Jun 16, 2019
Manual dissection of the Schistosoma mansoni and S. japonicum head and back end for transcriptomic analysis
Protocol Citation: Leandro Xavier Neves: Manual dissection of the Schistosoma mansoni and S. japonicum head and back end for transcriptomic analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.uq3evyn
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2018
Last Modified: June 16, 2019
Protocol Integer ID: 16891
Keywords: esophageal gland. schistosome esophagus. RNA-sequencing
Abstract
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 Schistosoma mansoni and S. japonicum 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 (Macaca mulatta), 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 S. japonicum in the Rhesus model shed light on the possible operating mechanisms that prevent parasite feeding on blood. 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 whole worm analyses are not sufficient for identification of genuine gland products. 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 Next Generation Sequencing. This methodology can be applied in the molecular characterisation of other schistosome organs and tissues that present a well-defined anatomic location.
Guidelines
This protocol will guide you through the dissection of the head and back end of adult schistosome worms (males or females). Although the recovery of adult worms by portal perfusion of infected animals is obviously indispensable this is not addressed in this protocol. Nevertheless, worm handling and fixation are utterly important and should be performed according to the following instructions:
- Perfuse 45-days infected animals using RPMI-1640 medium (without phenol red) buffered with 10mM HEPES, pH 7.4, containing 4 UI/mL of heparin.
- Wash the parasites in the same medium (pre-warmed at 37°C) until total removal of blood and tissue debris.
OBS.: always use pre-warmed medium to prevent getting worms too curled up as this makes the dissection procedure difficult.
- Carefully transfer the parasites, by pouring them with plenty of pre-warmed medium, into a Corning 50 mL conical tube.
- Discard the medium and add 5-6 volumes of RNAlater for instantaneous worm fixation.
- Keep the worms at 4°C until dissection.
- CRITICAL POINT Parasites respond to the ex vivo environment; thus it is mandatory to avoid a long waiting between the perfusion and fixation in RNAlater. The total procedure time (perfusion, washings and fixation) must not exceed 10 min.
Procedure overview
Once the parasites are fixed you are able to perform the dissection. In summary, the isolation of the schistosome head (HE) is achieved by cut along the line of the transverse gut. The posterior third of the parasite (BE) is also dissected and constitute a second sample for comparative analysis. This protocol will simultaneously illustrate the entire process in male and female worms however we strongly recommend to perform the dissection of different sexes separately.
- CRITICAL POINT The procedure must be entirely performed in ice-cold conditions. Keep HE and BE fragments on ice during the dissection, then store them in the fridge.
ALTERNATIVE METHOD APPLICATION: Once the set of stereomicroscope, scissors and tweezers are available, different incision points can be used for obtaining alternative body sections. It is important to consider that the precision decreases proportionally to the fragment size (e.g. the female head is too small so that it is difficult to cut exactly in the transverse gut). In our lab we have successfully dissected the whole head, esophagus, back end, ovary and ootype on either S. mansoni or S. japonicum male and female worms. Check the protocols available at our collection.
Materials
MATERIALS
RNAlaterSigma-aldrichCatalog #R0901-100ML
Safety warnings
Adult schistosomes are not an infective stage thus biological hazard is not evident. Nevertheless, follow the safety instructions of all chemicals used (e.g. RNAlater, heparin). Always wear gloves and lab coat.
Before start
Prepare your workplace
- Fill a Styrofoam box with crushed ice. This is where you keep the worms and dissected fragments.
- Wear powder-free nitrile gloves.
- Add approximately 100 microlitres of RNAlater to a 1.5 mL microtube and keep it in the ice. Label this tube 'HE'.
- Add approximately 500 microlitres of RNAlater to a second 1.5 mL microtube and keep it in the ice. Label this tube 'BE'.
- Add approximately 1 mL of RNAlater to a 2 mL microtube and keep it in the ice. Label this tube 'Dissected body'.
- Fill a 50 mL beaker with deionized water. You may have to immerse the scissor blades and the tweezers into the water periodically to get rid of salt crystals formed during the procedure. Make sure you have easy access to soft and lint-free paper to dry out the tools.
- Fill to half depth a small glass Petri dish (~4.5 cm of diameter) with RNAlater. Let it chill on the ice a few minutes.
- Turn on the stereomicroscope and switch on the bottom light only; this usually gives the best contrast during the dissection.
- Get notepad and pen to make notes about the number of HE and BE fragments you have dissected.
- Get a laboratory timer.
- Ensure you have a P20 micropipette and disposable tips with you during the procedure.
OBS.: eventually you need to cut the tip to open its orifice. Use ordinary scissors to do that.
- Put few worm pairs in the Petri dish (10-15). Avoid a huge number of parasites in the dish as this complicates the procedure.
- Finally, take the scissors and tweezers and a comfortable chair. Make sure all the items above are reachable while seated.
Place the Petri dish with worms on the microscope
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.
Equipment
new equipment
NAME
Stereo microscope EZ4, Leica
BRAND
10447197
SKU
Leica EZ4 educational stereomicroscope.
SPECIFICATIONS
Start a 10 min countdown
Start a 10 min countdown
This will help you to control how long the Petri dish is out of the ice.
Zoom in at the head of a worm
Zoom in at the head of a worm
Look for a 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.
Equipment
new equipment
NAME
Biology Tweezer 115 mm, Ideal Tak
BRAND
7SG.CX.0
SKU
Biology Tweezer 115 mm (Ideal Tak Chiasso, Switzerland). Manufacturer Part Number: 7SG.CX.0
SPECIFICATIONS
Equipment
new equipment
NAME
Vannas scissors, John Weiss
BRAND
0103123
SKU
Vannas scissors (John Weiss, Milton Keynes, UK). Straight with sharp tips.
SPECIFICATIONS
Make the first incision at the line of the transverse gut
Make the first incision at the line of the transverse gut
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) along the transverse gut (i.e. where the dark pigmentation of hemozoin starts; indicated by the red arrow in the illustration below).
Collect the HE fragment using a P20 micropipette
Collect the HE fragment using a P20 micropipette
Once the HE fragment is detached it floats around. Aspirate it with the RNAlater solution using the P20 micropipette then dispense it inside the HE microtube. Always keep the tube on ice.
CRITICAL STEP do not aspirate any other fragment or particulate material with the HE.
Record your progress in the notepad
Record your progress in the notepad
Cut off the back end
Cut off the back end
The final cut (#2) 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
Record your progress in the notepad
Find another parasite and repeat steps 3 to 9
Find another 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.
When the 10 minutes timer runs out place the Petri dish back in 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
Start a new round of dissection
Restart from Step 1.