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Aug 2017

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Assessment of Caenorhabditis elegans Competitive Fitness in the Presence of a Bacterial Parasite
寄生菌存在情况下秀丽隐杆线虫的竞争适应性测定   

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Abstract

Accurate measurements of an organism’s fitness are crucial for measuring evolutionary change. Methods of fitness measurement are most accurate when incorporating an individual’s survival and fecundity, as well as accounting for any ecological interactions or environmental effects experienced by the organism. Here, we describe a protocol for measuring the relative mean fitness of Caenorhabditis elegans populations, or strains, through an assay that accounts for individual survival, fecundity, and intraspecific competitive ability in the presence of a bacterial parasite. In this competitive fitness assay nematodes from a focal population or strain are mixed with a GFP-marked tester strain in equal proportions, the mixture of nematodes are then exposed to a parasite, and the relative competitive fitness of the focal strain is determined by measuring the change in the ratio of focal nematodes to GFP-marked nematodes after one generation. Specifically, this protocol can be implemented to measure changes in nematode host fitness after experimental evolution by determining the relative competitive fitness of evolved versus ancestral nematode populations.

Keywords: Fitness assay (适应性测定), Experimental evolution (实验进化), C. elegans (秀丽隐杆线虫), S. marcescens (粘质赛氏杆菌), Competition (竞争)

Background

Accurate measurements of fitness and changes in fitness over time are critical for determining a population’s response to natural selection. Nonetheless, fitness is notoriously difficult to measure because it incorporates an individual’s survival, fecundity, reproductive timing, and must account for ecological and environmental effects on individuals. Although no protocol for measuring fitness is optimal under all possible conditions, measures of fitness that account for survival and fecundity, while holding ecological and environmental effects constant, are likely to provide reliable overall estimates of fitness for a given scenario. Here we describe a protocol for measuring the relative fitness differences between C. elegans populations or strains and for determining the change in relative fitness over evolutionary time in the presence of a bacterial parasite. We utilized the gram-negative bacterium, Serratia marcescens, as a virulent parasite when consumed by C. elegans. Especially, S. marcescens strain SM2170 is capable of killing C. elegans hosts within 24 to 48 h of ingestion (Penley et al., 2017). This procedure makes use of Competitive Fitness Assays (CFAs) (Lenski et al., 1991; Wiser and Lenski, 2015), utilizing intraspecific competition to compare the relative fitness between different nematode populations or strains (Morran et al., 2009). Measurements of relative fitness, as determined via the CFA, incorporate survival and reproduction with intraspecific competition in a controlled environment to provide a comprehensive measure of fitness (Penley et al., 2017).

Relative fitness in the presence of the bacterial parasite is determined by competing a focal strain with an isogenic GFP-labeled tester strain over the course of one generation and measuring the reproductive success of the focal strain against the tester strain (Morran et al., 2009). Thus, this CFA accounts for survival against the parasite and host reproduction over the course of the nematode’s lifecycle. One single tester strain is used to measure the relative fitness of each focal nematode population or strain to facilitate comparisons of relative fitness between populations or strains. Importantly, the tester is marked with pharyngeal GFP to allow easy visualization of tester strain offspring versus focal population or strain offspring after one generation of competition. CFAs are initialized with a 50:50 mix of focal and tester strain individuals, and therefore, any deviation from 50:50 mix in the offspring indicates unequal competitive fitness between the focal and tester strains. An increase in the proportion of focal nematodes in the offspring indicates greater competitive fitness relative to the tester, while a decrease indicates reduced competitive fitness relative to the tester. The proportion of focal hosts in the offspring can be compared across multiple populations to measure the relative competitive fitness between focal strains or populations of interest. Importantly, competitive fitness measures are most effective when competing approximately equal numbers of individuals between two populations or strains with minor to moderate differences in competitive fitness. Uneven or variable starting ratios of strains or populations can confound measurements of relative fitness, while major differences in fitness between competing strains or populations are often difficult to accurately quantify (Wiser and Lenski, 2015).

This protocol is particularly useful for measuring evolutionary change after experimental evolution of C. elegans hosts in the presence of a bacterial parasite. First, the relative fitness of experimental host populations can be directly compared with the relative fitness of the ancestral population. Ancestral C. elegans populations can be stored at -80 °C during experimental evolution and then revived for CFAs to assess changes in the experimental population fitness over time (Gray and Cutter, 2014; Teotonio et al., 2017). Second, during experimental evolution, hosts may adapt to parasite exposure through altered life histories and/or increased levels of host defense. Therefore, measuring only survival in the presence of the parasite may not fully account for changes in host fitness. This CFA can account for changes in both life history and resistance that alter reproductive output in the presence of the parasite. Importantly, this procedure was originally developed to measure the change in C. elegans’ competitive fitness after multiple generations of evolution in presence of the bacterial parasite, Serratia marcescens (Morran et al., 2009; Morran et al., 2014; Parrish et al., 2016; Penley et al., 2017). Nonetheless, this protocol can be adapted to measure the relative competitive fitness of any two or more C. elegans populations or strains in the presence of any relevant bacterial parasite. Further, it can be used to measure the change in relative competitive fitness over the course of experimental evolution for any C. elegans populations evolved in the presence of a bacterial parasite.

Materials and Reagents

  1. 1.5 ml micro-centrifuge tube (MIDSCI, catalog number: MID15C )
  2. 1,000 μl pipette tips (MIDSCI, catalog number: AVR4 )
  3. 200 μl pipette tips (MIDSCI, catalog number: AVR1 )
  4. 1,000 μl wide bore pipette tips (Genesee Scientific, catalog number: 22-426 )
  5. 200 μl wide bore pipette tips (Genesee Scientific, catalog number: 22-423 )
  6. Microscope slides (Fisher Scientific, catalog number: 12-550-19 )
  7. Semimicro spatula (Fisher Scientific, catalog number: 14-374 )
  8. Disposable inoculating loops, 10 μl (VWR, catalog number: 12000-810 )
  9. 100 x 15 mm Petri dishes (Tritech Research, catalog number: T3301 )
  10. 0.22 μm sterile syringe filter (Spectrum Chemical Manufacturing, catalog number: 882-66597 )
  11. Disposable plastic syringe (Thermo Fisher Scientific, catalog number: S7510-10 )
  12. Serratia marcescens strain SM2170, BSL2 (Sue Katz, Rogers State University) 
  13. Escherichia coli strain OP50, BSL1 (Caenorhabditis Genetics Center)
  14. GFP-labeled C. elegans (strain JK2735) (Caenorhabditis Genetics Center)
  15. Nematode Growth Media Lite powder (United States Biological, catalog number: N1005 )
  16. LB granules (Fisher Scientific, catalog number: BP9723-500 )
  17. Potassium Phosphate Monobasic (KH2PO4) (Fisher Scientific, catalog number: P288-100 )
  18. Sodium Chloride (NaCl) (Fisher Scientific, catalog number: S671-500 )
  19. Sodium Phosphate Dibasic Anhydrous (Na2HPO4) (Fisher Scientific, catalog number: S374-500 )
  20. Magnesium Sulfate Anhydrous (MgSO4) (Fisher Scientific, catalog number: M65-500 )
  21. Ampicillin Sodium Salt (Dot Scientific, catalog number: DSA40040-25 )
  22. Household bleach 
  23. LB Broth (see Recipes)
  24. NGM Lite plates (see Recipes)
  25. Escherichia coli (OP50)-seeded NGM Lite plates (see Recipes)
  26. M9 Buffer (see Recipes)
  27. 1 M MgSO4 Solution (see Recipes)
  28. Ampicillin 200 mg/ml (see Recipes)

Equipment

  1. Hand tally counters (United Scientific Supplies, catalog number: HTCP01 )
  2. 2 L flask (Corning, PYREX®, catalog number: 5320-2L
  3. P1000 μl pipetman (Eppendorf, model: Research® plus, catalog number: 3121000120 )
  4. P100 μl pipetman (Eppendorf, model: Research® plus, catalog number: 3121000074 )
  5. -80 °C freezer (Eppendorf, New BrunswickTM, model: Innova® U725 )
  6. Tabletop centrifuge for 1.5 ml micro-centrifuge tubes (Eppendorf, model: 5424 )
  7. 20 °C controlled environment chamber (Percival Scientific, model: I36NLC8 )
  8. 28 °C shaker incubator (Eppendorf, New BrunswickTM, model: Innova® 42R , catalog number: M1335-0010)
  9. Stereomicroscope (Olympus, model: SZX16 )
  10. LED transmitted light illumination base (Olympus, model: SZX2-ILLT )
  11. GFP filter for stereomicroscope (Olympus, model: SZX2-FGFP )
  12. Stereomicroscope objective 7x-115x (Olympus, model: SDFPLAPO1XPF )
  13. Fluorescence illumination lamp (Excelitas Technologies, model: X-Cite® 120Q )
  14. X-Cite® Liquid Light Guide (Bulbtronics, Excelitas Technologies, model: 805-00038 )
  15. Autoclave (STERIS, model: SG-120 )
  16. Chemical fume hood (Kewaunee Scientific, model: H05 )

Software

  1. JMP Pro 12.0.1 (SAS Institute Inc., Cary, NC)

Procedure

Notes:

  1. This protocol specifically describes measuring the change in a population’s competitive fitness after experimental evolution in the presence of Serratia marcescens (SM2170). However, it can be adapted to measure relative fitness between any two or more nematode populations of interest in the presence of any parasite that can grow and transmit under the conditions described. No specific experimental nematode strains or experimental evolution protocols are necessary to use this protocol simply as a means of measuring fitness. However, a clearly marked GFP strain is required.
  2. Before experimental evolution, the generation 0 ancestor nematodes should be frozen at -80 °C. Similarly, at the end of experimental evolution, it will be necessary to freeze the experimentally evolved populations for use in these and any other subsequent assays. The freezing protocol can be found in Stiernagle (2006) (https://www.ncbi.nlm.nih.gov/books/NBK19649/).
  3. This procedure spans over ~2 weeks. See Table 1 for an example of the procedure schedule.

Table 1. Example of Procedure Daily Schedule


  1. Thawing experimental and tester populations
    1. Remove one frozen tube for each of the following strains from the -80 °C freezer: 
      1. Generation 0 ancestor 
      2. Experimentally evolved population of interest 
      3. GFP-labeled tester strain (JK2735) 
      Note: If measuring relative competitive fitness, thaw 2 or more nematode populations of interest and the GFP-labeled tester strain.
    2. Allow the tubes to thaw at room temperature.
    3. After thawing, gently invert the tubes several times to mix the solution.
    4. Pour the entire contents of the tube onto a labeled 100 mm E. coli OP50-seeded NGM Lite plate.
    5. Allow the plates to absorb all the liquid content. If the liquid content is not fully absorbed, dry the plates briefly in the chemical fume hood with lids off.
    6. Incubate these plates at 20 °C for 24 h.
    7. Examine the plates to see that at least 50 nematodes survived thawing. If so, incubate the plates at 20 °C to allow the population to recover from thawing and expand (about 1 week). If not, thaw another freezer tube for the population of interest and repeat the thawing process.

  2. Prepping for Synchronization
    1. Once the generation 0 ancestor, experimentally evolved, and GFP-labeled tester populations have expanded to the point that hundreds of individuals are observed on each plate, you may proceed.
    2. For each population, use a flame-sterilized metal spatula to cut approximately 2.5 cm2 of NGM Lite from the plate containing nematodes.
    3. For each population, remove this piece of NGM Lite from the original plate and place it face down onto a new 100 mm OP50-seeded NGM Lite plate.
    4. Incubate these new plates at 20 °C for 48 h.
    5. After 48 h, view the plates from Step B4 under the microscope to check for eggs. If at least ~1,000 eggs are observed on the surface of the plate, proceed to Procedure C. If not, incubate the plates at 20 °C and check the next day. If the plate is exhausted of OP50, the nematodes will no longer produce eggs. In this case, repeat Steps B2-B5.

  3. Synchronization
    Note: The synchronization protocol is adapted from Stiernagle (2006). The purpose of synchronization is to kill all nematodes, leaving only the eggs that have been deposited on the plate to survive. This will age synchronize the population at the egg stage. 
    1. Working one plate at a time, add 2 ml of M9 buffer to the plate.
      Note: Some of the M9 buffer will be absorbed into the plate. Therefore, adding 2 ml of M9 buffer to the plate will result in ~1 ml to be collected from the plate.
    2. Tilt the plate side to side to wash the entire surface of the plate with M9 buffer.
      Note: If the bacterial lawn on the plate appears thick and sticky, use a pipette tip to gently scrape the surface of the plate. This will help loosen the eggs.
    3. Tilt the plate to allow all of the M9 buffer to pool in one area. 
    4. Use a pipette to remove the M9 buffer and move it into a 1.5 ml micro-centrifuge tube.
    5. Repeat Steps C1-C4 so that each population is in its own micro-centrifuge tube.
    6. Centrifuge tubes at 845 x g for 30 sec to form a pellet.
    7. Remove the supernatant from each tube, using a new pipette tip between tubes.
    8. Add 800 μl M9 buffer to each tube containing the pellets.
      Note: Steps C9-C17 should be completed in 3 min or less to prevent the bleach from damaging the eggs.
    9. Add 200 μl household bleach to each centrifuge tube containing the pellets and 800 μl M9 buffer.
      Note: This will yield a 20% household bleach solution in each tube.
    10. Close and invert the tubes several times to mix the bleach and M9 buffer, and to loosen the pellet.
    11. Centrifuge the tubes at 845 x g for 30 sec.
    12. Remove the supernatant from each tube, using a new pipette tip between tubes.
    13. Wash each pellet with 1 ml M9 buffer. Close and invert the tubes several times to loosen the pellet.
    14. Centrifuge the tubes at 845 x g for 30 sec.
    15. Remove supernatant from each tube, using a new pipette tip between tubes.
    16. Repeat Steps C13-C15.
      Note: You will have washed the pellet twice.
    17. Pipette the entire pellet from each tube onto its own new 100 mm OP50-seeded NGM Lite plate, being sure to change tips between tubes.
    18. Incubate the plate at 20 °C for 48 h, or until the nematodes are at larval stage L4.

  4. Preparation of Serratia Selection Plates (SSPs)
    1. On the same day of synchronization (see Table 1–Day 10), inoculate OP50 and SM2170 (or relevant parasite) each into 5 ml LB. Incubate overnight at 28 °C, shaking at 160 rpm.
    2. The next day, on the bottom of a 100 mm Petri dish filled with 25 ml NGM Lite, draw a ~2.5 cm stripe across the middle of the dish, dividing the dish into 3 sections (see Figure 1).
      Notes:
      1. It is important to use plates that are not damp. In order to dry the surface of the plates, they can be placed in a chemical fume hood with lids off for ~20 min. Be sure to remove excess condensation from the plate lid as well. 
      2. Make enough SSPs to give proper replication of the ancestor and each experimentally evolved population. We recommend at least 3 replicates for each.


      Figure 1. View of the bottom of a 100 mm Petri dish with ~2.5 cm strip drawn across the middle

    3. Pipette 35 μl of OP50 (~ 2.9 x 109 CFU/ml) onto one of the far thirds of the NGM Lite and use a sterile inoculation loop to evenly spread the OP50 within the section. It is important to plate the OP50 first to prevent SM2170 from contaminating and being spread in this section of the plate. When pipetting the bacteria onto the SSP, discharge only to the first stop of the pipette to prevent splattering on the plate.
    4. On the opposite far third of the NGM Lite, pipette 35 μl of SM2170 (~ 7.8 x 108 CFU/ml) and use a sterile inoculation loop to evenly spread the bacteria within this section. Again, be sure to discharge to only the first pipette stop. The middle third will be left blank.
    5. Keep the plates level to prevent bacteria from spreading into other sections, incubate the SSPs overnight at 28 °C.

  5. Calculating Nematode Concentration
    1. 48 h after bleach synchronizing the nematode populations, add 2 ml M9 buffer to the plates containing synchronized nematodes.
      Notes: 
      1. Nematodes should be at L4 larval stage.
      2. Some of the M9 buffer will absorb into the plate. Therefore, adding 2 ml of M9 buffer to the plate will result in approximately 1 ml to be collected from the plate.
    2. Tilt the plate with M9 buffer side to side to wash the entire surface of the plate.
    3. Keeping the plate tilted to pool the M9 buffer, and using a new wide bore pipette tip between populations, collect each population of nematodes into its own 1.5 ml micro-centrifuge tube.
      Note: Wide bore tips are used when transferring live nematodes to avoid damaging the C. elegans and to increase the precision of measurements.
    4. Centrifuge the tubes containing nematodes at 94 x g for 30 sec to form a pellet.
    5. Wash the pellet 2 times with M9 buffer (as described in Steps C12-C16, except use 94 x g when centrifuging).
    6. After washing the populations twice, resuspend the nematodes in 1 ml M9 buffer.
    7. Invert the micro-centrifuge tube several times to mix.
    8. Working one population at a time, and using a wide bore pipette tip, take 3-20 μl samples, and place onto a microscope slide (see Figure 2).


      Figure 2. Microscope slide with 3-20 μl samples of nematodes in M9 buffer

    9. Under the microscope, count the number of nematodes in each 20 μl sample using a hand tally counter.
      Note: In order to count accurately, there should be approximately 30-50 nematodes in each sample. If the number of nematodes is outside this range, appropriately dilute or concentrate the centrifuge tubes containing the nematodes, and take 3 new 20 μl samples onto a microscope slide. This may need to be done more than once to get the correct approximate range.
    10. Calculate the average of the 3 samples and divide by 20 to get the average concentration of nematodes per 1 μl.
    11. Use the following equation to calculate the volume needed to give 100 nematodes:
      Volume = 100/x
      where x is the average concentration of nematodes per 1 μl, calculated in Step E10.
    12. Repeat Steps E7-E11 for the generation 0 ancestor, each experimentally evolved C. elegans population and the GFP-labeled tester strain.
      Note: The nematode population of interest will be competed against the GFP-labeled tester strain on the same plate. Therefore, if the calculated volumes of the focal strain and the GFP-tester strain total to more than ~150 μl, it will be necessary to concentrate the nematodes, repeat Steps E7-E11. 

  6. Competing either ancestral or experimental “focal” populations vs. a tester strain on the SSP
    1. Prior to introducing nematodes to the SSP, add a stripe of ampicillin across the middle section of the plate. (20 μl of 200 mg/ml ampicillin) (see Figure 3)
      Note: SM2170 is very motile. Ampicillin prevents SM2170 from spreading into the OP50 portion of the SSP.


      Figure 3. Serratia selection plate (SSP) design including ampicillin

    2. To the parasite side of the SSP, add both the focal host population of interest and the GFP-labeled tester strain in the volumes calculated in Step E11. Replicate as needed for each experimental population and the ancestral population to generate multiple pseudo replicate CFAs for each population.
    3. Pipette onto 3 OP50-seeded NGM Lite plates the volume calculated in Step E11 for the following:
      1. The generation 0 ancestor
      2. The experimentally evolved strain
      3. The GFP-labeled tester strain
      Note: These plates will be counted in Procedure G to determine the actual average number of hosts added to the SSP assay plates.
    4. Allow the SSPs to remain level and dry at room temperature for approximately 20 min.
    5. Incubate SSPs at 20 °C for 48-72 h, or enough time for the competed populations to reproduce and for the offspring to be at larval stage L1/L2.

  7. Scoring the SSP assays
    Note: It is necessary to use the GFP filter with the stereomicroscope and fluorescence illumination lamp in order to visualize the pharyngeal GFP marker. 
    1. Within the OP50 third of the SSP, count the number of focal and GFP-tester offspring out of a sample of 200 total offspring in a cross-section of the SSP (see Figure 4).
      Note: It is necessary to alternate between high and low backlighting during counting. High background light will allow the counter to visualize all the offspring, while low background light will allow the counter to differentiate between focal and GFP-labeled strains.


      Figure 4. Comparing focal and GFP-tester strains. A. Example of how both non-GFP and GFP expressing C. elegans individuals will appear under high backlight conditions. Scale bar = 0.1 mm. B. Example of GFP expressing C. elegans under low backlight conditions. Focal strains do not express GFP. Scale bar = 0.1 mm.

    2. Count the number of adult hosts on each of the 3 OP50-seeded plates for each population from Step F3. Take the average of the 3 counts and use this number to calculate the initial ratio of focal to tester strain parents.

  8. Calculate the mean relative fitness and the percent change in fitness after experimental evolution 
    1. The relative competitive fitness of a focal population or strain (experimental population or ancestral population) to the tester strain can be calculated as the relative change in the ratio of non-GFP to GFP expressing individuals over the course of the assay. Specifically, this is determined as:



      where, WX is the relative fitness of the focal strain, x is the proportion of focal strain offspring, and y is the proportion of focal strain parents initially plated on the SSP. See Supplementary Data for a sample data set from competitive fitness assays conducted on C. elegans host populations evolved in the presence of SM2170 (Evolution) and host populations evolved in the presence of heat-killed SM2170 (Control) for 30 generations. Relative fitness values greater than one indicate greater fitness for the focal strain relative to the tester strain. Whereas, relative fitness values of less than one indicate greater tester strain fitness relative to the focal strain. Finally, calculate the mean relative fitness for each focal strain by averaging the relative fitness values across each pseudo replicate.
    2. After experimental evolution, the percent change in mean competitive fitness for an experimental population can be calculated as:



      where, W ̅E is the mean relative fitness of the experimental population across all pseudo replicates and W ̅A is the relative fitness of the ancestral population across all pseudo replicates (Supplementary Data). Positive percent change values indicate that the experimental population increased in fitness during experimental evolution. Conversely, negative percent change values indicate a loss of fitness in the experimental population. Importantly, the percent change in mean fitness should be calculated separately for each independently evolved replicate population. We found a greater percent change in mean fitness in populations evolved under the Evolution treatment than those evolved under the Control treatment (Supplementary Data and Figure 5).

Data analysis

These analyses were previously described in Penley et al. (2017) with the exception that the mean proportion of experimental to tester strain (GFP) offspring was analyzed as opposed to the relative competitive fitness measure. All analyses were performed in JMP Pro 12.0.1.
The values of mean relative competitive fitness and the percent change in mean competitive fitness can be compared between particular strains, populations, or treatments using Analysis of Variance (ANOVA) and subsequent least squares mean linear contrast tests to test factors within different fixed effects (0.05 alpha value). However, the data must conform to the ANOVA assumptions of normality and homogeneity of variance. Shapiro-Wilk test (normality) and Levene’s test (homogeneity of variance) can be applied to the dataset to test the appropriateness of using ANOVA to analyze the data. If the data do not conform to the assumption of ANOVA despite transformation, then the data can be analyzed via a nonparametric Kruskal Wallis test. We tested the main effect of treatment (Evolution vs. Control) on values of the percent change in mean competitive fitness (Table 2), and found a significant treatment effect (Table 2 and Figure 5).

Table 2. ANOVA table. Testing the main effect of treatment (Evolution vs. Control) on the mean percent change in competitive fitness.



Figure 5. Sample data results using example data from Supplementary Data

It is important to note that independent replicate populations within experimental evolution treatments serve as the true replicates for analysis. Thus, the mean of all pseudo replicates is used for statistical analysis as the value representing each independently evolved replicate population. Accordingly, replicate population serves as a random effect and is nested with the relevant treatment in the ANOVA model.

Notes

The GFP marker carried by the tester strain is dominant. This CFA works most effectively when assaying only C. elegans hermaphrodites from both the focal strain/population and the test strain. C. elegans males can outcross with hermaphrodites. Focal population males may outcross with tester strain hermaphrodites. If this occurs the offspring from these hybrid crosses will express GFP, resulting in underestimates of the focal strain relative fitness.

Recipes

  1. LB Broth (200 ml)
    1. Add 5 g LB granules to 200 ml dH2O and stir until dissolved
    2. Aliquot 5 ml into each test tube and add a cap to each
    3. Autoclave for 20 min and store at 4 °C for up to 3 months
  2. NGM Lite plates (1 L)
    1. Add 29 g NGM Lite powder to 2 L flask containing 1 L dH2O
    2. Cover with aluminum foil and autoclave for 20 min
    3. Once cooled enough to touch, pour ~25 ml NGM Lite into 100 mm Petri dishes
    4. Store at 4 °C for up to 3 months
  3. Escherichia coli (OP50)-seeded NGM Lite plates 
    1. Inoculate E. coli OP50 in LB, incubate at 28 °C overnight, shaking at 160 RPM
    2. The next day add 200 μl OP50 culture to each NGM Lite plate
    3. Spread the culture over the entire surface of the plate
    4. Incubate at 28 °C overnight
    5. Store at 4 °C for up to 1 month
  4. M9 Buffer (1 L) (adapted from Stiernagle, 2006)
    1. Add 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, and 1 ml 1 M MgSO4 solution to a graduated cylinder
    2. Add dH2O to 1 L and stir until dissolved
    3. Aliquot into bottles and keep lids loose
    4. Autoclave for 20 min
    5. Store at room temp for up to 6 months
  5. 1 M MgSO4 Solution (100 ml)
    1. Add 12.037 g MgSO4 to 100 ml dH2O
    2. Stir until dissolved and store at room temp for up to 1 year
  6. Ampicillin 200 mg/ml (10 ml)
    1. Add 2 g ampicillin sodium salt into a 15 ml tube and fill dH2O up to 10 ml
    2. Stir until dissolved and filter-sterilize (0.22 μm)
    3. Store in 1 ml aliquots at -20 °C for up to 1 year

Acknowledgments

This protocol was adapted from previous work (Morran et al., 2009; Morran et al., 2011; Morran et al., 2014; Parrish et al., 2016; Penley et al., 2017). We thank S. Scholz, M. Parmenter, J. Anderson, and P. Phillips for assistance in developing this protocol. Additionally, we thank R. Parrish II, O. Schmidt, M. Allen, and A. Khalid for help in refining the protocol. We also thank three anonymous reviewers for improving this manuscript. Funding for this work was provided by Emory University to LTM. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR).

Competing interests

The authors declare that they have no conflicts of interest or competing interests.

References

  1. Gray, J. C. and Cutter, A. D. (2014). Mainstreaming Caenorhabditis elegans in experimental evolution. Proc Biol Sci 281(1778): 20133055.
  2. Lenski, R. E., Rose, M. R., Simpson, S. C. and Tadler, S. C. (1991). Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000 generations. American Naturalist 138(6): 1315-1341. 
  3. Morran, L. T., Parmenter, M. D. and Phillips, P. C. (2009). Mutation load and rapid adaptation favour outcrossing over self-fertilization. Nature 462(7271): 350-352.
  4. Morran, L. T., Parrish, R. C., 2nd, Gelarden, I. A., Allen, M. B. and Lively, C. M. (2014). Experimental coevolution: rapid local adaptation by parasites depends on host mating system. Am Nat 184 Suppl 1: S91-100. 
  5. Morran, L. T., Schmidt, O. G., Gelarden, I. A., Parrish, R. C., 2nd and Lively, C. M. (2011). Running with the Red Queen: host-parasite coevolution selects for biparental sex. Science 333(6039): 216-218.
  6. Parrish, R. C., 2nd, Penley, M. J. and Morran, L. T. (2016). The integral role of genetic variation in the evolution of outcrossing in the Caenorhabditis elegans-Serratia marcescens host-parasite system. PLoS One 11(4): e0154463. 
  7. Penley, M. J., Ha, G. T. and Morran, L. T. (2017). Evolution of Caenorhabditis elegans host defense under selection by the bacterial parasite Serratia marcescens. PLoS One 12(8): e0181913.
  8. Stiernagle, T. 2006. Maintenance of C. elegans in The C. elegans research community, ed. WormBook: the online review of C. elegans biology.
  9. Teotonio, H., Estes, S., Phillips, P. C. and Baer, C. F. (2017). Experimental evolution with Caenorhabditis nematodes. Genetics 206(2): 691-716.
  10. Wiser, M. J. and Lenski, R. E. (2015). A comparison of methods to measure fitness in Escherichia coli. PLoS One 10(5): e0126210.

简介

准确测量生物体的适应度对于衡量进化变化至关重要。当结合个体的生存和繁殖力以及考虑生物体所经历的任何生态相互作用或环境影响时,适合度测量的方法是最准确的。在这里,我们描述了一种用于测量秀丽隐杆线虫种群或菌株的相对平均适合度的方案,该方案通过在细菌寄生虫存在下计算个体存活率,繁殖力和种内竞争能力的测定。在这种竞争性适应性测定中,来自焦点种群或菌株的线虫与GFP标记的测试菌株以相等比例混合,然后将线虫的混合物暴露于寄生虫,并且通过测量来确定焦点菌株的相对竞争适合度。一代后,焦点线虫与GFP标记的线虫的比例发生变化。具体地,可以实施该协议以通过确定进化线虫与祖先线虫群体的相对竞争适合度来测量实验进化后线虫宿主适应度的变化。

【背景】 准确测量适应度和适应度随时间的变化对于确定群体对自然选择的反应至关重要。尽管如此,健康是众所周知难以衡量的,因为它包含了个人的生存,繁殖力,繁殖时间,并且必须考虑到对个体的生态和环境影响。尽管在所有可能的条件下没有用于测量适合度的协议是最佳的,但是考虑到生存和繁殖力的适应度的测量,同时保持生态和环境影响不变,可能提供对给定场景的适合性的可靠总体估计。在这里,我们描述了一个用于测量 C之间相对适应度差异的协议。线虫种群或菌株,用于确定细菌寄生虫存在时进化时间内相对适合度的变化。我们利用革兰氏阴性细菌粘质沙雷氏菌作为 C消耗的有毒寄生虫。线虫。特别是, S. marcescens 菌株SM2170能够杀死 C.线虫在摄取24至48小时内寄主(Penley et al。,2017)。该程序利用竞争健康测定(CFAs)(Lenski et al。,1991; Wiser和Lenski,2015),利用种内竞争来比较不同线虫种群或菌株之间的相对适合度(Morran et al。,2009)。通过CFA确定的相对适合度的测量结果将生存和繁殖与受控环境中的种内竞争相结合,以提供综合的适应度量(Penley et al。,2017)。

在细菌寄生虫存在下的相对适合度通过在一代的过程中将焦点菌株与同基因GFP标记的测试菌株竞争并测量局部菌株对测试菌株的繁殖成功来确定(Morran 等。,2009)。因此,该CFA解释了在线虫生命周期中寄生虫和宿主繁殖的存活率。一个单一的测试菌株用于测量每个焦点线虫群体或菌株的相对适合度,以促进群体或菌株之间相对适合度的比较。重要的是,测试者用咽GFP标记,以便在一代竞争后容易地观察测试菌株后代与焦点种群或菌株后代的关系。 CFA用50:50的焦点和测试菌株个体混合进行初始化,因此,后代中50:50混合物的任何偏差表明焦点和测试菌株之间的竞争适应性不相等。后代中局灶性线虫比例的增加表明相对于测试者具有更大的竞争适应性,而减少表明相对于测试者的竞争适应性降低。可以在多个群体中比较后代中的宿主宿主的比例,以测量焦点菌株或目标群体之间的相对竞争适合度。重要的是,竞争健身措施在两个人群或菌株之间竞争大致相同数量的个体时最有效,其中竞争适应性具有轻微到中等差异。菌株或群体的不均匀或可变起始比率可能混淆相对适合度的测量值,而竞争菌株或群体之间的适合度的主要差异通常难以准确量化(Wiser和Lenski,2015)。

该协议对于测量 C的实验演化后的进化变化特别有用。线虫在细菌寄生虫存在下寄主。首先,可以直接将实验宿主种群的相对适合度与祖先种群的相对适合度进行比较。祖先 C.线虫种群可以在实验进化过程中储存在-80°C,然后恢复为CFA,以评估实验种群适应度随时间的变化(Gray and Cutter,2014; Teotonio et al。 ,2017)。其次,在实验进化期间,宿主可通过改变的生活史和/或增加的宿主防御水平来适应寄生虫暴露。因此,仅测量寄生虫存在下的存活可能不能完全解释宿主适应性的变化。该CFA可以解释在寄生虫存在下改变生殖输出的生活史和抗性的变化。重要的是,此程序最初是为测量 C的变化而开发的。在细菌寄生虫存在下进行多代进化后的线虫竞争适应性粘质沙雷氏菌(Morran et al。,2009; Morran et al 。,2014; Parrish et al。,2016; Penley et al。,2017)。尽管如此,该协议可以适用于测量任何两个或更多 C的相对竞争适应度。线虫种群或菌株存在任何相关的细菌寄生虫。此外,它可以用于测量任何 C的实验演化过程中相对竞争适应度的变化。线虫种群在细菌寄生虫的存在下进化。

关键字:适应性测定, 实验进化, 秀丽隐杆线虫, 粘质赛氏杆菌, 竞争

材料和试剂

  1. 1.5毫升微量离心管(MIDSCI,目录号:MID15C)
  2. 1,000μl移液器吸头(MIDSCI,目录号:AVR4)
  3. 200μl移液器吸头(MIDSCI,目录号:AVR1)
  4. 1000μl宽口径移液器吸头(Genesee Scientific,目录号:22-426)
  5. 200μl宽口径移液器吸头(Genesee Scientific,目录号:22-423)
  6. 显微镜载玻片(Fisher Scientific,目录号:12-550-19)
  7. Semimicro刮刀(Fisher Scientific,目录号:14-374)
  8. 一次性接种环,10μl(VWR,目录号:12000-810)
  9. 100 x 15 mm培养皿(Tritech Research,目录号:T3301)
  10. 0.22μm无菌注射器过滤器(Spectrum Chemical Manufacturing,目录号:882-66597)
  11. 一次性塑料注射器(Thermo Fisher Scientific,目录号:S7510-10)
  12. Serratia marcescens 菌株SM2170,BSL2(Sue Katz,罗杰斯州立大学) 
  13. 大肠杆菌菌株OP50,BSL1(Caenorhabditis遗传中心)
  14. GFP标记的 C.线虫(株JK2735)(Caenorhabditis遗传中心)
  15. 线虫生长培养基Lite粉末(美国生物,目录号:N1005)
  16. LB颗粒(Fisher Scientific,目录号:BP9723-500)
  17. 磷酸二氢钾(KH 2 PO 4 )(Fisher Scientific,目录号:P288-100)
  18. 氯化钠(NaCl)(Fisher Scientific,目录号:S671-500)
  19. 磷酸氢二钠无水(Na 2 HPO 4 )(Fisher Scientific,目录号:S374-500)
  20. 无水硫酸镁(MgSO 4 )(Fisher Scientific,目录号:M65-500)
  21. 氨苄青霉素钠盐(Dot Scientific,目录号:DSA40040-25)
  22. 家用漂白剂 
  23. LB肉汤(见食谱)
  24. NGM Lite板材(见食谱)
  25. 大肠杆菌(OP50) - 种子NGM Lite板(见食谱)
  26. M9缓冲液(见食谱)
  27. 1 M MgSO 4 溶液(见食谱)
  28. 氨苄西林200 mg / ml(见食谱)

设备

  1. 手工计数器(United Scientific Supplies,目录号:HTCP01)
  2. 2升烧瓶(Corning,PYREX ®,目录号:5320-2L) 
  3. P1000μl移液器(Eppendorf,型号:Research ® plus,目录号:3121000120)
  4. P100μl移液器(Eppendorf,型号:Research ® plus,目录号:3121000074)
  5. -80°C冰箱(Eppendorf,New Brunswick TM ,型号:Innova ® U725)
  6. 用于1.5 ml微量离心管的台式离心机(Eppendorf,型号:5424)
  7. 20°C受控环境室(Percival Scientific,型号:I36NLC8)
  8. 28°C摇床培养箱(Eppendorf,New Brunswick TM ,型号:Innova ® 42R,目录号:M1335-0010)
  9. 立体显微镜(奥林巴斯,型号:SZX16)
  10. LED透射光照明底座(奥林巴斯,型号:SZX2-ILLT)
  11. 用于立体显微镜的GFP过滤器(Olympus,型号:SZX2-FGFP)
  12. 立体显微镜物镜7x-115x(Olympus,型号:SDFPLAPO1XPF)
  13. 荧光照明灯(Excelitas Technologies,型号:X-Cite ® 120Q)
  14. X-Cite ®液体光导(Bulbtronics,Excelitas Technologies,型号:805-00038)
  15. 高压灭菌器(STERIS,型号:SG-120)
  16. 化学通风橱(Kewaunee Scientific,型号:H05)

软件

  1. JMP Pro 12.0.1(SAS Institute Inc.,Cary,NC)

程序

注意:

  1. 该协议专门描述了在粘质沙雷氏菌(SM2170)存在下实验进化后测量群体竞争适应度的变化。然而,它可以适于在存在任何可以在所述条件下生长和传播的寄生虫的情况下测量任何两种或更多种感兴趣的线虫群体之间的相对适合度。没有特定的实验线虫菌株或实验进化方案是使用该方案简单地作为测量适合度的手段所必需的。但是,需要明确标记的GFP菌株。
  2. 在实验进化之前,0代祖先线虫应在-80°C冷冻。类似地,在实验进化结束时,有必要冻结实验进化的群体以用于这些和任何其他后续测定。冻结协议可以在Stiernagle(2006)(https://www.ncbi.nlm.nih.gov/books/NBK19649/)中找到。
  3. 此过程持续约2周。有关程序计划的示例,请参阅表1.

表1.程序每日时间表示例


  1. 解冻实验和测试人群
    1. 从-80°C冰箱中取出以下每种菌株的一个冷冻管: 
      1. 第0代祖先 
      2. 实验进化的感兴趣的人群 
      3. GFP标记的测试菌株(JK2735) 
      注意:如果测量相对竞争适合度,解冻2个或更多感兴趣的线虫群体和GFP标记的测试菌株。
    2. 让管在室温下解冻。
    3. 解冻后,轻轻颠倒管几次以混合溶液。
    4. 将管的全部内容物倒入标记的100mm E中。大肠杆菌 OP50种子NGM Lite平板。
    5. 让板吸收所有液体成分。如果液体含量未被完全吸收,请在化学通风橱中将盖板短暂干燥并盖上盖子。
    6. 将这些板在20℃孵育24小时。
    7. 检查平板,看到至少50个线虫在解冻后存活。如果是这样,将平板在20°C温育,使群体从解冻中恢复并扩张(约1周)。如果没有,为感兴趣的人群解冻另一个冷冻管并重复解冻过程。

  2. 准备同步
    1. 一旦生成0祖先,实验进化,并且GFP标记的测试者群体已经扩展到每个平板上观察到数百个个体的点,您可以继续。
    2. 对于每个群体,使用火焰灭菌的金属刮刀从含有线虫的平板上切下约2.5cm 2 的NGM Lite。
    3. 对于每个人群,从原始板上取下这片NGM Lite并将其面朝下放在新的100 mm OP50种子NGM Lite板上。
    4. 将这些新板在20°C孵育48小时。
    5. 48小时后,在显微镜下观察步骤B4的板以检查蛋。如果在平板表面上观察到至少约1,000个卵,则进行程序C.如果不是,则将平板在20℃下孵育并在第二天检查。如果平板耗尽OP50,线虫将不再产卵。在这种情况下,请重复步骤B2-B5。

  3. 同步
    注意:同步协议改编自Stiernagle(2006)。同步的目的是杀死所有线虫,只留下沉积在平板上的卵子才能存活。这将使蛋阶段的人口同步年龄。 
    1. 一次加工一个平板,在平板上加入2毫升M9缓冲液。
      注意:一些M9缓冲液将被吸收到板中。因此,向板中加入2ml M9缓冲液将导致从板中收集~1ml。
    2. 将板平移到一侧,用M9缓冲液清洗板的整个表面。
      注意:如果平板上的细菌草坪看起来厚而粘,请用移液器吸头轻轻刮去平板表面。这有助于放松鸡蛋。
    3. 倾斜平板以允许所有M9缓冲区在一个区域中汇集。 
    4. 用移液管移除M9缓冲液并将其移入1.5 ml微量离心管中。
    5. 重复步骤C1-C4,使每个群体都在自己的微量离心管中。
    6. 将离心管在845 x g 下离心30秒以形成沉淀。
    7. 使用管之间的新移液管尖端从每个管中除去上清液。
    8. 在含有颗粒的每个管中加入800μlM9缓冲液。
      注意:步骤C9-C17应在3分钟或更短时间内完成,以防止漂白剂损坏鸡蛋。
    9. 在每个含有颗粒和800μlM9缓冲液的离心管中加入200μl家用漂白剂。
      注意:这将在每个管中产生20%的家用漂白剂溶液。
    10. 关闭并倒置管几次以混合漂白剂和M9缓冲液,并松开颗粒。
    11. 将管在845 x g 下离心30秒。
    12. 使用管之间的新移液管尖端从每个管中除去上清液。
    13. 用1ml M9缓冲液洗涤每个沉淀。关闭并倒置管几次以松开颗粒。
    14. 将管在845 x g 下离心30秒。
    15. 使用管之间的新移液管尖端从每个管中去除上清液。
    16. 重复步骤C13-C15。
      注意:您将洗涤沉淀两次。
    17. 将每个管子的整个颗粒吸移到自己的新的100 mm OP50种子NGM Lite板上,确保更换管子之间的尖端。
    18. 将平板在20°C孵育48小时,或直到线虫处于幼虫期L4。

  4. Serratia 选择板(SSPs)的制备
    1. 在同步的同一天(参见表1-第10天),将OP50和SM2170(或相关的寄生虫)各自接种到5ml LB中。在28℃下孵育过夜,以160rpm振荡。
    2. 第二天,在装有25毫升NGM Lite的100毫米培养皿的底部,在培养皿中间划出约2.5厘米的条纹,将培养皿分成3个部分(见图1)。
      注意:
      1. 使用非潮湿的板材很重要。为了干燥板的表面,可以将它们放置在化学通风橱中,盖子关闭约20分钟。务必同时清除印版盖板上多余的冷凝水。
      2. 制作足够的SSP以正确复制祖先和每个实验进化的群体。我们建议每个至少重复3次。


      图1. 100毫米培养皿底部的视图,中间有~2.5厘米的条带

    3. 移取35μlOP50(~2.9×10 9 CFU / ml)到NGM Lite的远端三分之一上,并使用无菌接种环将OP50均匀地扩散到切片内。首先镀OP50非常重要,以防止SM2170污染并在板的这一部分扩散。将细菌移液到SSP上时,只能排放到移液器的第一个位置,以防止溅到板上。
    4. 在相反的三分之一的NGM Lite上,移取35μl的SM2170(~7.8×10 8 CFU / ml)并使用无菌接种环在该部分内均匀地扩散细菌。再次,确保只排放到第一个移液器停止。中间三分之一将留空。
    5. 保持平板水平以防止细菌扩散到其他部分,在28°C下将SSP孵育过夜。

  5. 计算线虫浓度
    1. 在漂白剂同步线虫种群后48小时,向含有同步线虫的平板中加入2ml M9缓冲液。
      注意: 
      1. 线虫应该处于L4幼虫阶段。
      2. 一些M9缓冲液将吸收到板中。因此,向板中加入2ml M9缓冲液将导致从板中收集约1ml。
    2. 将M9缓冲液从一侧到另一侧倾斜,以清洗板的整个表面。
    3. 保持平板倾斜以汇集M9缓冲液,并在群体之间使用新的大口径移液管尖端,将每个线虫群收集到其自己的1.5毫升微量离心管中。
      注意:在转移活线虫时使用大口径吸头以避免损坏秀丽隐杆线虫并提高测量精度。
    4. 将含有线虫的管在94 x g 离心30秒以形成沉淀。
    5. 用M9缓冲液洗涤沉淀2次(如步骤C12-C16中所述,除了在离心时使用94 x g )。
    6. 将种群洗涤两次后,将线虫重悬于1ml M9缓冲液中。
    7. 将微型离心管反转几次以混合。
    8. 一次操作一个群体,并使用大口径移液器吸头,取3-20μl样品,放在显微镜载玻片上(见图2)。


      图2.在M9缓冲液中含有3-20μl线虫样品的显微镜载玻片

    9. 在显微镜下,使用手动计数器计数每个20μl样品中的线虫数。
      注意:为了准确计数,每个样本中应该有大约30-50个线虫。如果线虫的数量超出该范围,适当稀释或浓缩含有线虫的离心管,并将3个新的20μl样品取出到显微镜载玻片上。这可能需要不止一次才能获得正确的近似范围。
    10. 计算3个样本的平均值并除以20,得到每1μl线虫的平均浓度。
    11. 使用以下等式计算给出100个线虫所需的体积:
      体积= 100 / x
      其中x是每1μl线虫的平均浓度,在步骤E10中计算。
    12. 对第0代祖先重复步骤E7-E11,每个实验进化 C.线虫种群和GFP标记的测试菌株。
      注意:感兴趣的线虫群体将在同一平板上与GFP标记的测试菌株竞争。因此,如果计算的焦点菌株体积和GFP测试菌株总量超过~150μl,则需要浓缩线虫,重复步骤E7-E11。

  6. 竞争祖先或实验“焦点”人群与SSP上的测试人员菌株
    1. 在将线虫引入SSP之前,在板的中间部分添加氨苄青霉素条带。 (20μl200mg / ml氨苄西林)(见图3)
      注意:SM2170非常运动。氨苄青霉素可防止SM2170扩散到SSP的OP50部分。


      图3.沙雷氏菌选择板(SSP)设计包括氨苄青霉素

    2. 在SSP的寄生虫侧,在步骤E11中计算的体积中添加目标聚焦宿主群和GFP标记的测试菌株。根据需要复制每个实验群体和祖先群体,以为每个群体生成多个伪复制CFA。
    3. 移取到3个OP50播种的NGM Lite板上,计算步骤E11中计算的体积,如下所示:
      1. 第0代祖先
      2. 实验进化的菌株
      3. GFP标记的测试菌株
      注意:这些平板将在程序G中计算,以确定添加到SSP测定板的实际平均宿主数。
    4. 使SSP保持水平并在室温下干燥约20分钟。
    5. 将SSP在20°C孵育48-72小时,或足够时间让竞争群体繁殖,并使后代处于幼虫期L1 / L2。

  7. 评分SSP分析
    注意:必须将GFP过滤器与立体显微镜和荧光照明灯一起使用,以便可视化咽部GFP标记。 
    1. 在SSP的OP50中,在SSP的一个横截面中,从200个总后代的样本中计算焦点和GFP测试子代的数量(见图4)。
      注意:在计数期间必须在高亮度和低亮度之间交替。高背景光将使计数器可视化所有后代,而低背景光将允许计数器区分焦点和GFP标记的菌株。


      图4.比较局灶性和GFP测试菌株。 A.非GFP和GFP表达 C的示例。线虫个体将出现在高背光条件下。比例尺= 0.1毫米。 B.表达GFP的GFP的实例。在低背光条件下的线虫。局灶性菌株不表达GFP。比例尺= 0.1 mm。

    2. 从步骤F3计算每个种群的3个OP50接种平板中每一个上的成体宿主数。取3个计数的平均值,并使用此数字计算焦点与测试菌株亲本的初始比率。

  8. 计算实验进化后的平均相对适应度和适应度变化百分比
    1. 可以将焦点群体或菌株(实验群体或祖先群体)对测试菌株的相对竞争适合度计算为在测定过程中非GFP与GFP表达个体的比率的相对变化。具体而言,这被确定为:



      其中WX是局部应变的相对适合度,x是局部应变后代的比例,y是最初在SSP上铺板的局部应变亲本的比例。有关竞争健身的样本数据集,请参见补充数据在 C上进行的分析。线虫宿主群体在SM2170(进化)存在下进化,宿主群体在热灭活的SM2170(对照)存在下进化30代。大于1的相对适合度值表明相对于测试菌株的焦点应变的适应性更大。然而,相对适合度值小于1表示相对于焦点应变的更大的测试者应变适应度。最后,通过平均每个伪复制品的相对适合度值来计算每个焦点菌株的平均相对适合度。
    2. 在实验演变之后,实验人群的平均竞争适应度的百分比变化可以计算为:



      其中,W̅E是所有伪复制品中实验群体的平均相对适应度,W̅A是所有伪复制品中祖先种群的相对适应度(补充资料)。阳性百分比变化值表明实验人群在实验进化期间的适应度增加。相反,负百分比变化值表明实验人群的适应度下降。重要的是,对于每个独立进化的重复种群,应分别计算平均适合度的百分比变化。我们发现,在进化治疗下进化的人群中平均适应度的变化百分比比在对照治疗下进化的人数更大(补充数据和图5)。

数据分析

这些分析先前在Penley 等人(2017)中描述,不同之处在于分析实验与测试菌株(GFP)后代的平均比例,而不是相对竞争适合度测量。所有分析均在JMP Pro 12.0.1中进行。
平均相对竞争适应度的值和平均竞争适应度的百分比变化可以使用方差分析(ANOVA)和随后的最小二乘平均线性对比度测试来比较特定菌株,群体或治疗,以测试不同固定效应内的因子(0.05)阿尔法值)。但是,数据必须符合方差的正态性和同质性的ANOVA假设。可以将Shapiro-Wilk测试(正态性)和Levene测试(方差同质性)应用于数据集,以测试使用ANOVA分析数据的适当性。如果数据不符合ANOVA的假设,尽管转换,则可以通过非参数Kruskal Wallis检验分析数据。我们测试了治疗(进化与对照)对平均竞争适应度变化百分比值的主要影响(表2),并发现了显着的治疗效果(表2和图5)。

表2. ANOVA表。测试治疗(进化与对照)对竞争健康的平均变化百分比的主要影响。


图5.使用补充数据的示例数据的示例数据结果

值得注意的是,实验进化处理中的独立复制群体可作为分析的真实重复。因此,所有伪重复的平均值用于统计分析,作为代表每个独立进化的重复群体的值。因此,重复群体充当随机效应并且与ANOVA模型中的相关治疗嵌套。

笔记

由测试菌株携带的GFP标记是显性的。当仅测定 C时,该CFA最有效。来自焦点菌株/群体和测试菌株的线虫雌雄同体。 ℃。线虫雄性可以与雌雄同体远交。焦点人群雄性可能与测试者菌株雌雄同体异常交配。如果发生这种情况,来自这些杂交杂交的后代将表达GFP,导致低估了局灶性菌株相对适合度。

食谱

  1. LB肉汤(200毫升)
    1. 将5g LB颗粒加入200ml dH 2 O中并搅拌直至溶解
    2. 将5ml等分到每个试管中,并为每个试管加盖
    3. 高压灭菌20分钟,在4°C下储存长达3个月
  2. NGM Lite板(1升)
    1. 将29g NGM Lite粉末加入到含有1L dH 2 O的2L烧瓶中
    2. 用铝箔和高压灭菌器覆盖20分钟
    3. 一旦冷却到足以触摸,将~25毫升NGM Lite倒入100毫米培养皿中
    4. 在4°C下储存长达3个月
  3. Escherichia coli (OP50) - 种子NGM Lite牌照 
    1. 接种 E.在LB中的大肠杆菌 OP50,在28℃下孵育过夜,以160RPM振荡
    2. 第二天,每个NGM Lite平板加入200μlOP50培养物
    3. 将培养物扩散到培养皿的整个表面上
    4. 在28°C孵育过夜
    5. 在4°C下储存长达1个月
  4. M9缓冲液(1升)(改编自Stiernagle,2006年)
    1. 加入3g KH 2 PO 4 ,6g Na 2 HPO 4 ,5g NaCl和1ml将1M MgSO 4 溶液加入量筒中
    2. 将dH 2 O加入1L并搅拌直至溶解
    3. 分装成瓶子并保持盖子松动
    4. 高压灭菌20分钟
    5. 在室温下储存长达6个月
  5. 1M MgSO 4 溶液(100ml)
    1. 将12.037g MgSO 4 加入100ml dH 2 O中
    2. 搅拌至溶解并在室温下储存长达1年
  6. 氨苄青霉素200毫克/毫升(10毫升)
    1. 将2 g氨苄青霉素钠盐加入15 ml管中,加入dH 2 O至10 ml
    2. 搅拌至溶解并过滤灭菌(0.22μm)
    3. 在-20°C下以1 ml等分试样储存长达1年

致谢

该协议改编自先前的工作(Morran et al。,2009; Morran et al。,2011; Morran et al。,2014; Parrish et al。,2016; Penley et al。,2017)。我们感谢S. Scholz,M。Parmenter,J。Anderson和P. Phillips协助制定该协议。此外,我们感谢R. Parrish II,O。Schmidt,M。Allen和A. Khalid帮助完善该协议。我们还要感谢三位匿名审稿人改进这份手稿。 Emory大学为LTM提供了这项工作的资金。本研究中使用的一些线虫菌株由Caenorhabditis遗传学中心提供,该中心由NIH国家研究资源中心(NCRR)资助。作者声明他们没有利益冲突或竞争利益。

参考

  1. Gray,J。C.和Cutter,A。D.(2014)。在实验进化中将 Caenorhabditis elegans 纳入主流。 Proc Biol Sci 281(1778):20133055。
  2. Lenski,R.E.,Rose,M.R.,Simpson,S.C。和Tadler,S.C。(1991)。 Escherichia coli的长期实验进化。 I.适应和2000年代的分歧。 美国博物学家 138(6):1315-1341。 
  3. Morran,L。T.,Parmenter,M。D.和Phillips,P。C.(2009)。 Mutation负荷和快速适应有利于自我受精的异交。 自然 462(7271):350-352。
  4. Morran,L。T.,Parrish,R。C.,2nd,Gelarden,I。A.,Allen,M。B. and Lively,C。M.(2014)。 实验共同进化:寄生虫的快速局部适应取决于宿主交配系统。 Am Nat 184 Suppl 1:S91-100。 
  5. Morran,L。T.,Schmidt,O。G.,Gelarden,I。A.,Parrish,R。C.,2nd and Lively,C。M.(2011)。 与红女王一起运行:寄主 - 寄生虫共同进化选择双亲性行为。 Science 333(6039):216-218。
  6. Parrish,R。C.,2nd,Penley,M。J. and Morran,L。T.(2016)。 遗传变异在 Caenorhabditis elegans异位进化中的不可或缺的作用 -Serratia marcescens 寄主 - 寄生虫系统。 P LoS 一个 11(4):e0154463  
  7. Penley,M。J.,Ha,G。T.和Morran,L。T.(2017)。 细菌寄生虫选择秀丽隐杆线虫宿主防御的演变 Serratia marcescens 。 PLoS One 12(8):e0181913。
  8. Stiernagle,T。2006. 维护 C. C中的线虫。线虫研究社区,编辑。 WormBook: C的在线评论。线虫生物学。
  9. Teotonio,H.,Estes,S.,Phillips,P。C. and Baer,C.F。(2017)。 Caenorhabditis 线虫的实验进化。 Geneti cs 206(2):691-716。
  10. Wiser,M。J.和Lenski,R。E.(2015)。 测量 Escherichia coli 中适合度的方法的比较。 PL oS 开启 e 10(5):e0126210。
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引用:Penley, M. J. and Morran, L. (2018). Assessment of Caenorhabditis elegans Competitive Fitness in the Presence of a Bacterial Parasite. Bio-protocol 8(16): e2971. DOI: 10.21769/BioProtoc.2971.
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