Microbial Ecology

TWiM #183: Two Symbioses

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 5:00 minutes

The Most Interesting Things (according to students)

A parasite of a protist lacks almost all independent metabolic capability and modifies the host mitochondria to provide all energy and biosynthesis needs.

“Most of the diversity of microbial life is not available in culture, and as such we lack even a fundamental understanding of the biological diversity of several branches on the tree of life. One branch that is highly underrepresented is the candidate phylum TM6, also known as the Dependentiae. Their biology is known only from reduced genomes recovered from metagenomes around the world and two isolates infecting amoebae, all suggest that they live highly host-associated lifestyles as parasites or symbionts. Chromulinavorax destructans is an isolate from the TM6/Dependentiae that infects and lyses the abundant heterotrophic flagellate, Spumella elongata. Chromulinavorax destructans is characterized by a high degree of reduction and specialization for infection, so much so it was discovered in a screen for giant viruses. Its 1.2 Mb genome shows no metabolic potential and C. destructans instead relies on extensive transporter system to import nutrients, and even energy in the form of ATP from the host. Accordingly, it replicates in a viral-like fashion, while extensively reorganizing and expanding the host mitochondrion. 44% of proteins contain signal sequences for secretion, which includes many proteins of unknown function as well as 98 copies of ankyrin-repeat domain proteins, known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus.” (Deeg et al 2018, no changes)

1.2. Main paper; discussion starts at 23:08 minutes

The Most Interesting Things (according to students)

Cicadas compensate for nutrient-poor diets with bacterial and fungal symbionts capable of offering the missing nutrients.  A fungal symbiont is replacing one of the older bacterial ones.

“Diverse insects are associated with ancient bacterial symbionts, whose genomes have often suffered drastic reduction and degeneration. In extreme cases, such symbiont genomes seem almost unable to sustain the basic cellular functioning, which comprises an open question in the evolution of symbiosis. Here, we report an insect group wherein an ancient symbiont lineage suffering massive genome erosion has experienced recurrent extinction and replacement by host-associated pathogenic microbes. Cicadas are associated with the ancient bacterial co-obligate symbionts Sulcia and Hodgkinia, whose streamlined genomes are specialized for synthesizing essential amino acids, thereby enabling the host to live on plant sap. However, our inspection of 24 Japanese cicada species revealed that while all species possessed Sulcia, only nine species retained Hodgkinia, and their genomes exhibited substantial structural instability. The remaining 15 species lacked Hodgkinia and instead harbored yeast-like fungal symbionts. Detailed phylogenetic analyses uncovered repeated Hodgkinia-fungus and fungus-fungus replacements in cicadas. The fungal symbionts were phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, identifying entomopathogenic origins of the fungal symbionts. Most fungal symbionts of cicadas were uncultivable, but the fungal symbiont of Meimuna opalifera was cultivable, possibly because it is at an early stage of fungal symbiont replacement. Genome sequencing of the fungal symbiont revealed its metabolic versatility, presumably capable of synthesizing almost all amino acids, vitamins, and other metabolites, which is more than sufficient to compensate for the Hodgkinia loss. These findings highlight a straightforward ecological and evolutionary connection between parasitism and symbiosis, which may provide an evolutionary trajectory to renovate deteriorated ancient symbiosis via pathogen domestication.” (Matsuura et al 2018, no changes)

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

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Information Flow and Genetics (V&C_IFG)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 12 (ASM_12):  Bacteria and Archaea exhibit extensive metabolic diversity, including nitrogen fixation, methane production, and anoxygenic photosynthesis, many of which are unique to these two domains.
  • Fundamental Statement 13 (ASM_13):  Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • 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 16 (ASM_16): Genetic variation can influence microbial structures and their functions.  
  • 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.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define the function of mitochondria.
 S L
  • Compare an organisms metabolic/physiological characteristics (or lack thereof) to those commonly attributed to pathogenic viruses and bacteria to argue whether a pathogen is more bacteria-like or more virus-like.
 S H
  • Define bacteriome.
  • List capabilities provided by insect endosymbionts.
 M L
  • Predict the evolutionary fitness of different mutant alleles on pathogen survival.
 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

  • Electron Microscopy (10:10): This is a high resolution microscopy technique that was used to see mitochondria surrounding replication body. 

4.2. Main Paper

  • Light Microscopy (29:18–29:44): This type of microscopy used visible light and revealed a fungal symbiont inside cicadas.
  • Phylogenetic Analysis (36:20–36:56): This technique is an analysis of the genetic similarity and differences among related species and was used to show how the Cicada-Fungus symbiosis happened independently 3 times. 

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

5.1. Snippet Paper

  • Pathogenesis (9:30–10:10): C. destructans targets host mitochondria for replication.
  • Bacterial Replication (10:20–10:50): New bacterial replicates burst the host cell ‘lytic bacteria; (11:20–11:35) compare to Bordetella, Vibrio and others.
  • Parasitism (13:15–13:40; 14:30–14:50): Parasites can lack metabolic pathways and thus must import most nutrients from host.
  • Senescence (15:15–16:30): Oxidative stress and cell senescence keeps C. destructans viable after 4 years in cold (4°C).
  • Evolution (18:35–19:00; 21:30–21:50): The podcasters discuss the possibility of DNA segments evolving into a virus.
  • Replication Cycles (19:00–21:00): Differences between viral and bacterial replication (binary fission versus assembling individual components) .

5.2. Main Paper

  • Symbioses (24:18–25:31; 34:15–34:45): Symbionts (both bacterial and fungal) provide cicadas with amino acids they cannot obtain from their xylem diet.
  • Genomics (25:33–27:09): Symbionts have tiny genomes, less than the minimal for independent life; (27:09  -28:20) Hodgkinia has various smaller molecules instead of one chromosome, meaning they can lose important functions.
  • Evolution (28:40–30:11): Cicadas acquired fungal symbiont (Ophiocordyceps) to replace Hodgkinia; (32:02–35:10) “tamed” fungus that is not pathogenic in cicadas – coevolution.
  • Mycology (30:10–32:01): The podcasters described features of fungus Ophiocordyceps.

6. Podcast Questions

  1. Mitochondrial activities include production of which of the following? Pick all that apply.
    1. oxygen
    2. ATP
    3. carbon dioxide
    4. water
    5. degradative enzymes

2. For each mutant phenotype and environment described below, predict whether the corresponding alleles that confer the phenotype are likely to have increased fitness (I) , decreased fitness (D) , or no effect (N).

    1. _______ Ability to ferment, Outside of host
    2. _______ Unregulated protein production, inside of host
    3. _______ Ability to inactivate reaction oxygen species, inside of host
    4. _______ Loss of ability to stimulate mitochondrial replication, outside of host
    5. _______ Loss of ability to scavenge iron, inside of host

3. Label the C. destrucans characteristics listed below as more similar to a virus (V) or a parasitic bacterium (B).

    1. _______ Has a reduced genome
    2. _______ Replicates by assembling
    3. _______ Has no metabolic potential
    4. _______ Has a distinct cell membrane
    5. _______ Shows cellular division before host cell lysis

4.  In the cicada-Sulcia–Hodgkinia symbiotic relationship, the bacteriome is ________. 

    1. a large bacterium found inside of the cicadas that are not found in other species
    2. a compartment within the bacterial endosymbiont in the cicadas
    3. a compartment within the cicadas where endosymbionts are typically found
    4. a cicada’s microbiome that includes all of the bacteria, viruses, and fungi
    5. The complete set of bacterial genes found within the cicada, including organelles

7. Figure Reading Exercises

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

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify representative components in a model figure.
  • Identify the relationship of the organisms in this interaction.
  • Describe how “per cell” DNA content changes can be used to determine intracellular pathogen replication
  • Predict how differences in host or pathogen would affect fitness.
Experimental Background (Deeg et al., Figure 5)

One of the most under-represented branches in the tree of life is  in the phylum Dependentiae or TM6.  Organisms in this branch are often parasites or symbionts of other species.  In this study, Deeg et al (2018) isolated an organism in this phylum from a heterotrphic flagellate, Spumella elongata, and characterized its life cycle and genome.  Their model of replication, which is a summary of their findings, is illustrated in Figure 5.

drawing of fungal symbionts
Figure 5: “Schematic representation of the inferred replication cycle of Chromulinavorax destructans. The host S. elongata cell is shown in green, with the mitochondrion in brown, nucleus in dark green, Golgi in orange, and vacuoles in light orange. C. destructans is depicted in blue throughout its replication cycle. “(Deeg et al, 2018, no changes)

7.1.2. Questions

  1. In this model, what do the blue circles represent?
    1. S. elongata mitochondria
    2. C. destructans mitochondria
    3. S. elgonata cells
    4. C. destructans cells
2. The authors provide a model (Figure 5) in which intracellular pathogen replication takes place inside a vacuole and induces replication of host mitochondria.  At 48 hours post-infection, how would the DNA content of the infected cell compare to an uninfected cell? The DNA content of the infected cell would be:
    1. more than an uninfected cell due to the increased number of mitochondria.
    2. the same as an uninfected cell due to no change in the DNA content.
    3. more than an uninfected cell due to the increased number of pathogen cells.
    4. less than the uninfected cell due to loss of the replicated pathogen cells through extrusion.
    5. more than an uninfected cell due to the increased number of pathogen cells and mitochondria.

3. Based on the model in figure 5, if you were to make a separate lysate of the same number of infected and uninfected cells and measure the ATP levels in each lysate, what would you find and why?

    1. More ATP in the uninfected cells due to the harmful effects on the infected cells.
    2. More ATP in the infected cells due to a larger number of mitochondria present in those cells.
    3. More ATP in the uninfected cells due to a larger number of mitochondria in those cells.
    4. More ATP in the infected cells due to a lack of kinase activity allowing buildup of ATP in those cells.

4. If you were to acquire two different strains of Chromulinavorax destructans that differed  by mutation in one gene and one strain (strain 1) was able to replicate more quickly and release propogated cells at hour 6 instead of hour 12 (strain 2, wild-type; see figure 5), which strain would have greater fitness and why?

    1. Strain 1 has greater fitness because its replication time is shorter and can infect more frequently over time.
    2. Strain 2 has greater fitness because it is wild-type and wild-type always has a greater fitness.
    3. Strain 1 has greater fitness because the reactive oxygen and ATP levels in strain 1 will be higher.
    4. Strain 2 has greater fitness because strain 1 will likely be metabolically deficient due to its short generation time.

5. Based on the model of replication shown in Figure 5, what relationship do Chromulinavorax destructans  and Spumella elongata  have?

    1. parasitic
    2. commensalistic
    3. mutalistic

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify relevant features in a phylogenetic tree.
  • Analyze the data to identify where specific symbionts are found and whether they were  present in a common ancestor.
  • Analyze the data to make conclusions about whether an organism is more or less closely related to other species.
  • Analyze the data to make conclusions about gain and loss of specific endosymbiont species and why gain or loss occurred.
  • Speculate why single symbionts are not observed.
Experimental Background (Matsuura et al., Figure 5)

Many insects have bacterial symbionts, including cicadas. Cicadas usually play host to two obligate symbionts, Sulcia and Hodgkinia.  Typically, a symbiont genome is reduced in size and retains features that benefit the symbiont and the host.  Matsuura et al. (2018) examined the symbionts of 20 Japanese cicadas and report both extreme genome reduction and instances of symbiont extinction, as well as replacement with pathogenic microbes. As a summary of their results, they produced a phylogenetic tree based on specific cicada genome sequences and mapped the symbionts present in each species (Matsuura et al, 2018; Figure 5).

phylogenetic tree of cicada endosymbionts

Figure 5 “Phylogenetic relationship of cicadas and their infection status with microbial symbionts. A maximum-likelihood phylogeny inferred from 15 mitochondrial gene sequences and 22 tRNAs (14,733 aligned nucleotide sites) of 20 Japanese cicada species, together with four previously studied American species (highlighted by #), is shown. Bootstrap support values are indicated on each node in the order of maximum-likelihood/neighbor-joining. Detected microbial symbionts are mapped on the right side of each species name with orange circles for Sulcia, green circles for Hodgkinia, and yellow circles for the yeast-like fungal symbiont. In the green circles of Hodgkinia, the number 1, 2, or 4 indicates the number of distinct Hodgkinia genomes that form a complex. C indicates highly fragmented Hodgkinia complexes in which the exact number of genomes could not be determined (51, 53). Colored triangles on the phylogeny indicate the estimated replacement events from Hodgkinia to the fungal symbionts. Selected images of adult cicadas are depicted to the right of the maximum-likelihood phylogeny. A. bihamatus, Auritibicen bihamatus; C. atrata, Cryptotympana atrata; E. chibensis, Euterpnosia chibensis; G. bimaculata, Graptopsaltria bimaculata; Ma. tredecim, Magicicada tredecim; Me. iwasakii, Meimuna iwasakii; Me. oshimensis, Meimuna oshimensis; Ta. japonensis, Tanna japonensis; Te. nigricosta, Terpnosia nigricosta; Te. vacua, Terpnosia vacua; Tet. ulnaria, Tettigades ulnaria; Tet. undata, Tettigades undata.” (Matsuura et al, 2018, no changes)

7.2.2. Questions

  1. What do the horizontal lines of the tree represent?
    1. time elapsed in cicada evolution
    2. genetic differences in the cicadas
    3. size differences in the cicadas
    4. color differences in the cicadas

2.  Which endosymbiont/s is/are found in all of the species?

    1. Yeast-like symbionts
    2. Sulcia
    3. Hodkinia
    4. Sulcia and Hodkinia
    5. Sulcia and Yeast-like symbionts

3. Based on the information provided in the tree, which endosymbionts were likely present in last common ancestor to these organisms? Pick all that apply.

    1. Sulcia
    2. Hodgkinia
    3. Yeast-like endosymbiont

4. The genomes present in the Hodgkinia symbiont were analyzed with 1, 2, and 4 different genomes present in some isolates (see numbers in the green circles; Figure 5) or a highly fragmented genome present in other isolates (see C in the green circles; Figure 5).  Based on the differences you see for Mo. minuta  compared  to its clade members, V. terminalis, Mu. kuroiwae, Ma. tredecim and and K. yezoensis, what might have occurred?

    1. Mo. minuta lost its Hodgkinia endosymbiont, probably because the symbiont lost the genes to make the nutrients it provided to the cicada, and gained the yeast-like symbiont to provide those nutrients.
    2. V. terminalis, Mu. kuroiwae, Ma. tredecim and and K. yezoensis lost their yeast-like endosymbiont, probably because the symbiont acquired mutations that made proteins that were toxic to the cicada, and then gained the Hodgkinia symbiont to fill the niche of the symbiont.
    3. Mo. minuta lost its Sulcia endosymbiont, probably because the symbiont lost the genes to make the nutrients it provided to the cicada, and gained the yeast-like symbiont to provide protection from other fungal pathogens.
    4. V. terminalis, Mu. kuroiwae, Ma. tredecim and and K. yezoensis lost their yeast-like endosymbiont, probably because the symbiont lost the genes to make the nutrients it provided to the cicada, and gained the Hodgkinia symbiont to provide those nutrients.

5. In Figure 5, we see that the authors have marked the instances where a yeast-like symbiont (YLS) was gained with a yellow triangle.  Based on the relationships noted in the tree, do you think that these organisms gained YLS independent of each other or from a common ancestor?  Why?

    1. They are independent since not all clades contain members with all three symbionts, Hodgkinia, Sulcia, and YLS and the earliest diverging has only Sulcia and Hodgkinia.
    2. They are all from a common ancestor since all clades except the earliest diverging pair of cicada species (Tet. unaria and Tet. undata) contain a YLS.
    3. They are independent  since all of these are found in different clades and most contain members with the Hodgkinia-Sulcia endosymbiosis and no YLS.
    4. They are all from a common ancestor that originally contained all three symbionts, Hodgkinia, Sulcia endosymbiosis, and YLS, but one of the three was lost in each cicada species.

6.  Cicada endosymbionts were always found as co-symbionts.  Why might single symbionts not appear?

    1. A single species shows little variation and is efficiently killed by the cicada immune system.
    2. A single species is not able to provide the entire complement of nutrients to the cicada.
    3. The pair of species cancel out the toxins produced by each other allowing them both to survive.
    4. The pair of species provide materials to each other so without its partner, it doesn’t survive.

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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