Posters
Presenting Author Academic/Professional Position
Sydney Do
Academic Level (Author 1)
Medical Student
Discipline/Specialty (Author 1)
Human Genetics
Academic Level (Author 3)
Faculty
Discipline Track
Biomedical Science
Abstract Type
Research/Clinical
Abstract
Background: RNA interference (RNAi) silences specific gene expression through the introduction of double-stranded RNA (dsRNA). In Tribolium castaneum, RNAi enables precise manipulation of gene activity, allowing researchers to study developmental processes and genetic interactions across all life stages. dsRNA is processed by ribonucleases into small interfering RNAs (siRNAs) or microRNAs (miRNAs), which are recruited by the RNA-induced silencing complex (RISC), which targets and degrades specific mRNA molecules, allowing the observation of loss-of-function phenotypes.
T. castaneum offers advantages as a model for RNAi studies, including a fully annotated genome, a strong RNAi response, ease of application, and gene expression dynamics similar to that of complex animals. Our research utilizes RNAi to investigate the roles of key developmental genes in embryonic patterning by systematically knocking down multiple segmentation genes and analyze their functions during early embryogenesis. Understanding these genes in model organisms provides insight into conserved genetic pathways that regulate body patterning, many of which are involved in human congenital disorders.
Methods: We injected female T. castaneum with double-stranded RNA (dsRNA) targeting specific segmentation genes. The injection mixture contained 1 µg/µL dsRNA, 2x injection buffer, and a green tracking dye. Adult beetles were immobilized with ether anesthesia, sexed using morphological features, and injected under a dissection microscope into the reproductive region until visible abdominal expansion confirmed successful injection. Post-injection, beetles were housed in flour for egg collection.
Embryos were harvested at 48-hour intervals and fixed for imaging. Fluorescent in situ hybridization (RNA-FISH) was performed to visualize the expression patterns of segmentation genes. Embryonic morphology was assessed at the germband stage using fluorescent microscopy.
Results: Knockdown of pair-rule genes in adult females led to notable embryonic phenotypes in their offspring. Wild-type embryos displayed typical sequential stripe expression of eve, run, and odd, consistent with the segmentation clock during germband elongation. In contrast, missing or malformed segments in knockdown embryos were observed, with notable differences from wild-type morphology highlighted by gaps in gene expression and structural discontinuities:
- Eve RNAi embryos showed disrupted eve stripe formation and a truncated posterior.
- Run RNAi embryos lacked characteristic run stripes, with fewer abdominal segments formed.
- Odd RNAi similarly revealed disrupted segmental patterning.
Conclusions:
This study characterized the functional effects of segmentation genes in T. castaneum by examining gene knockdown phenotypes inherited by daughter embryos. The exogenous introduction of dsRNA into adult female beetles significantly alters zygotic gene expression in offspring embryos, disrupting the sequential expression of these genes and terminating the spatiotemporal dynamics required for T. castaneum oscillatory patterning during germband elongation. Reduced pair-rule gene function resulted in offspring with missing abdominal segments and truncated bodies. This contributes to our understanding of conserved developmental gene networks, many of which are implicated in congenital disorders in humans. These findings also highlight the potential of insect models in refining RNAi-based gene therapies and understanding gene regulatory mechanisms relevant to developmental biology and disease.
Presentation Type
Poster
Recommended Citation
Do, Sydney; Garcia, J.; and El-Sherif, Ezzat, "Segmenting the Flour Beetle: RNAi Probes into Embryonic Gene Function in Tribolium castaneum" (2025). Research Colloquium. 104.
https://scholarworks.utrgv.edu/colloquium/2025/posters/104
Included in
Segmenting the Flour Beetle: RNAi Probes into Embryonic Gene Function in Tribolium castaneum
Background: RNA interference (RNAi) silences specific gene expression through the introduction of double-stranded RNA (dsRNA). In Tribolium castaneum, RNAi enables precise manipulation of gene activity, allowing researchers to study developmental processes and genetic interactions across all life stages. dsRNA is processed by ribonucleases into small interfering RNAs (siRNAs) or microRNAs (miRNAs), which are recruited by the RNA-induced silencing complex (RISC), which targets and degrades specific mRNA molecules, allowing the observation of loss-of-function phenotypes.
T. castaneum offers advantages as a model for RNAi studies, including a fully annotated genome, a strong RNAi response, ease of application, and gene expression dynamics similar to that of complex animals. Our research utilizes RNAi to investigate the roles of key developmental genes in embryonic patterning by systematically knocking down multiple segmentation genes and analyze their functions during early embryogenesis. Understanding these genes in model organisms provides insight into conserved genetic pathways that regulate body patterning, many of which are involved in human congenital disorders.
Methods: We injected female T. castaneum with double-stranded RNA (dsRNA) targeting specific segmentation genes. The injection mixture contained 1 µg/µL dsRNA, 2x injection buffer, and a green tracking dye. Adult beetles were immobilized with ether anesthesia, sexed using morphological features, and injected under a dissection microscope into the reproductive region until visible abdominal expansion confirmed successful injection. Post-injection, beetles were housed in flour for egg collection.
Embryos were harvested at 48-hour intervals and fixed for imaging. Fluorescent in situ hybridization (RNA-FISH) was performed to visualize the expression patterns of segmentation genes. Embryonic morphology was assessed at the germband stage using fluorescent microscopy.
Results: Knockdown of pair-rule genes in adult females led to notable embryonic phenotypes in their offspring. Wild-type embryos displayed typical sequential stripe expression of eve, run, and odd, consistent with the segmentation clock during germband elongation. In contrast, missing or malformed segments in knockdown embryos were observed, with notable differences from wild-type morphology highlighted by gaps in gene expression and structural discontinuities:
- Eve RNAi embryos showed disrupted eve stripe formation and a truncated posterior.
- Run RNAi embryos lacked characteristic run stripes, with fewer abdominal segments formed.
- Odd RNAi similarly revealed disrupted segmental patterning.
Conclusions:
This study characterized the functional effects of segmentation genes in T. castaneum by examining gene knockdown phenotypes inherited by daughter embryos. The exogenous introduction of dsRNA into adult female beetles significantly alters zygotic gene expression in offspring embryos, disrupting the sequential expression of these genes and terminating the spatiotemporal dynamics required for T. castaneum oscillatory patterning during germband elongation. Reduced pair-rule gene function resulted in offspring with missing abdominal segments and truncated bodies. This contributes to our understanding of conserved developmental gene networks, many of which are implicated in congenital disorders in humans. These findings also highlight the potential of insect models in refining RNAi-based gene therapies and understanding gene regulatory mechanisms relevant to developmental biology and disease.
