Transplantation of schistosome sporocysts between host snails: A video guide

Schistosomiasis is an important parasitic disease, touching roughly 200 million people worldwide. The causative agents are different Schistosoma species. Schistosomes have a complex life cycle, with a freshwater snail as intermediate host. After infection, sporocysts develop inside the snail host and give rise to human dwelling larvae. We present here a detailed step-by-step video instruction in English, French, Spanish and Portuguese that shows how these sporocysts can be manipulated and transferred from one snail to another. This procedure provides a technical basis for different types of ex vivo modifications, such as those used in functional genomics studies.

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Introduction
Schistosomiasis is an acute and chronic parasitic disease caused by blood fluke trematodes of the genus Schistosoma. Estimates show that at least 206.5 million people required preventive treatment in 2016 (WHO, 2017). This disease is spread throughout tropical and subtropical areas of Africa, South America, Middle-East and Asia and, more recently, it represents an emerging risk in Europe (Holtfreter et al., 2014).
The schistosome lifecycle includes two obligatory hosts ( Figure 1a): (I) A freshwater snail, in which stages of the parasite, called sporocysts, multiply asexually and produce the mammalian infecting larvae, known as the cercariae, and (II) A mammalian host in which the parasites (males and females) develop to sexual maturity, pair, mate and produce eggs.
Eggs are excreted from the mammalian host in faeces or urine, and if they reach bodies of freshwater a second free-swimming larval stage, called the miracidium, hatches and infects the snail by active penetration through the integument. After penetration, the miracidium loses the ciliated plates, and develops in less than 18 hours into a mother sporocyst. Daughter sporocysts develop inside the mother sporocyst and eventually escape to invade the snail tissues and organs, mainly the ovotestis and the hepathopancreas. In each daughter sporocyst, germinal cells (similar to stem cells) divide and differentiate to produce either cercariae (cercariogenous sporocysts), or further daughter sporocysts (sporocystogenous sporocysts).
The asexual multiplication of the daughter sporocysts continues throughout the time that the parasite remains resident in the snail (Théron, 1981). Several generations of daughter sporocysts may occur in the same individual snail, because daughter sporocysts can undergo a re-differentiation into new daughter sporocysts, whether they were sporocystogenous or cercariogenous (Combes et al., 1983). We exploited this reversible developmental plasticity to design a method, called "sporocyst transfer" or "transplantation", generating a direct lifecycle (snail to snail transmission) and bypassing the mammalian host and free-swimming larval stages (Jourdane et al., 1984) (Figure 1b). This technique, first developed by Chernin (1966) for Schistosoma mansoni, consists of transplanting schistosome sporocysts from one donor snail to recipient snails. It was improved in our laboratory for S. mansoni with the snail Biomphalaria glabrata as host (Jourdane, 1977;Jourdane & Théron, 1980;Jourdane & Combes, 1989). We also adapted the procedure to other species of schistosomes like S. haematobium and the snail Planorbarius metidjensis (Kéchemir & Combes, 1982), and S. bovis in Bulinus truncatus (Jourdane et al., 1984). We describe here, in a video documentary, a detailed and improved method that enables the generation of clonal populations of schistosomes.
The technique of microsurgical transplantation requires a precise protocol and specific skills in snail handling and surgery that can be quite difficult to establish in a laboratory. Therefore, the aim of this publication is to provide for the scientific community a technical video in order to make this technique more broadly available.

Design and development of the video on the transplantation technique
The video explains all the steps, equipment, reagents and manipulations necessary for the technique to be applied by researchers in their own laboratories. It is provided in French (Video 1), English (Video 2), Spanish (Video 3) and Portuguese (Video 4). There are seven obligatory steps (see protocol in Supplementary File 1). The first step explains the preparation of the donor snail, using an antibiotic solution. The second step explains how to prepare the recipient snails, using an anesthetic solution. The third step provides information on how to prepare the work space and which equipment is needed. The fourth step details the dissection of the donor snail. The fifth step explains how to isolate the sporocyst grafts. The sixth step shows the different manipulations necessary to conduct the transplantation technique properly. The seventh and last step explains how to maintain the recipient snails after the transplantation. The manufacturing process of the two specific tools necessary for the sporocysts transfer, i.e. the microretractor and the glass microsyringe, is presented in Video 5 and Supplementary File 2. Housing, feeding and animal care followed the national ethical standards established in the writ of February 1st, 2013 (NOR: AGRG1238753A). The French Ministère de l'Agriculture et de la Pêche and the French Ministère de l'Education Nationale de la Recherche et de la Technologie provided permit A66040 to the laboratory for animal experiments and certificate to the experimenters (authorization 007083, decree 87-848). All efforts were made to ameliorate any suffering of animals. For details see Supplementary File 1 and the video on sporocyst transfer.

Discussion
The technical video was designed to make the technique of transplantation of schistosome sporocysts widely available so that it can be used by researchers from different scientific backgrounds, with different levels of research experience and from different countries, including those that speak English, French, Spanish and Portuguese. Many applications of microsurgical transplantation of sporocysts have been highlighted previously (Jourdane & Combes, 1989) such as: (i) Selection and maintenance of "pure" and stabilised strains with high compatibility to snails (cloning avoids genetic recombination), resistance to certain antihelmintics, or with specific chronotypes, (ii) Avoidance of genetic drift (for example in S. haematobium where there is a loss of compatibility towards the snail after 2 or 3 passages in the mammal), (iii) Compensation for the very low success rate of monomiracidial infections required to produce single sexes of the parasite, (iv) Maintenance of male and female clones without being forced to identify the sex, (v) Constitution of genomic libraries, (vi) Improving the ability to conduct schistosome geneticbased studies, or (vii) Studying mollusc immunology.
Additional uses of the sporocyst transplantation technique are envisioned based on recent molecular breakthroughs afforded by RNA interference gene knockdown- (Alrefaei et al., 2011;Mann et al., 2010) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene knockout- (Jinek et al., 2012) strategies. Use of the sporocyst transplantation technique in combination with these targeted manipulation strategies will assist the scientific community in functional genomics-based studies of schistosome biology directly in the snail host.
The conditions for successful transplantation are the following: proper anesthesia of the recipient snails; obtaining highest prevalence and post-transplantation survival of the recipient snails; ability of the daughter sporocyst grafts to produce new generation of daughter sporocysts; ability of the new generation of daughter sporocysts to produce enough cercariae (or sporocysts); ability of these cercariae to infect the mammal definitive host (infectivity) and produce eggs with viable and infective miracidia; and the ability to successfully perform several consecutive transplantations. produce new generation = produce new generation a

VIDEO
The high quality of the video is noted. It is appreciated that modification of the commentary may be, at this stage, challenging. The only version that I have checked is the English one. It is noted that the phrases accompanying the relaxation protocol include this: "Five hours later the snails are asleep, released and ready to…" "released" should be "relaxed". This is not an essential change. I have some concern over the use of PBS. Is this mammalian PBS? Is there a formulation (that can be included in the publication) for a PBS that is physiological for pulmonate snails and their sporocysts? Perhaps the authors use such a formulation. If so, this needs to be clear as some others may assume that mammalian PBS is suitable. And, in the big picture, the techniques work, so a snail PBS may not be essential. But the success rate may be improved if a non-physiological PBS is replaced by a physiological one. The osmotic concentrations of and mammals are markedly different, as described by