Hello, my name is Immo Hansen, from the Molecular Vector Physiology laboratory at New Mexico State University. The mosquito fat body culture system we will demonstrate today is an in vitro organ culture system that we use in our laboratory to study physiology and metabolism of live fat body tissue attached to the abdominal body walls of mosquito. Insect fat body is an organ in which many metabolic processes take place.
Its functions mirror those of liver and fat tissues in vertebrates. We have used this system to study the regulation of yolk protein production and the mechanisms of nutrient uptake in the yellow fever mosquito, Aedis aegypti. Aedi aegypti is a principal vector of arbovirus like dengue, chikungunya and zika.
The mosquito fat body is the sole source of yolk proteins in mosquitoes. This video protocol demonstrates an in-vitro mosquito fat body culture system and shows several experimental applications of the system. The protocol that we will be demonstrating is set up in multiple parts.
First, we will be preparing the solutions and reagents necessary for the fat body culture system. Second, we will perform mosquito dissections to isolate fat bodies. Then, last thing, we will transfer isolated fat bodies to an amino acid enriched medium to activate yolk protein synthesis.
There are several downstream applications that can be performed following fat body culture. Today we will be performing fat body culture followed by next-generation sequencing. We will compare two samples, an unstimulated sample with one sample stimulated with amino acids and ecdysone.
Additional materials and equipment that will be needed include an aspirator with collection vial for mosquito collection, CO2 fly pad hooked up to CO2 gas bottle, ethanol to clean the fly pad, stereo microscope with light source, dissection micro scissors, fine-tipped tweezers, and 96 well plates. To prepare for fat body culture, three stock solutions and reagents must be prepared, the amino stock solution, Aedes physiological saline stock solution, and calcium stock solution. The Aedes physiological saline stock solution and calcium stock solution are combined to make the fat body culture media.
Mosquitoes used for this experiment should be at least three days old after emergence. Using a battery powered aspirator, collect the mosquitoes into a collection vial. Place the collection vial with your sample onto a CO2 pad to anesthetize the mosquitoes.
Alternatively, ice can be used to anesthetize the mosquitoes. Discard all males before beginning dissections. First focus the dissecting microscope using a test mosquito to ensure visibility.
Once you have focused the microscope, place a concave slide on the microscope and place two drops of APS in the center. The APS assists in keeping the mosquito elevated and in collecting various mosquito organs. Select the mosquito with the forceps, picking the mosquito up by the legs.
Remove the legs. While holding the mosquito by the thorax with your left hand, use the other forceps to grasp the mosquito by the last two or three segments of the abdomen and gently pull to remove. This action should remove the ovaries, malpighian tubules, hind gut, midgut and the crop.
Once the organs are removed, use the spring scissors to finish the dissection of the fat body. Insert one blade into the hole that was created from removing the last two to three segments of the abdomen and cut the abdomen lengthwise until it reaches the segment below the thorax. After the incision, the abdomen should expand outward.
Another incision is made laterally across the top of the abdomen where your first incision ended. After the incisions, the abdomen open up and float with the cuticle side up and the fat bodies resting within the APS. Once the fat bodies have equilibriated on the APS solution for at least half an hour, they can then be transferred to the fat body culture media.
In order to stimulate the fat bodies to begin yolk protein gene expression, we transfer them to a media with high levels of free amino acids and the insect steroid hormone, 20-hydroxyecdysone. Once transferred, the fat bodies are incubated for six hours in the media. After the incubation period, the fat bodies are transferred to an eppendorf tube with a buffer appropriate for the desired analyses.
Depending on the experimental goal, the fat bodies can be used in several subsequent protocols such as qPCR, Western blots, proteomics, metabolomics, or RNA-seq. As an example, we have performed a fat body culture experiment and stimulated isolated fat bodies by incubating them on a solution containing a balanced mixture of all 20 naturally occurring amino acids and the insect steroid hormone 20-hydroxyecdysone for six hours as control fat bodies were incubated on a PS for an equal amount of time. This figure shows the results of an RNA-seq analysis of our experiment.
The heat map indicates gene expression levels of 1256 genes expressed in cultured fat bodies under two different treatments. All of these genes have significantly changed expression levels between the two treatments. This table shows the ten most up regulated genes after activation of cultured fat bodies with amino acids and 20-hydroxyecdysone.
As expected, most of these genes are yolk protein genes. Thank you for your interest in mosquito fat body culture.
