FlyMet offers a comprehensive tissue-specific metabolomics resource for Drosophila .

FlyMet: Experimental Protocols
The experimental protocols employed throughout this project can be found below:

Drosophila Dissection Protocols:

The basic protocol used for all dissections is described below. In addtion, tissue specific methods are described for the following adult and larvae specfic tissues:


General Dissection Protocol

Precool a 2.0 ml screw-cap tube in an ethanol-dry ice bath and carry out the dissections in cooled PBS. Flies/larvae were anesthetized by placing them on ice and the dissection dish was kept on ice throughout the procedure. Quickly dissect a small number of flies/larvae and transfer to the precooled tube in the ethanol-dry ice bath. At the end of the process store the dissected tissues were in the tube at -80 C.


Adult Accessory Gland

To dissect the accessory glands, initially remove the thorax from the abdomen and gently cut open the abdomen with forceps. Subsequently, remove the testes and accessory glands from the abdomen and the testes from the accessory glands. Finally, collect the accessory glands and place them into the precooled 2.0 ml tube in an ethanol-dry ice bath.


Adult Brain

To dissect the adult brain initially remove the abdomen from the body of the fly and with the dorsal side of the thorax against the dissecting dish, remove the proboscis from rest of the head. With the opening generated, place forceps on either side of the head (NOT on the eye) near the antenna and gently pull the head capsule from the body (hopefully exposing the brain). Separate the brain from the thoracic-abdominal ganglion. If the brain remains in the head capsule, carefully pull open the capsule to expose the brain. Finally, collect the brain and place it into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath.


Larval Brain

To dissect the larval brain, hold the larva about two-thirds of the way down from the mouth with forceps. Grab and pull the mouthpieces with another set of forceps, the brain and imaginal discs will separate from the rest of the larva. Remove the imaginal discs and any other gross tissue as these are potential contaminants. Finally, collect the brain and place them into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath.


Adult Carcass

To dissect the adult carcass, remove the head, legs and wings from the body of the fly. Discard the head and collect the legs and wings. Next, remove the abdomen and with the dorsal side against the dissecting dish and gently cut open the abdomen towards the genital region to open it up. Remove the digestive system and reproductive system and as much of the trachea as possible. Scrape the cuticle with the forceps to remove the fat body, heart, nephrocytes and trachea. Collect the cuticle. Open the thorax and remove as much of the muscle and other tissue. Collect the thoractic cuticle. There will be contamination from the tissues closely associated with the cuticle.


Larval Carcass

To dissect the larval carcass, gently tear an opening in the larva near the spiracles. Using this opening, cut down the cuticle with forceps towards the mouthpieces, gaining access to the larval organs. Remove the fat body and then the digestive system with the tubules attached. Scrape the cuticle to remove associated tissues. There will be contamination from the tissues closely associated with the cuticle


Adult Crop

To dissect the adult crop, initially remove the head from the fly and gently separate the abdomen from the thorax. Grab the fly’s genital region and pull gently to separate the digestive system from rest of the fly. With care, remove the fore/midgut with the crop still attached and cut away the crop from the gut (try to ensure the foregut is not included). Finally, collect the crop and place it on ice


Adult Eye

To dissect the adult eyes, initially remove the abdomen from the rest of the body. With the dorsal side of the thorax placed against the dissecting dish, remove the proboscis. Next, place forceps on either side of the head (NOT on the eye) near the antenna and gently pull the head capsule off the body. Once the head capsule removed, carefully remove the eyes by gently cutting them out and place them into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath. Potential contaminants are from cuticle, antenna, and fat.


Adult Fat Body

To dissect the adult fat body, initially remove abdomen from rest of body. Then, using forceps, grip the genital region and pull to remove the digestive and reproductive systems. With the dorsal side of the abdomen against the dissecting dish, gently cut open the abdomen towards the genital region to open the abdomen up. Remove any remaining parts of digestive and reproductive systems and as much of the trachea as possible. Collect the fat body and place them into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath. It can be helpful to break each segment from the main part of the abdomen to expose the fat body better. Try to avoid the heart, trachea, and nephrocytes as they are potential contaminants of fat body samples


Larval Fat Body

To dissect the larval fat body initially grab the larva by the mouth hooks. Gently pull on the mouth hooks to generate a hole/opening and carefully cut the cuticle open with forceps. Take out the fat body and remove the gonads, collecting the remaining fat body and place it into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath.


Adult Head

To dissect the adult head, gently pull the head away from the body with forceps in one hand while holding the body with another set of forceps. Collect the head and place it into the precooled 2.0 ml tube in an ethanol-dry ice bath. The head samples also contain the eyes and brain.


Adult Heart

To dissect the adult heart, initially remove abdomen from rest of body. Then, using forceps, grip the genital region and pull to remove the digestive and reproductive systems. With the dorsal side of the abdomen against the dissecting dish, gently cut open the abdomen towards the genital region to open the abdomen up. Remove any remaining parts of the digestive system, reproductive system, and as much of the trachea and fat body as possible. The heart is tube running down the dorsal side of the abdomen – the fat body extends from the heart so be gentle when removing the fat body. Collect the heart and place it into a precooled 2.0 ml tube in an ethanol-dry ice bath. There will be potential contamination from the fat body and nephrocytes.


Adult Hemolymph

To dissect adult hemolymph, use a 0.2 mm insect pin to poke a hole into the fly at the thorax/abdomen junction (under the scutellum). The fly is then put into a 0.5 ml tube with a small hole in the bottom, which is subsequently placed in a 1.5 ml tube to collect the hemolymph. The double-tube is then centrifuged at 5 rpm for 5 minutes at 4 C and the hemolymph collected and place it into the precooled 2.0 ml tube in an ethanol-dry ice bath


Larval Hemolymph

To dissect larval hemolymph, use a 0.2 mm insect pin to poke a hole into the larva before placing it into the lid of a 0.5ml tube (with a hole in the bottom). Place the lid into a 1.5 ml tube to collect the hemolymph. Centrifuge at 5 rpm for 5 minutes at 4 C and the hemolymph collected and place the sample into the precooled 2.0 ml tube in an ethanol-dry ice bath.


Adult Hindgut

To dissect the adult hindgut, initially remove the head of the fly from the body and gently separate the abdomen form the thorax. Grab the fly’s genital region and pull gently to remove the digestive system from rest of the fly. Cut away the tubules, midgut and rectal pad from the hindgut. Collect the hindgut and place them into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath. Bacteria and food are potential contaminants for hindgut samples.


Larval Hindgut

To dissect the larval hindgut, gently tear an opening in the larva near the spiracles. Using this opening, gently cut down the cuticle with forceps towards the mouthpieces, gaining access to the larval organs. Next, remove the fat body and cut away the gut from the mouthpieces at the top of the proventriculus. Finally, cut the tubules and midgut away from the hindgut before placing them into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath. Bacteria and food are potential contaminants for larval hindgut samples


Adult Midgut

To dissect the adult midgut, initially remove the head of the fly from the body and gently separate the abdomen form the thorax. Next, grab the fly’s genital region and pull gently to remove the digestive system from rest of the fly. Finally, cut away the tubules, hindgut, foregut and crop from the midgut and place the midguts into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath. Bacteria and food are potential contaminants of adult midgut samples


Larval Midgut

To dissect the larval midgut, carefully tear an opening near the spiracles. Using this opening, gently cut down the cuticle with forceps towards the mouthpieces, gaining access to the larval organs. Next, remove the fat body and cut the gut away from the mouthpieces by the top of the proventriculus. Finally, cut the tubules and hindgut away from the midgut and place the midgut into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath. Bacteria and food are potential contaminants of larval midgut samples.


Adult Ovary

Adult Ovary To dissect an adult ovary, initially remove the head of the fly from the body and gently separate the abdomen form the thorax. Gently cut open the abdomen with forceps and remove the ovaries from the abdomen. Include the oviduct, but not the uterus and place the ovaries into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath.


Adult Rectal Pad

To dissect adult rectal pads, initially remove the head of the fly from the body. Holding the fly at its thorax/abdomen boundary, grab the fly’s genital region and pull gently to expose the digestive system. Pull on the anus of the fly and the rectal pad with a portion of the hindgut can be removed. Cut away the hindgut from the base of the rectal pad. Finally, remove any cuticle that is attached to the rectal pad and place into the precooled 2.0 ml tube in an ethanol-dry ice bath. Bacteria and food are potential contaminants of adult rectal pad dissections


Adult Salivary Glands

To dissect the adult salivary glands initially remove the legs and wings from the fly. Then, holding the thorax, gently remove the abdomen by pulling with forceps. With the dorsal side of the thorax held against the dissection dish, carefully cut open the thorax, removing muscle and digestive tissues. The salivary glands will appear near the foregut-midgut-crop junction tightly with the midgut. Carefully remove them and place them into the precooled 2.0 ml tube in an ethanol-dry ice bath.


Larval Salivary Glands

To dissect the larval salivary glands, hold the larva about two-thirds of the way down from the mouth with forceps. Grab and pull the mouthpieces with another set of forceps. The salivary glands will usually pull out with the brain and imaginal discs. Remove as much of the fat connected to them as possible and collect the salivary glands into a precooled tube in an ethanol-dry ice bath


Adult Spermatheca

To dissect adult spermatheca, initially remove the head from the abdomen. Next, holding onto the abdomen with forceps grab the genital region of the fly and pull to expose the uterus and spermatheca. Finally remove the fat surrounding the spermatheca before removing the spermatheca itself and placing into an ethanol-dry ice bath.


Adult Testes

To dissect adult testes, initially remove the head from the body of the fly and the abdomen from the thorax. Gently cut open the abdomen with forceps and remove the testes and accessory glands from the abdomen. Finally, remove the testes from the accessory glands and collect the testes into a tube in a ethanol-dry ice bath.


Adult Thoracicoabdominal Ganglion

To dissect adult thoracicoabdominal ganglion, initially remove the legs and wings from the fly. Then, holding the thorax, gently remove the abdomen with forceps. With the dorsal side of the thorax held against the dissection dish, carefully cut open the thorax, removing muscle and digestive tissues. Finally, separate the thoracicoabdominal ganglion from the brain and place it into the pre-cooled 2.0 ml tube in an ethanol-dry ice bath.


Larval Trachea

To dissect larval tracea, gently, but firmly, pull on the larval spiracle. This should yield most of the trachea, which should be collected. To collect any remaining trachea, open the larva up by cutting down the larval cuticle towards the mouth. Isolate any additional parts of the trachea and place into the precooled 2.0 ml tube in a ethanol-dry ice bath.


Adult Malpighian Tubules

To dissect adult Malpighian tubules, gently tug the head away from the rest of the fly. This breaks the connection of the midgut with the foregut. With forceps, grip the genital region and gently pull to remove the digestive system. This should bring the tubules out too. Finally, with forceps, disconnect the tubules from the gut. Collect the tubules into a precooled tube and place into an ethanol-dry ice bath


Larval Malpighian Tubules

To dissect larval Malpighian tubules, gently tear an opening near the spiracles. Using this opening, gently cut the cuticle with forceps towards the mouthpieces, gaining access to the larval organs. Remove the fat body and the digestive system with the tubules attached. With forceps, disconnect the tubules from the gut and collect the tubules into a pre-cooled tube and place into an ethanol-dry ice bath


Whole Fly Samples

To prepare whole fly samples a dissection dish is placed on ice and the flies anesthetized by placing them on the dish. Five flies of the same sex were then collected in a precooled tube and placed into an ethanol-dry ice bath.


Whole Larvae Samples

To prepare whole larvae samples, crawling third instar larvae were removed from a fly vial and transferred into a small cooled small Petri dish, placed on ice and filled with 1x PBS. The whole larvae were then washed with the PBS and then transferred into another dish filled with PBS. Five larvae were then collected in a precooled tube and placed into an ethanol-dry ice bath.