Abstract

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Highlights

  • Mosquitoes are vectors of devastating human diseases, including malaria, dengue fever, chikungunya, and Zika[1,2,3]

  • We demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika

  • During the process of vitellogenesis, the mosquito fat body uses an array of high-specificity amino acid transporters to import blood meal-derived amino acids from the hemolymph to synthesize large quantities of yolk proteins[12,19,35,36]

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Summary

Introduction

Mosquitoes are vectors of devastating human diseases, including malaria, dengue fever, chikungunya, and Zika[1,2,3]. The mosquito fat body plays a central role in nutrient storage, metabolic homeostasis, reproduction, and xenobiotic catabolism[9,10,11,12] It is the main storage organ for triglycerides, glycogen, and amino acids in form of storage proteins. The mosquito fat body culture system presented here was developed in the '70s and remains a powerful tool for studying fat body physiology[10], especially in combination with current analysis technologies. The foundation of this technique is based on the isolation of the abdominal body walls and the associated fat body tissue. We analyzed and compared the differential gene expression of not-activated versus activated fat bodies via next-generation sequencing analysis

Preparing the Solutions and Reagents
APS Note
Preparing for Dissection
Fat Body Dissection
Fat Body Culture
Representative Results
Discussion
Full Text
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