TWiM #185: There’s No Moa Moa

1. Paper Abstracts

1.1. Snippet paper; discussion starts at [3:05] minutes

“Over the past 50,000 y, biotic extinctions and declines have left a legacy of vacant niches and broken ecological interactions across global terrestrial ecosystems. Reconstructing the natural, unmodified ecosystems that preceded these events relies on high-resolution analyses of paleoecological deposits. Coprolites are a source of uniquely detailed information about trophic interactions and the behaviors, gut parasite communities, and microbiotas of prehistoric animal species. Such insights are critical for understanding the legacy effects of extinctions on ecosystems, and can help guide contemporary conservation and ecosystem restoration efforts. Here we use high-throughput sequencing (HTS) of ancient eukaryotic DNA from coprolites to reconstruct aspects of the biology and ecology of four species of extinct moa and the critically endangered kakapo parrot from New Zealand (NZ). Importantly, we provide evidence that moa and prehistoric kakapo consumed ectomycorrhizal fungi, suggesting these birds played a role in dispersing fungi that are key to NZ’s natural forest ecosystems. We also provide the first DNA-based evidence that moa frequently supplemented their broad diets with ferns and mosses. Finally, we also find parasite taxa that provide insight into moa behavior, and present data supporting the hypothesis of coextinction between moa and several parasite species. Our study demonstrates that HTS sequencing of coprolites provides a powerful tool for resolving key aspects of ancient ecosystems and may rapidly provide information not obtainable by conventional paleoecological techniques, such as fossil analyses.” (Boast et al. 2018)

1.2. Main paper; discussion starts at 25:04 minutes

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 8.2.

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

3.  Potential Learning Objectives for the Podcast

¹ Papers: 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

• Aseptic Technique (8:00–17): This technique involves procedures to reduce contamination. The study of the DNA found in coprolites is best performed in a clean room, as any DNA or other microbial contamination could be amplified and contaminate the entire sample.
• Comparative Genomics/High Throughput Sequencing (9:58–11:25): High throughput sequencing (HTS) is a technique used to sequence whole genomes and/or environmental samples in a large batch. It can be used to compare sequence similarity across whole or partial genomes (comparative genomics). Using high throughput sequencing, the Moa DNA sourced from ancient coprolites was compared to modern day New Zealand birds, and were found to be genetically distinct. This HTS was also used to reconstruct the biological and ecological aspects of the coprolites.

4.2. Main Paper

• Comparative Resistance Test (37:11–40:16): This is a general test of resistance to something. Here, 139 different Enterococcus faecium isolates were separated into three groups: 1997-2003, 2004-2009, 2010-2015. Isolates were exposed to isopropanol for 5 minutes and the reduction was recorded
• Sensitivity Assay (43:40–44:09): A sensitivity assay was performed in order to determine the alcohol concentration tolerance of Enterococci. The higher the concentration, the more sensitive an organism is.
• Colonizing Dose (CD50) (44:12): Similar to infectious dose–the number of microbes required to infect an individual–the colonizing dose is the number of bacteria required with colonizing a surface. When performing the sensitivity assay for microbial tolerance to 23% isopropanol, they discovered than the CD50 was much higher for microbes that were sensitive to 23% isopropanol than those that were tolerant
• Comparative Genomics (49:09–51:49): Sequences of genomes can be compared. Here, bacterial genomic signatures of adaptation were examined as well as the extra chromosomal content. Allelic exchange to make targeted mutants in an isopropanol tolerant isolate was used to validate the bacterial genetic markers linked to the set alcohol tolerance threshold.
• Gradient Plates (53:25–54:27): This technique involves agar with a continuous gradient of selective agent. To make these agar plates, the petric dish containing liquid agar with the selective agent is solidified at an angle, then agar without the selective agent is plated to level out the angle. Mutagenized microbes are spread on the plate and a gradient of growth appears based on resistance or sensitivity to the selective agent concentration.

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

5.1. Snippet Paper

• Coprolite (3:35–4:18; 5:00–5:17): Fossilized feces often examined for prehistoric parasites, DNA, and more. In the case of the extinct New Zealand bird, the Moa, the coprolites revealed the ecological interactions between the bird and its environment.
• Fungal Reproductive Cycle (11:48–13:38): Some fungi grow and spread spores via their gills, and others (i.e. truffles) spread spores by animal digestion which are then spread through feces. Some mushrooms do not ‘open’, and when eaten by birds are spread very far.

5.2. Main Paper

• Hospital Acquired Infection/Bacteremia (27:00–27:34; 30:50–31:00): A bacterial infection of the bloodstream obtained while in the hospital. Approximately 700 people (the capacity of a large plane) die of hospital acquired infections every day.
• Cell Lysis (29:18–29:37): The alcohol in a sanitizer gel dehydrates a microbial cell and the cells burst (lyse).
• Methicillin-resistant Staphylococcus aureus (MRSA) (29:39–30:09): The hand-gel alcohols have been effective at limiting spread of multidrug resistant organisms such as MRSA (methicillin resistant Staphylococcus aureus).
• Sepsis (31:14–31:48): An intense response by the immune system to an infection that leads to bodily damage or death.
• Intrinsic Resistance (31:52–32:12): A microbes resistance against bile, and many other natural host defenses. Hospital acquired enterococcal infections are very difficult to treat due to this intrinsic resistance.
• Antimicrobial Resistance (32:12; 33:49–34:04): Often acquired resistances to a variety of antimicrobial drugs as seen with penicillin and more.
• Metabolic Diversity (Alcohol Catabolism/Metabolism) (32:52–33:29): The metabolic method by which tolerant microbes break down the alcohol found in hand gels into carbohydrates.
• Exposure Pressure (33:33–33:49; 38:25–38:57): As the use of alcohol based sanitizing gels has increased since 1997, organisms are much more exposed. Similarly to selective pressure where an organism must develop resistance to an antibiotic in order to survive, as the organism is continually exposed to the alcohol based hand-gels, they must develop a method of survival.
• Tolerance vs. Resistance (45:25–45:42; 49:24–50:16; 54:28–54:45; 55:23–53:47): When an organism develops resistance to an antimicrobial, it is ‘immune’ to the effects of that substance, whereas when an organism expresses tolerance to an antimicrobial substance, it is merely able to withstand a certain concentration. Several microbes are showing tolerance to low levels of alcohol concentration, but are still susceptible to higher concentrations.

6. Podcast Questions

1. A coprolite is fossilized _________ and can be used to examine ________ of extinct species.
   1. bone; bone density and general health
   2. soft tissue; metabolic rates
   3. feces; diet and parasite load
   4. brain tissue; cognitive development
2. The earliest coprolites come from the Cambrian period. What feature of the “materials” that became fossils likely allowed them to be fossilized more often?
   1. The components of the fossil reacted quickly with the silt covering them.
   2. The calcium rich bones and teeth decay more slowly than soft tissue.
   3. The materials included highly resistant fibers such as lignin and hemi-cellulose.
   4. Chemical composition of the materials favored mineral replacement.
3. Elio discusses the different ways that fungal spores are dispersed, particularly mushrooms with lamellae and truffles. If a modern truffle was to lose its odor, what would you predict for its success in spore dispersal?
   1. Spore dispersal would increase due to a predator’s increase in appetite for them.
   2. Spore dispersal would be the same because odor is not a defining feature of dispersal.
   3. Spore dispersal would stop because the genes for odor are linked to spore formation.
   4. Spore dispersal would decrease due to predators not being able to find and eat them.
4. True/False: Hand sanitizer use is more effective than hand washing with soap for preventing hospital acquired infections (HAI).
5. Elio makes the point consistently in the podcast that the researchers started with identifying isolates with tolerance to 23% isopropanol, but clinical settings use 70% isopropanol. Which experimental design would be good for testing whether tolerance to 23% correlates with tolerance to 70%?
   1. Use all isolates and test for survival/transmission using the contaminated cage with water, 23%, and 70% isopropanol treatments. If 23% and 70% correlate, you would find similar amounts of transmission for these two treatments for each isolate. If 23% and 70% do not correlate, you would find different isolates have differing tolerances.
   2. Use isolates tolerant to 23% and test for survival/transmission using bacterial exposure to sterile water, 23%, and 70% isopropanol treatments. If 23% and 70% correlate, survival would be identical for the two alcohol treatments and different for water. If 23% and 70% do not correlate, you would find all the treatments would be the same.
   3. Use isolates tolerant to 70% and test for survival/transmission using bacterial exposure to sterile water, 23%, and 70% isopropanol treatments. If 23% and 70% correlate, survival would be identical for the 23% alcohol and water and different for 70%. If 23% and 70% do not correlate, you would find both the alcohol treatments would be the same.
   4. Use all isolates and test for survival/transmission using the contaminated cage with 23%, and 70% isopropanol treatments. If 23% and 70% correlate, you would find all isolates survive both the 23% and 70% alcohol treatments. If 23% and 70% do not correlate, you would find different isolates have differing tolerances and the highest survival frequency for 70%.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 3) and one from the main paper (Figure 2c).

7.1. First Figure Reading Exercise

7.1.2. Questions

1. Which taxon abundance is represented by bright orange bars?
   1. Trematoda
   2. Nematoda
   3. Eimeriorina
   4. Amoebozoa
   5. Embryophyta
   6. Fungi
2. Which of the following sites is not a Southern Beech ecology?
   1. Mt. Nicholas
   2. Euphrates C.
   3. Hodge Creek
   4. Shepherd’s C.
3. Across all Moa and kakapo copralites samples, which known taxon is most highly abundant and the most common food source for Moa and kakapo?
   1. Trematoda
   2. Nematoda
   3. Eimeriorina
   4. Amoebozoa
   5. Embryophyta
   6. Fungi
4. Which group of those listed below appears to have the highest diversity of taxa in the coprolite samples?
   1. L. Bush Moa
   2. Giant Moa
   3. H.-footed Moa
   4. Kakapo
   5. Modern animals
5. Trematoda, Nematoda, and Eimeriorina Alveolata are taxa with many common parasitic species. Which Moa shows the lowest parasite load?
   1. L. Bush Moa
   2. Giant Moa
   3. Upland Moa
   4. H.-footed Moa
6. What differences are most apparent when considering gut composition differences between the extinct Moa and modern flightless birds?
   1. Moa likely had low parasite load, but zoo-bound flightless birds do not.
   2. Moa did not eat mostly segmented worms, but modern flightless birds do.
   3. Moa likely ate mostly fungi, but zoo-bound modern flightless birds do not.
   4. Moa were nearly exclusively herbivores and zoo-bound modern birds are not.
7. If the researchers were to sample coprolites from another Grassland/Shrubland site, what level of taxa diversity would you predict based on these data? What is your evidence?
   1. Low diversity; all of the grassland/shrubland sites have low diet diversity with almost exclusively Fungi.
   2. Moderate diversity; the grassland/shrubland sites in these data have diet diversity in the moderate range.
   3. High diversity; all of the grassland/shrubland sites have diet high diversity including nearly all Fungi.

7.2. Second Figure Reading Exercise

7.2.2. Questions

1. Which patterns in the bar charts identify the results for water-based cage disinfection for a VR bacterium that is tolerant to 23% alcohol?
   1. Diagonal lines in the bar and green triangle symbols (Ef_aus0226)
   2. Diagonal lines in the bar and red square symbols (Ef_aus0233)
   3. No lines in the bar and purple triangle symbols (Ef_aus0099)
   4. No lines in the bar and blue circle symbols (Ef_aus0004)
2. What controls did the researchers use in this transmissibility experiment?
   1. The water treatment is the control for all strains.
   2. The sensitive bacteria (on the right) are the controls.
   3. The water-treated for the resistant bacteria are the controls.
   4. The resistant bacteria (on the left) are the controls.
3. For the transmission experiment (panel C), the dependent variable is ______ and the independent variable is _______.
   1. Cage treatment; bacterial genotype
   2. Alcohol tolerance; mouse colonization
   3. Mouse colonization; cage treatment
   4. Bacterial genotype; alcohol tolerance
4. The researchers aimed to test whether VR bacteria that were tolerant to 23% alcohol (aus0233) had different transmissibility to VR bacteria that were not tolerant to 23% alcohol (aus0004). Which statement best describes their results?
   1. There was no difference between VR bacteria and VS bacteria, as well as no difference in alcohol-tolerant and alcohol-sensitive.
   2. VR bacterial strains colonized mice more often than VS bacteria strains colonized mice following alcohol cleaning.
   3. VR, alcohol-tolerant bacteria colonized mice more often than VS, alcohol-tolerant bacteria following alcohol cleaning.
   4. VR, alcohol-tolerant bacteria colonized mice more often than VR, alcohol-sensitive bacteria following alcohol cleaning.
5. Based on the data presented in the figure, should hospitals be equally, less, or more concerned about alcohol tolerance in vancomycin-sensitive E. faecium as vancomycin-resistant E. faecium?
   1. No. Vancomycin-resistant E. faecium is more resistant to alcohol treatment.
   2. Yes. Vancomycin-sensitive E. faecium can be resistant to alcohol treatment.
   3. No. Vancomycin-sensitive E. faecium is shown to be sensitive to alcohol treatment.
   4. Yes. Vancomycin-resistant E. faecium is shown to be intolerant to alcohol treatment.

8. Paper Information and Licensing

8.1. Snippet paper

• Boast AP, Weyrich LS, Wood JR, Metcalf JL, Knight R, Cooper A. 2018. Coprolites reveal ecological interactions lost with the extinction of New Zealand birds. Proc Natl Acad Sci U S A. 115(7):1546-1551. https://doi.org/10.1073/pnas.1712337115
• This article is licensed for Creative Commons use using CC BY 4.0, per the PNAS licensing for 2018, which allows re-use and adaptation with proper attribution and notation of any changes. See https://www.pnas.org/doi/10.1073/pnas.1712337115

8.2. Main paper

• Pidot SJ, Gao W, Buultjens AH, Monk IR, Guerillot R, Carter GP, Lee JYH, Lam MMC, Grayson ML, Ballard SA, Mahony AA, Grabsch EA, Kotsanas D, Korman TM, Coombs GW, Robinson JO, Gonçalves da Silva A, Seemann T, Howden BP, Johnson PDR, Stinear TP. 2018. Increasing tolerance of hospital Enterococcus faecium to handwash alcohols. Sci Transl Med. 10(452):eaar6115. doi: 10.1126/scitranslmed.aar6115
• This article is not licensed for Creative Commons use; See https://www.science.org/doi/10.1126/scitranslmed.aar6115. Thus, the abstract and figures cannot be copied here. It is behind a paywall and not available on PubMed, so we suggest you contact the authors for a copy.