By Kelsi Elkins, Emily Virgin, Dr. Susannah French

A Pilot Study Comparing Maternal Immunity, Egg Yolk Immunity, and Offspring Wound Healing in Side-Blotched Lizards (Uta Stansburiana) Following An Immune Challenge

A Pilot Study Comparing Maternal Immunity, Egg Yolk Immunity, and Offspring Wound Healing in Side-Blotched Lizards (Uta Stansburiana) Following an Immune Challenge PDF File

Introduction

  • Maternal allocation of physiological factors before birth can drastically alter offspring phenotype or quality.
  • In vertebrates, mothers can invest antibodies or immune factors via egg yolk or placenta to provide protection from pathogens.
  • Infection during reproduction can alter maternal investment into offspring.
  • Maternal investment of immune components can reduce the physiological costs of an infection.
  • It is unclear if this occurs in reptilian species.
A lizard like reptile crawling on the ground.

Research Questions

  1. Do immune challenged females alter egg size or egg yolk investment of bactericidal factors?
  2. Are offspring from immune challenged females larger? Do they have better wound healing ability?

Hypotheses

Experiment 1:

  • Offspring of immune challenged females will have larger egg size and more egg yolk investment than those of non-immune challenged females.

Experiment 2:

  • Offspring of immune challenged females will be larger and have quicker wound healing ability than the offspring on non-immune challenged females.

Methods and Design

Field Capture and Housing

  • Reproductive female lizards were captured from the St. George area and then processed and housed at Utah State until oviposition.

Immune Challenge and Assay

  • Females were injected with lipopolysaccharide (LPS) to mimic a bacterial infection, saline (PBS), or no injection (NI) 24 hours after being housed.
  • Blood samples were collected 72 hours aftertreatment.
  • Yolk samples and female plasma samples were assayed ex vivo to measure bactericidal ability following exposure to E. coli.

 

A flow chart depicting how "PBS or No Injection" and "LPS Injection" causes biological changes.

Wound Analysis

  • ImageJ was used to analyze the area of the open wound from photographs of hatchlings.
  • Percent healed was determined by comparing wound area on day 1 to day 7.
Timeline of Wounding and Measurements of Hatchlings
Day 0 (hatch day) Take photo, measure mass and SVL
Day 1 Administer 1 mm wound
Day 3 Take photo, measure mass and SVL
Day 5 Take photo, measure mass and SVL
Day 7 Take photo, measure mass and SVL

Statistical Analyses

  • Mixed effects Analysis of Variance

    Experiment 1:

      Dependent Variable: yolk immunity, egg size

      Independent Variable: Treatment

      Random Effect (Intercept): Maternal ID and Site

    Experiment 1 –Egg Yolk Immunity (Fig. 1) and Egg Mass
    (Fig. 2) Do Not Differ Across Maternal Treatments

    A box graph with the treatment for the x-axis and the Bactericidal Capacity for the y-axis.
    Figure 1: When controlling for maternal ID and site, egg yolk BKA does not differ among treatment groups and is not significantly different from the null model.
    (ANOVA between hypothesized and null model: p=0.13)
    A box graph with the treatment for the x-axis and the Bactericidal Capacity for the y-axis.
    Figure 2 When controlling for maternal ID and site, egg
    mass does not differ among treatment groups and is not
    significantly different from the null model.
    (ANOVA between hypothesized and null model:
    p=0.48)

    Experiment 2:

      Dependent Variable: hatchling immunity and size

      Independent Variable: Treatment

      Random Effect (Intercept): Maternal ID 

    Experiment 2 –Offspring Wound Healing Ability (Fig. 3) and Size (Fig. 4) Do Not Differ Across Maternal Treatments

    A box graph with the treatment for the x-axis and the Bactericidal Capacity for the y-axis.
    Figure 3: When controlling for maternal ID, hatchling immunity does not differ among treatment groups and is not significantly different from the null model.
    (ANOVA between hypothesized and null model: p = 0.47)
    A box graph with the treatment for the x-axis and the Bactericidal Capacity for the y-axis.
    Figure 4: When controlling for maternal ID, hatchling SVL differs between LPS and NI treatment, but model fit is not significantly different
    (ANOVA between hypothesized and null model: p = 0.01, but model fit (BIC) is the same)

  • Determine whether hypothesized model is significantly different (p < 0.05) from null model

A bucket with sand and eggs in it.
A plateau with shrubs and mountains in the distance.
A lizard crawling on a rock.

Conclusion

  • We saw no statistical difference in egg and hatchling characteristics across maternal treatment groups, apart from hatchling size differing between the LPS and NI group.
  • Differences in hatchling size between LPS and NI group could be due to low sample sizes, since model fit did not differ between the hypothesized and null model.
  • Increasing the sample sizes of eggs and hatchlings may provide more statistical power and help us explore trends that began to emerge.
  • It is possible that the timing of treatment might not have been sufficient to alter maternal investment into eggs and hatchlings.
  • It is possible that lizards invest only pathogen-specific antibodies as opposed to investing innate immune components into the egg.
  • Future studies should include physiological assays that measure both innate and adaptive immune factors.

Literature Cited

  • Groothuis, T. G., Hsu, B. Y., Kumar, N., & Tschirren, B (2019). Philosophical Transactions of the Royal Society B, 374(1770), 20180115.
  • Grindstaff, J. L., Hasselquist, D., Nilsson, J. Å., Sandell, M., Smith, H. G., & Stjernman, M (2006). Proceedings of the Royal Society B: Biological Sciences, 273(1600), 2551-2557.
  • Bowers, E. K., Bowden, R. M., Sakaluk, S. K., & Thompson, C. F (2015). The American Naturalist, 185(6), 769-783.
  • Grindstaff, J. L. (2008). Journal of Experimental Biology, 211(5), 654-660.
  • French, S. S., & Neuman-Lee, L. A. (2012). Biology Open, 1(5), 482-487.