Intra-species variation in body color maturation in the red flour beetle, Tribolium castaneum

Student Presenter

Abstract

Introduction and Aims

Pigmentation Pathway and Pigment Mutants

Methods and Techniques

Results

Conclusions

Acknowledgements

NACE Career Readiness Competencies

Student Presenter

This poster was created in collaboration with Yoshinori Tomoyasu (Mentor) and Jacob Mohn (Graduate Student).

Lillian Shelley

Biology and Psychology Majors; Pre-Medicine and Neuroscience Co-Majors; Molecular Biology and Medical Humanities Minors

Graduating May 2027

Abstract

The insect exoskeleton provides structural support and protection while also displaying a remarkable diversity in coloration. Although the genetic pathways involved in exoskeleton formation and pigmentation have been well characterized in model insects, natural variation in these processes within a single species remains poorly understood. This project investigates intra-species variation in body color maturation in the red flour beetle, Tribolium castaneum. We hypothesize that different beetle strains exhibit variation in both the timing of body color maturation and the expression of genes involved in exoskeleton formation. To test this hypothesis, we will first quantify differences in the timing of body color maturation among multiple Tribolium strains originating from geographically distinct areas, as well as several body color mutants. Adult beetles will be monitored after eclosion, and body coloration will be quantitatively measured. In the second aim, we will measure expression levels of the sclerotization-related gene ebony. Gene expression will be quantified using droplet digital PCR (ddPCR), allowing absolute measurement of transcript abundance across strains. Together, these experiments will reveal the variation in body color maturation as well as the differences in ebony gene expression among Tribolium populations. This work will contribute to a deeper understanding of how variation in gene expression shapes phenotypic diversity and will provide broader insight into the molecular mechanisms underlying genome evolution and diversification. These findings will advance our general understanding of how genetic variation generates biological diversity and may also impact research in areas such as genetics, evolution, and biomedical sciences. Furthermore, this project will contribute to my educational development by providing a deeper understanding of how genes and genomes function across organisms, including humans. This knowledge will be essential as I pursue a future career in medicine, while also engaging in research at the interface of biomedical science and human health.

Introduction and Aims

Diversity is fundamental to life.
The genetic mechanism underlying this diversity is still elusive.
There is a vast array of color patterns in organisms, making pigmentation crucial to diversity.
Coleoptera makes up 25% of all described species, with a variety of pigmentation patterns, making this clade a great model to study.
In Tribolium castaneum, a genetic model organism, there are several body color mutants caused by mutations in the pigmentation pathway.
Understanding the molecular and developmental basis of these mutants can provide insight into the evolution of pigmentation.

Aim 1 (Morphological): Quantify differences in the timing of body color maturation among Tribolium strains.

Aim 2 (Molecular): Quantify the difference in ebony gene expression among Tribolium strains.

Subaim: Identify most suitable reference for Tribolium gene using ddPCR analysis.

Pigmentation Pathway and Pigment Mutants

Photos taken from https://www.sciencedirect.com/science/article/pii/S0021925818930863 and directly photographed by Tomoyasu Lab members.

Diagram of the insect cuticle pigmentation and sclerotization pathway, showing the biosynthesis of melanins, NADA, and NBAD from tyrosine via dopamine in epidermal cells.

Photographs of five adult beetles comparing wild type and four pigmentation mutants, showing variation in cuticle color from reddish-brown to dark gray/black.

Methods and Techniques

Differential Assessment of Relative Chromaticity (DARC) Test

- Collect pupae in petri dish
- Check daily and collect eclosed adults
- Stage and freeze adults based on developmental time course (Days 1, 2, 3, 4, 5, 10, etc. after eclosion)
- Image using a microscope with 5 beetles next to 3 yellow pins used as color controls
- Convert image to grayscale
- Analyze using ImageJ
  - 3 technical replicates per beetle
- Compare using Student t-test

ddPCR

- Digital droplet PCR is a highly sensitive PCR that separates the DNA sample into thousands of droplets, allowing absolute quantification without standard curves.

Results

Aim 1 Results

A graph showing the chromaticity of Red Flour Beetles over a period of 30 days.

Aim 2 Results

Data table showing semi-quantitative PCR expression values for two genes across two *Tribolium castaneum* strains.

Data table showing ddPCR expression levels of five candidate reference genes across two wild-type *Tribolium castaneum* strains, *Ga1* and *Pu11*.

Conclusions

Aim 1 Findings/Conclusion: Despite similar time courses of color development, there are overall coloration differences in Tribolium strains. cola, a known mutant, is the darkest strain examined while Pu11, a wild-type strain, is the lightest. Ga1 is also a wild-type strain and has intermediate coloration compared to the other two.
Aim 2 Findings/Conclusion: There is a difference in ebony expression among Pu11 and cola. This supports the idea that cola is a regulatory mutant decreasing ebony expression.
- Subaim: There is a large difference in expression of RPS3e and ELF1a among WT straints, showing that these are not suitable reference genes.
Next Steps:
- Examine new strains of Tribolium using the DARC test to determine the timing of body wall coloration.
- Use ddPCR to explore differences in ebony expression among strains.
- Explore reference genes in relation to other wild-type Tribolium strains.

Academic research poster titled "Intra-species variation in body color maturation in the red flour beetle, *Tribolium castaneum*" by Lillian Shelley

Acknowledgements

This project would not have been possible without the support of Miami University and all past and present members of the Tomoyasu Lab.

NACE Career Readiness Competencies

Technology - I demonstrated use of both ddPCR and modern microscopy through this project, fundamental technology used in biomedical research.

Leadership - I was the sole undergraduate on this project, taking on a large role as a leader.

Career + Self-Development - This project is fundamental to gaining skills I will use in the future as I pursue an MD/PhD, such as the technology mentioned above.

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