:57–18:30): Microflora coevolves with their host, genome of microbes like Coxiella is often reduced since host provides most things needed for survival. Microbe in turn provides essential nutrients to the host.

5.2. Main Paper

  • Antibiotic Resistance & Stewardship (21:33–29:27): Worrisome trends of increased frequency and strength of antimicrobial resistance.  Resistance develops in very short time scales.
  • Fitness (29:30–35:10):  Ability to pass on genetic instructions to new generations. Tradeoffs usually imply costs to the organism to achieve fitness gains.
  • Metagenomics vs “Pure Culture” Microbiology (35:17–36:20): Alluding to the idea that we need to move away from studying microbes in pure culture, and start looking at the entire community using techniques such as metagenomics.
  • Pharmacokinetics & Pharmacodynamics (41:15–43:02): Clinical pharmacology analysis that enables studies of drug effects and determinants of drug action.
  • Zoonosis (48:26–48:48): transfer of wild animal viruses to susceptible human populations, usually through intrusion into ecological settings or contact with wildlife.

6. Podcast Questions

  1. True/False:
    1. _______ Ticks can remain attached for weeks.
    2. _______ Ticks can consumer 1000 times their body weight.
    3. _______Treatment with tetracycline affects feeding behavior because it depletes Coxiella.
    4. _______Ticks will produce more serotonin without Coxiella endosymbionts.
  2. Based on experimental evidence, which of the following best describes how Coxiella affects tick hosts?
    1. Ticks become infected with Coxiella and are too sick to obtain good blood meals.
    2. Coxiella activate amino acid biosynthesis pathways of the host tick.
    3. Coixella provide chorismate, a precursor for the production of serotonin.
    4. Coxiella encode for the shikimate pathway that reduces amino acid biosynthesis.
  3. Coxiella and the tick are in an endosymbiotic relationship. Which of these best describes the effect of the tick on Coxiella?
    1. Coxiella are targeted by the tick immune system, triggering the stress response.
    2. Coxiella benefit from the presence of blood and iron in the tick midgut.
    3. Coixella grow faster in the tick, since they get more high energy nutrients.
    4. Coxiella benefit by receiving essential nutrients they lack from the tick.
  4. How did experimental results differ in ticks that are treated or untreated with tetracycline but supplemented with chorismate?
    1. Tetracycline treated ticks will feed poorly independent of chorismate supplement.
    2. Chorismate supplemented ticks will feed poorly independent of tetracycline treatment.
    3. Chorismate supplemented ticks will feed well independent of tetracycline treatment.
    4. None of the above.
  5. The researchers identify the shikimate pathway as a contributor to tick feeding behavior.  The best experiment would compare all four conditions: (1) Coxiella wild-type, (2) no Coxiella (tetracycline treated ticks), (3) Coxiella mutants lacking  individual shikimate pathway genes, and (4) shikimate pathway genes expressed on a plasmid in the Coxiella mutants (a mutation rescue). What feeding behavior would you expect for ticks that are colonized with Coxiella mutants in the shikimate pathway?  Why do you expect this result?
    1. decreased feeding in mutants and antibiotic-treated compared to wild-type and rescued strain; chorismate is synthesized by the shikimate pathway
    2. increased feeding in mutants and antibiotic-treated compared to wild-type and rescued strain; chorismate synthesis is blocked by the shikimate pathway
    3. decreased feeding in mutants and wild-type compared to the antibiotic-treated and rescued strain; serotonin is synthesized by the shikimate pathway
    4. increased feeding mutants and wild-type compared to the antibiotic-treated and rescued strain; serotonin synthesis is blocked by the shikimate pathway
  6. How long is typical for resistance to be observed once an antibiotic starts clinical use?
    1. 1 year
    2. 5 years
    3. 10 years
    4. 15 years
  7. What did the podcasters conclude about the reason antibiotic resistance in human pathogens is on the rise, but is not observed in frequency or strength in natural populations?
    1. Microbes evolve slowly under pressure from antibiotics, so resistance is rarely a concern.
    2. Microbes are destroyed completely by antibiotics, so resistance is not a major problem.
    3. Microbes avoid contact with antibiotics naturally, so resistance does not often occur.
    4. Microbes manage antibiotic use better than humans, so resistance doesn’t usually develop.
  8. The podcasters note that a seven day course of antibiotics is often used simply because a week has seven days and not because this was empirically determined to be the best treatment time. You could test the optimal treatment time for an antibiotic using a model infection model.  What factors do you think would affect this optimal time/dose? [pick all that apply]
    1. the specific pathogen strain
    2. the route of antibiotic delivery
    3. the specific antibiotic mechanism
    4. the tissue of infection
    5. the other bacteria present
    6. the specific mouse strain used

7. Figure Reading Exercises

The following are two figure reading exercises from the snippet paper (Figure 1 and Figure 2AB).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features in strip plots.
  • Identify experimental design features, such as controls and variable types.
  • Evaluate the data to make conclusions regarding antibiotic treatment on Coxiella and other bacteria.
  • Evaluate the data to make conclusions about the effect of Coxiella on tick feeding behavior.
  • Predict the outcome of a similar experiment using a different antibiotic.
Experimental Background (Zhong et al., Figure 1)

Insect-borne diseases cause more than 700,000 deaths each year.  Expanding insect habitats due to climate changes are thought to be responsible for the near doubling of infections in the last 20 years.  Ticks are a common insect vector, carrying a number of pathogens, including Lyme disease, that are transferred to the host during feeding. Ticks also harbor specific bacterial endosymbionts such as Coxiella (CHI) that can provide essential vitamins and cofactors to ticks, but the impact of these endosymbionts on tick feeding behavior is largely unknown.  In this study Zhong et al. (2021) aimed to investigate the relationship between Coxiella symbiosis and tick feeding using the Asian longhorned tick, Haemaphysalis longicornis).  The researchers were first interested in how the presence of Coxiella affected tick feeding behaviors, so they eliminated CHI from ticks using the antibiotic tetracycline, which should be specific for Coxiella.  First, to confirm this, they treated ticks with TET and quantified both CHI bacterial load (panel A) and total bacteria (panel B) using quantitative polymerase chain reaction (qPCR). To assess the feeding behavior changes, they compared the weights and appearance of untreated, TET-treated, and control-treated nymphs that had been allowed to feed for 4 days (panel C). Next, to determine whether the effects they observed were specific to tetracycline or due to general antibiotic treatment, they treated ticks with an antibiotic cocktail including penicillin, streptomycin, and gentamycin, and again assayed CHI bacterial load (panel D) total bacteria abundance (panel E), and tick feeding behavior (panel F).

  • The abstract and figures cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

7.1.2. Questions

  1. Match the treatment with the acronym used in the graphs.
Acronym Treatment
a. ___3_____ TET 1. 0.9% saline
b. ___1_____ CT 2. penicillin, streptomycin, gentamycin
c. ___2_____ PSG 3. tetracycline
d. ____4____ N 4. untreated
  1. In each panel the data for a single treatment are displayed as symbols and a horizontal bar.  What do these represent, respectively?
    1. mean; individual data points
    2. median; individual data points
    3. mode; standard deviation
    4. individual data points; mean
  2. Which sample in panels A and B shows the smallest variability?  What feature in the data points tells you this?
    1. CT at day 2; the loose distribution of data points
    2. TET at day 2; the tight clustering of data points
    3. CT at day 4; the high fold change for this treatment
    4. TET at day 4; the low fold change for this treatment
  3. The purpose for the use of 0.9% NaCl in these experiments is to                          .
    1. feed the ticks.
    2. feed the Coxiella.
    3. kill the Coxiella.
    4. serve as a control.
  4. Ticks were treated with tetracycline or saline and counts of Coxiella and total bacteria were quantified (panels A and B).  What can you conclude from these data?
    1. Tetracycline reduces both Coxiella and total microbiota.
    2. Tetracycline reduces Coxiella but not total microbiota.
    3. Tetracycline reduces total microbiota but not Coxiella.
    4. Tetracycline has no effect on Coxiella or total microbiota.
  5. Ticks were treated with an antibiotic cocktail or saline and counts of Coxiella and total bacteria were quantified (panels D and E). What can you conclude from these data?
    1. The antibiotic cocktail reduces total microbiota, but not Coxiella.
    2. The antibiotic cocktail reduces both Coxiella and total microbiota.
    3. The antibiotic cocktail reduces Coxiella, but not total microbiota.
    4. The antibiotic cocktail has no effect on Coxiella or total microbiota.
  6. Young tick (nymph) feeding behaviors for untreated, tetracycline-treated, and saline-treated ticks were quantified by weighing nymphs after feeding (panel C).  What can you conclude from these data?
    1. Tetracycline slightly increased feeding in tick nymphs when compared to untreated and control.
    2. Tetracycline significantly increased feeding in tick nymphs when compared to untreated and control.
    3. Tetracycline had no effect on feeding in tick nymphs when compared to untreated and control.
    4. Tetracycline significantly decreased feeding in tick nymphs when compared to untreated and control.
    5. Tetracycline slightly decreased feeding in tick nymphs when compared to untreated and control.
  7. Young tick (nymph) feeding behaviors for untreated, antibiotic cocktail-treated, and saline-treated ticks were quantified by weighing nymphs after feeding (panel C).  What can you conclude from these data?
    1. Antibiotic cocktail treatment slightly increased feeding in tick nymphs when compared to untreated and control.
    2. Antibiotic cocktail treatment increased feeding in tick nymphs when compared to untreated and control.
    3. Antibiotic cocktail treatment had no effect on feeding in tick nymphs when compared to untreated and control.
    4. Antibiotic cocktail treatment decreased feeding in tick nymphs when compared to untreated and control.
    5. Antibiotic cocktail treatment slightly decreased feeding in tick nymphs when compared to untreated and control.
  8. If the researchers treated nymph ticks with ciprofloxacin, a broad-spectrum antibiotic, that experimentally is not very effective against Coxiella burnetii, how do you think this would affect Coxiella abundance, microbiota abundance, and tick feeding? Ciprofloxacin will likely                           .
    1. Only reduce the microbiota of the tick and increase feeding.
    2. Reduce the microbiota and Coxiella,and reduce feeding.
    3. Only reduce the Coxiella significantly and not alter feeding.
    4. Only reduce the microbiota and not alter feeding or Coxiella.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in a volcano plat and strip plot.
  • Analyze gene expression data features of a volcano plot.
  • Evaluate an enrichment plot to make conclusions about the pathways most affected and patterns amongst them.
  •  Predict gene expression changes for particular changes to the Coxiella-tick interaction.
Experimental Background (Zhong et al., Figure 2AB)

Insect-borne diseases cause more than 700,000 deaths each year.  Expanding insect habitats due to climate changes are thought to be responsible for the near doubling of infections in the last 20 years.  Ticks are a common insect vector, carrying a number of pathogens, including Lyme disease, that are transferred to the host during feeding. Ticks also harbor specific bacterial endosymbionts such as Coxiella (CHI) that can provide essential vitamins and cofactors to ticks, but the impact of these endosymbionts on tick feeding behavior is largely unknown.  In this study Zhong et al. (2021) aimed to investigate the relationship between Coxiella symbiosis and the Asian longhorned tick, Haemaphysalis longicornis) by identifying which tick genes are expressed differently in the presence and absence of their Coxiella endosymbiont.  To do this RNA sequencing, and subsequent analysis, was performed on RNA isolated from young ticks (nymphs) treated with saline (CT) or tetracycline (TET) to reduce Coxiella. A comparison of each tick gene’s expression (fold change of log2(TET/CT) is displayed as a volcano plot with the log2 fold change on the x-axis (panel A).  For example, if a gene has a mathematical fold change of 2, the log2 value is 1 (upregulated; twice as much gene X RNA in TET vs. CT) or -1 (downregulated; half as much gene X RNA in TET vs CT).  For large data experiments such as this an adjusted p value (also known as an adj p or q value) is calculated rather than a standard p value; adj p < 0.05 is considered statistically significant.  Fold changes between 2 and -2 are not considered biologically relevant (between the two dashed lines; panel A).  Having identified differentially expressed genes (DEGs), the authors next wanted to know the function of these genes and which biological pathways were most affected.  To do this, they used a set of bioinformatic analyses to identify “enriched” pathways of genes that were down-regulated in the absence of Coxiella (TET). The top 20 enriched pathways are shown in the pathway enrichment plot (panel B).  The “rich factor” noted here is a measure of the fraction of pathway genes that are in the DEG list; using a proportional measure ensures that a fair comparison is made for pathways with few and many genes.

  • The abstract and figures cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

7.2.2. Questions

  1. If a gene’s data dot has a red color (panel A), what can you say about its expression when Coxiella is absent (TET) vs. present (CT)?
    1. At least twice as much RNA for this gene is found in TET-treated ticks compared to CT-treated ticks and the difference is statistically significant.
    2. Less than half as much RNA for this gene is found in TET-treated ticks compared to CT-treated ticks and the difference is statistically significant.
    3. There is no difference in the RNA for this gene is found in TET-treated ticks compared to CT-treated ticks and any minor difference is not significant.
    4. There is a difference in the RNA for this gene is found in TET-treated ticks compared to CT-treated ticks but the difference is not biologically relevant.
  2. There are several red points and one green point that are high on the y-axis.  What does this indicate about these genes?
    1. They are outliers that should be eliminated.
    2. They are more up or downregulated than other genes.
    3. It is very likely that they are due to random errors.
    4. It is very unlikely that they are due to random variation.
  3. What can you conclude about the impact of Coxiella presence on tick gene expression (panel A)?
    1. There were no down-regulated tick genes; all were upregulated in the absence of Coxiella.
    2. There were no up-regulated tick genes; all were downregulated in the absence of Coxiella.
    3. More tick genes were up-regulated than downregulated in the absence of Coxiella.
    4. More tick genes were down-regulated than upregulated in the absence of Coxiella.
  4. The pathway enrichment for one group of DEG is shown as a strip plot with size and color differences for the circles (panel B). What do these represent?
    1. number of down regulated genes; number of DEG in the pathway
    2. statistical significance; number of upregulated DEG in the pathway
    3. number of up regulated genes; number of down regulated genes
    4. number of down regulated DEG in the pathway; statistical significance
  5. Which metabolic pathway is most enriched the DEG group analyzed (panel B)?
    1. Arginine and proline metabolism
    2. Isoquinoline alkaloid biosynthesis
    3. Steroid hormone biosynthesis
    4. Phenylalanine metabolism
  6. The researchers identified a specific pattern in the enrichment data and identified it using red text. What are they identifying?
    1. These are pathways related to amino acid metabolism.
    2. These are pathways that are highly enriched and significant.
    3. These are pathways that are related to drug metabolism.
    4. These are pathways that are involved in bacterial defense.
  7. If the Coxiella bacteria were providing excess of some nutrient (nutrient X) to the tick that the tick could make itself, what would you expect to happen to the genes encoding for the synthesis of nutrient X when Coxiella bacteria are eliminated by tetracycline (TET)?  The genes for synthesis of nutrient X would most likely be                                    .
    1. Up-regulated immediately
    2. Down-regulated immediately
    3. Up-regulated over time
    4. Down-regulated over time

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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