Semester

Summer

Date of Graduation

2021

Document Type

Thesis

Degree Type

MS

College

Davis College of Agriculture, Natural Resources and Design

Department

Division of Plant and Soil Sciences

Committee Chair

Teiya Kijimoto

Committee Member

Vagner Benedito

Committee Member

Yong-Lak Park

Abstract

The origin of the evolutionary new trait (evolutionary novelty) and its subsequent evolution is of great interest for biologists in various fields, and beetle horns have been used to address this fundamental biological question. Previous studies on one of the horned beetle species, Onthophagus taurus, that utilized comparative gene expression analyses, suggested legs to be a strong candidate of the origin of horns. At the same time, their horns are secondary sexual traits whose development is regulated by the same gene (doublesex) as genitalia, which also originates from paired appendages such as legs. However, little is known about the similarity or difference in the gene expression pattern between horns and genitalia. At the same time, the horn shows an extreme form of plasticity influenced by nutrition uptake in males of O. taurus (polyphenism). This polyphenic status might have been acquired when (or after) the horn was invented. However, it is unclear if the same set of genes indicate sensitivity to nutritional input during the development of legs, genitalia, and horns. To address these questions, we utilized RNAseq with four tissues. We highlighted the gene expression pattern in polyphenic head horns (where nutritional conditions drastically affect its size), non-polyphenic thoracic horns (where nutritional conditions have moderate effects on size), non-horn appendages, legs and genitalia (where nutritional conditions do not affect the size) in two beetle developmental stages, prepupa and pupa.

Our transcriptomic data indicated a clear difference between the two developmental stages (pupa and prepupa). Furthermore, Spearman’s correlation showed less correlation between horns and genitalia, inferring the close relatedness of horns and legs more than genitalia. We also detected more differentially expressed (D.E.) genes between horns and genitalia than legs, suggesting legs to be still the most likely candidate of the origin of horns. Our transcriptomic data provided us with a list of genes. Among many, we detected genes that are involved in the wingless signaling pathway such as Wnt-1 (Wingless in Drosophila), frizzled-2, frizzled-4, and low-density lipoprotein receptor-related protein 6 (arrow in Drosophila) whose developmental function in the horn is not studied well. Wg pathway is known to interact with Hedgehog pathway that is also known to be involved in both horn development and polyphenism. Thus, we took a targeted gene approach where RNAi was utilized to knockdown the gene arrow to examine its horn developmental role. Our data suggest that arrow inhibits horn development in a dosage-dependent manner. Our results also showed the effects of arr knockdown in other tissues such as wings and legs. Thus, my research successfully integrated transcriptomic study and targeted gene approach to shed light further into the genetic underpinnings of the origin and evolution of the novel trait, beetle horns.

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