Manual dissection of the Schistosoma mansoni esophagus and back end for proteomic analysis v1
Authors: Xavier Neves Leandro , Alan Wilson R. , de Castro Borges William 
Published: June 2019

Schistosomes are intravenous parasiteswith 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 morphologyofSchistosoma mansoniandS. japonicumhas 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 vertebratehosts 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 detailedinvestigation using S. japonicumin 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 imperativeif one intends to emulate the Rhesus self-cure response for therapeutic purposes. However, this is not a trivial task aschallenges 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 aS. mansonipreparation failed to detect the presence of gland products attesting for their low abundance in the whole worm. Although optimized protocols for chemical/enzymatic dissection arereported forisolation of testes and ovary of adult worms, no methodhas 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 S. mansoni 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.