The overall goals of this procedure are to show how to maintain cultures of parasitoid wasps on drosophila hosts and how to dissect lymph gland and fat body. This is accomplished by first culturing wasps on drosophila larvae. Animals are then dissected to isolate either the larva lymph glands, which involves peeling off the cuticle and removing the fat bodies and other finer structures to isolate the lymph glands.
Likewise, the fat bodies themselves can be isolated using a similar procedure using immuno cyto chemistry and fluorescence microscopy. Gene expression and cell specific changes are visualized in the immune tissues. After wasp infestation, We study a unique system of infection and immunity that utilizes drosophila hose and its parasitic wasp.
Our lab is one of few where life cultures of drosophila parasitoids are maintained. Thus, we are often asked to share our techniques. Parasitoids are natural enemies of many insects.
The species we study attack the larval stages of many drosophila species. In the lab, we use drosophila melan gastro to understand how the immune system of insects are eff affected by parasitoid attacked. Secondly, we want to discover the strategies that are used by wass to overcome host defense.
Generally, individuals who are new to dissections for fat body and lymph gland struggle because while oph larvae are easy to grow, they are fairly small in size, and it takes considerable familiarity to routinely dissect these laval organs with ease. After discovering that constitutive activation of NF kappa B signaling leads to an autoimmune like encapsulation of cell tissues, we became curious about how this reaction would occur in oph fla mutants. So we hypothesized that was induced encapsulation is its natural counterpart and develop these standard protocols that we will demonstrate Today, I will now demonstrate how to prepare and maintain wasp cultures.
Begin by preparing a fresh fly food vial with a small dab of freshly prepared yeast paste. Transfer 50 to 60 young wild type flies to the vial to lay eggs for 48 hours at 24 degrees Celsius. After two days of egg laying, remove the flies from the vial and add a drop of honey to the buzz plug.
And finally, recap the vial. Lower the flow of carbon dioxide prior to anesthetizing wasps because they're more sensitive to the gas than flies. Once ready to anesthetize wasps sort out six to eight female and six to eight male wasps and add them to the vial.
With the drosophila eggs and larvae. Gently place the vial on its side until the wasps wake up. Then place the vial in a 24 degrees Celsius incubator.
Unaffected gilo host and hosts that successfully defend themselves will pup paint at the normal development time point. They can be removed prior to the wasps e explosion. Wasps usually AC close around 25 to 30 days to prepare wasps at the same stage of development.
Rear the wasps with host eggs laid over a two to six hour period instead of a 48 hour period to harvest infected larvae for dissections of immune tissues or parasites. Transfer the contents of the fly vials to a small glass or plastic Petri dish. Then using a stereo microscope and find forceps carefully pick six to 10 larvae.
Place the larvae into a well with one XPBS first, then transfer them into water, 70%ethanol water and one XPBS successively. The purpose of these steps is to free the animals of the fly food and thoroughly clean and sterilize their surface. On the dissecting stage of a Zes 1000 dissecting microscope, place a light box that allows incident light to be transmitted through the sample.
Using fine forceps, select a cleaned wandering third instar larvae from the PBS. Place the larvae onto a microscope slide. Using forceps position the animal with the ventral side up.
Tiled the light source away from the sample so that the organism appears transparent and the internal organs are visible. As you transfer the larvae onto the slide, you want to make sure that there is no excess PBS as this can sometimes make the section more difficult. Using two pairs of forceps, hold the animal at either side of the posterior end and gently pull the cuticle to introduce a small superficial tear in the cuticle.
The hemo lymph containing he cytes gut and fat bodies will begin to escape from the body cavity. The hemolymph can be taken up with a 10 microliter pipette man to make smears if needed, placing both forceps to the left side of the animal just beneath the mouth hooks carefully. Make a gentle tear in the cuticle.
Move both forceps on the right side of the animal just beneath the mouth hooks and carefully make another small tear. Now while holding the mouth hooks of the animal, gently peel back the cuticle towards the posterior end of the animal. As the cuticle is pulled back, the brain fat body imaginal discs, salivary glands and proven ventricular will become exposed.
The detached cuticle at the posterior end has the dorsal vessel with lymph gland attached to it. The lymph gland is underneath the brain. Cut the proven ventricular and move it away from the lymph gland area.
Carefully tease away fat, body, gut, salivary glands, and other structures from the lymph gland. Be careful not to detach the limp gland from the ring gland and dorsal vessel located posterior to the brain. Position the lymph gland flat against the microscope slide and carefully remove all additional tissues until the entire gland unfolds from the anterior lobes to the final set of pericardial cells.
The gland is now ready to be fixed and stained on the dissecting stage of a Zes 1000 dissecting microscope. Place a light box that allows incident light to be transmitted through the sample. Position a cleaned third instar larva on a microscope.
Slide in 200 microliters of one XPBS. Tilt the light source away from the sample so that the organism appears transparent and the internal organs are visible. Using forceps, position the animal so that the anterior end is away from you.
In the next step, it is important to note where you position your forceps to ensure that you tear only the cuticle without touching the internal organs. With one pair of forceps, hold the cuticle of the larvae on left side of the mouth, hooks with the other forceps. Gently tear the cuticle on other side down to the posterior end.
Stop before tearing the cuticle away from the body at the posterior end, very gently start removing the gut to one side. Gently remove the brain imaginal discs and the ring gland with the lymph gland running through the dorsal vessel. Finally, gently remove the cuticle on either side of the fat body.
Do not remove the salivary glands since there are fat bodies on either side of these glands. Before proceeding with fixing and staining the tissue, be sure to remove the PBS. A well dissected fat body sample should maintain normal cell contacts and should have minimal fat globules around the dissected sample.
The effect of wasp infestation on gene expression is visualized in vivo. Using a GFP reporter linked to Theso Mycin promoter in uninfected control animals, GFP expression was not detected. Whereas the GFP signal is clearly detected in the cytoplasm of fat bodies dissected from animals infected with l Victoria.
Lymph glands were dissected to visualize the cellular changes induced in the anterior lobes after El Victoria infection. Newly differentiated cytes marked with GFP driven by the misshapen enhancer are seen at the periphery of the lobes. Normally, the glands surround the lobe continuously.
However, the integrity of the anterior lobes was disrupted in these glands as the basement membrane was discontinuous after wasp infection from these same dissections. Cytes in the hemolymph were visualized in smears. Some lymphocytes were engaged in the capsule form to block parasite development to detect betzel expression.
The dissected lymph glands. The lymph glands were stained with polyclonal anti schpeil antibodies in vivo. Schpeil levels increased in lymph gland cells after wasp infection as compared to uninfected controls.
While attempting this procedure, it is important to prepare esophagal cultures at the desired biological age. Treat them gently so that manual handling does not induce immune reactions. Following this procedure, we can use the dissected organs, the fat body, and lymph gland to extract RNA and protein using the extracted RNA or protein.
We can perform PCR or Westin analysis to study the gene and protein expression. After watching this video, you should have a good understanding of how to grow. Parasitic was on drosophila hosts and perform dissections of immune tissues for immunohistochemistry and other applications.
