TITLE: Manual dissection of the Schistosoma mansoni esophagus and back end for proteomic analysis
AUTHORS: Leandro Neves, R. Alan Wilson, William de Castro Borges

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Schistosomes are intravenous parasites with ability to survive in the mammalian host for decades, using its blood as a source of nutrients. The feeding process is multistep and takes place along the worm’s alimentary tract, which comprises an (i) oral cavity opening to a short (ii) esophagus that is connected to the (iii) the gut caecum. The ultrastructureal morphology of </span><span style = "font-style:italic;">Schistosoma mansoni </span><span>and </span><span style = "font-style:italic;">S. japonicum </span><span>has revealed the existence of two secretory cell masses surrounding the esophagus tube, referred to as the anterior and posterior esophageal glands (antESO and postESO, respectively). We recently established that the esophageal glands have a pivotal role in the first steps of blood processing. For instance, erythrocytes and leucocytes are quickly processed along the esophagus before they are propelled to the lower parts of the intestines for further digestion nutrient uptake. We propose that incorrect functioning of alimentary tract is associated with worms death by starvation. This was first observed in the self-cure response of Rhesus macaque (</span><span style = "font-style:italic;">Macaca mulatta</span><span>), one of few known vertebrate hosts capable of combating the disease through worm elimination once the infection is stablished. Classical immunoproteomics (2D-PAGE and Western blotting) has revealed potential targets in both exposed tegument and secreted gut proteins. Recently, a more detailed investigation using </span><span style = "font-style:italic;">S. japonicum</span><span> in the Rhesus model shed light on the possible operating mechanisms that prevent parasite feeding on blood. In addition, ultrastructural studies and immunocytochemistry on surviving worms indicated the esophageal lumen and the gland secretions as the primary targets of a potent and protective humoral immune response that ultimately disrupts the esophageal functions. Therefore, the molecular characterisation of the esophageal gland constituents is imperative if one intends to emulate the Rhesus self-cure response for therapeutic purposes. However, this is not a trivial task as challenges are multiple. Perhaps, the most important caveat is that both anterior and posterior parts of the oesophageal gland represent a minor fraction of the whole parasite body (or even of its head), meaning that dominant constituents, such as those derived from muscle tissues, suppress the identification of a unique set of gland products. Indeed, our recent investigation on the soluble protein composition of a </span><span style = "font-style:italic;">S. mansoni</span><span> preparation failed to detect the presence of gland products attesting for their low abundance in the whole worm. Although optimized protocols for chemical/enzymatic dissection are reported for isolation of testes and ovary of adult worms, no method has proved feasible for gastrodermal epithelium and the esophageal gland cells. We tackled these challenges by developing a dissection technique on worms preserved in RNAlater solution aided by an essential set of scissors and tweezers that delivers adequate precision during the procedure. The method herein described is compatible with downstream proteomic analyses using mass spectrometry and has permitted a large-scale characterisation of proteins expressed in the </span><span style = "font-style:italic;">S. mansoni </span><span>esophagus and gastrodermis. This methodology can be applied in the molecular characterisation of other schistosome organs and tissues that present a well-defined anatomic location.</span></div></div>

[STEPS]
?. [Place the Petri dish with worms on the microscope]
Take the Petri dish out of the ice bath, touch it against a paper to remove the excess of water, place it under the stereomicroscope and adjust the focus.
?. [Zoom in at the head of a male worm]
Look for a male parasite with uncurled head/esophagus. Increase the lenses magnification and use your dissecting tools (tweezers and scissors) to keep the head in the field-of-view.
?. [Use the scissors to make the first incision at the posterior of the oral sucker]
Hold the parasite carefully with the tweezers, positioning it in an angle that allows you to clearly see the suckers (i.e. lateral view, or suckers facing up), then make the first incision (#1) at the posterior of the oral sucker. This  releases the oral sucker; this fragment is composed mostly by muscle tissue and usually has limited utility. If you don't need the oral sucker just leave it floating around.
?. [Cut off the esophagus (ESO) along the transverse gut]
The second incision (#2) must be performed along the transverse gut (i.e. where the dark pigmentation of hemozoin starts; indicated by the red arrow in the illustration below). Be sure you are not simultaneously cutting the ventral sucker off.
?. [Collect the ESO fragment using a P20 micropipette]
Now, the ESO fragment should be detached and floating around. Aspirate it with the RNAlater solution using the P20 micropipette then dispense it inside the ESO microtube. Always keep the tube on ice. CRITICAL STEP do not aspirate any other fragment or particulate material with the ESO.
?. [Record your progress in the notepad]
?. [Cut off the back end]
The final cut (#3) is considerably easier and usually does not require high magnification. Simply cut the posterior third of the parasite body. Collect the BE fragment with the tweezers, transfer it to the BE microtube and keep in the ice box.
?. [Record your progress in the notepad]
?. [Find another male parasite and repeat steps 3 to 9]
If needed, you can immerse the tweezers and scissors in the beaker with deionized water to get rid of salt crystals. Dry your tools in a soft and lint-free paper before continuing the dissection.
?. [Start a 10 min countdown]
This will help you to control how long the Petri dish is out of the ice.
?. [When the 10 minutes timer runs out place the Petri dish back in the ice]
In Step 2 you started a 10 minutes countdown. You can dissect as many worms as you can in this time, but it is important to chill the Petri dish every 10 minutes to preserve your material. ATTENTION This is an opportunity to transfer the dissected worms to the "dissected  body" microtube then add new  ones to the Petri dish for the next round. In addition, if you notice that salt crystals are accumulating in the RNAlater solution discard it, wash the Petri dish with deionized water, dry it with lint-free paper and add fresh RNAlater solution. PAUSE POINT If you wish, store the dissected fragments in the fridge and continue the procedure another time.
?. [Start a new round of dissection]
Restart from Step 1.