Dynamics of Schistosoma Mansoni Development in The Intermediate Host, Biomphalaria Glabrata, A Freshwater Snail

Background Schistosomiasis is one of the most signicant and prevalent waterborne parasitic diseases. Even though many studies have been reported about schistosomiasis, the dynamics of schistosome in intermediate host snails is little known. In the present study, the dynamics of Schistosoma larvae in infected snails was histologically investigated.


Abstract
Background Schistosomiasis is one of the most signi cant and prevalent waterborne parasitic diseases. Even though many studies have been reported about schistosomiasis, the dynamics of schistosome in intermediate host snails is little known. In the present study, the dynamics of Schistosoma larvae in infected snails was histologically investigated.

Methods
To examine the localization of Schistosoma mansoni (S. mansoni) parasites in the snails, Biomphalaria glabrata snails infected with miracidia were harvested and examined by stereoscopic observation. Then, frozen sections were prepared and stained with H&E. Furthermore, immunohistochemical detection of parasites was performed using anti-S. mansoni antibody, and their localization in the snails was analyzed.

Results
Snails infected with S. mansoni miracidia were harvested at 10 and 56 days post-infection (DPI) and analyzed. In the stereoscopic observations, white spots were observed at 56 DPI, while no spots were observed at 10 DPI. However, histological investigations visualized the larvae speci cally in the head-foot area of the snail at 10 DPI. Further, it was observed that the larvae relocated to the hepatopancreas and ovotestis areas at 56 DPI.

Conclusions
The present study revealed the dynamics of Schistosoma larvae in intermediate snails, shown as the differential localization of S. mansoni larvae at early and late infection stages.

Background
Schistosomiasis is one of the most signi cant and prevalent waterborne parasitic diseases, as it affects more than 240 million people worldwide (WHO 2020) [1,2]. Six Schistosoma species (Schistosoma haematobium, S. mansoni, S. japonicum, S.intercalatum, S. guineensis, S. mekongi) are pathogenic in humans and freshwater snails are known to be their intermediate hosts [3,4]. Following excretion of schistosome eggs from an infected human or animal, miracidia hatch from the eggs and infect intermediate host snails, with developing into cercariae in the snails [5,6]. After cercariae are released from the snails, they percutaneously infect humans and animals, then become schistosomulae, migrate into the portal vein via the bloodstream, and mature into adult worms [5,7]. Combinations of male and female adult worms lay eggs in intestinal or pelvic veins, and the schistosome lifecycle is repeated [8].
A large number of studies have been reported about schistosomiasis, including case reports and developments of new drugs and vaccination methods [9][10][11][12][13][14]. On the other hand, few studies that focused on intermediate hosts have been presented [15][16][17]. Notably, the dynamics of schistosome in intermediate host snails is little known, though it has been well known that the infection begins with miracidia invasion and ends with explosively increased production of cercariae. Since Schistosoma cercariae infect the nal host, it is crucial to suppress outbreak production of cercariae to control the prevalence of schistosomiasis.
In the present study, the dynamics of Schistosoma larvae in infected snails was histologically visualized and we revealed that the larvae initially invade the surface of the head-foot area of the snail and localize there in the early period of their development, then relocate to the hepatopancreas and ovotestis areas in the late period of the infection. Thus, it is elucidated that the process of Schistosoma larvae growth and development is related to the selection of preferential locations inside intermediate host snails.

Ethical statement
All of the present experiments using Schistosoma mansoni (S. mansoni) parasites, Biomphalaria glabrata (B. glabrata) snails, and mice were approved by the Institutional Animal Research Committee of Nara Medical University (#12813, 12776), and performed according to Japanese statutes regarding Humane Treatment and Management of Animals.

Animals and parasites
Female ICR mice (eight weeks old) were purchased from Japan SCL (Hamamatsu, Japan). A strain of S. mansoni (NIH-Sm-PR-1) was maintained at the animal facilities of Nara Medical University by passage through albino B. glabrata snails (Newton's NIH Puerto Rican/Brazilian M-line strain) and ICR mice [18]. Mice were subcutaneously infected with 250 S. mansoni cercariae, then the livers were harvested eight weeks after initiation of infection. Parasite eggs were separated from the livers and collected following treatments with actinase and collagenase. Miracidia were then hatched from the eggs in sterile water and used to infect B. glabrata snails (5-mm shell size). Each snail was infected with ve miracidia of S. mansoni. They were placed in 24-well plates containing tap water for 24 hours at 28 o C, then transferred into a 15-L aquarium. The release of cercariae was observed by illumination with an LED lamp from four weeks up to 12 weeks after infection.

Post-infection histological analysis of B. glabrata in snails
To examine the localization of the parasites in the snails, B. glabrata snails infected with miracidia were harvested at 10 and 56 days post-infection (DPI) (Fig. 1). After removing the shell, each snail was xed with 4% PFA for two days at 4˚C, followed by 20% sucrose in PBS for 12 hours. Next, the specimens were embedded in an OTC compound, then sections (10 µm thick) were prepared using a cryostat (Thermo) and stained with hematoxylin-eosin (H&E) using a standard protocol. Immunohistochemical detection of S. mansoni parasites was performed in each of 10 sections and their localization in the snails was analyzed. For immunohistochemistry, the sections were permeabilized with 0.2% Triton X-100 in PBS (TPBS) containing 1% BSA, followed by blocking with 5% normal goat serum, then treated with a mouse anti-S. mansoni monoclonal antibody (BGN/1A7, dilution 1:100, Novus Biologicals). The following incubation at 4˚C overnight and washing three times with TPBS, an AlexaFluor 546 conjugated goat antimouse secondary antibody (dilution 1:200, Molecular Probes, Invitrogen) was used to detect primary antibodies. All nuclei were stained with DAPI (Dojin), followed by washing three times with TPBS.

Results
Localization of S. mansoni larvae in infected-B. glabrata snails Snails infected with S. mansoni miracidia were harvested at 10 and 56 DPI and analyzed. At 10 DPI (early infection), no gross abnormalities were observed in the infected snails as compared with uninfected snails ( Fig. 2A). At 56 DPI (late infection), white spots were noted in the infected snails (Fig. 2B, 2F-2J), whereas those were not observed in uninfected snails at any time ( Fig. 2A-2E), indicating that the spots were formed where S. mansoni sporocysts or cercariae existed. The snails were xed and sliced, then the sections were subjected to H&E staining. In sections from the head-foot (HF) area at 10 DPI, deep purple colored structures were detected, in which the presence of S. mansoni larvae was suspected (Fig. 3A-3C, 3E, 3F), while it was not detected in the hepatopancreas (HP) area (Fig. 3D). At 56 DPI, S. mansoni larvae were detected in the deep purple colored structures in the sections of the HP area (Fig. 3G), while more precise observations revealed their distribution in other organs, including the ctenidium (Ct), and ovotestis (OT) (Fig. 3I-3K), but not in HF area (Fig. 3H).
After serial sections were stained by H&E (Fig. 4A, 4B, 4N, 4O), immunohistochemical analysis was conducted using the anti-S. mansoni antibody that reacts to both S. mansoni miracidia and cercariae ( Supplementary Fig. S1). Figures A, B, N and O in Figure 4 are same respectively to Figures A, B, G and J in Figure 3. S. mansoni larvae were immunohistochemically con rmed to be present in the HF area in the early period of their development (10 DPI) (Fig. 4A-4M). However, they were predominantly present in the HP areas in the late period of their development (56 DPI) (Fig. 4N-4R).

Mapping of S. mansoni larvae in B. glabrata snails
We attempted to produce a map showing where S. mansoni larvae existed in B. glabrata snails (Fig. 5A). Immunohistochemical analysis of serial sections of S. mansoni-infected B. glabrata snails at 10 DPI showed larvae in 30 out of 45 sections from the HF area (66%) (Fig. 5B), while those were observed in only 7 of 45 sections from other areas (15%), such as Hr and Ct. At 56 DPI, immunopositivity for S. mansoni larvae was observed in all 15 successive specimens from the HP and OT areas, which was in contrast to sparse distribution in the other areas, indicating accumulation of S. mansoni larvae in the HP + OT (HPOT) area (Fig. 5C). These ndings indicate that S. mansoni larvae become located in the HF area during the early infection period, then move to the HPOT area by the time of late infection, and suggest the presumable location-speci c assistance.

Discussion
Schistosome infects intermediate host snails with miracidia, then miracidia become cercariae by approximately four weeks [5,19]. Although it is known that the intra-snail development process includes mother sporocysts, daughter sporocysts, and cercariae [4,7], their distributions in snails have not been analyzed in detail. In the present study, localization of S. mansoni larvae during the early (10 DPI) and late (56 DPI) development periods in intermediate host B. glabrata snails was investigated, and t the distribution of S. mansoni larvae was clearly demonstrated to be different between those early and late periods, as the predominant location at 10 DPI was the head-foot (HF) area, while the hepatopancreas (HP) and ovotestis (OT) areas were predominant at 56 DPI. However, the larval developmental stage was not determined due to the limitation that anti-S. mansoni monoclonal antibody used in the present study recognizes both S. mansoni miracidia and cercariae ( Supplementary Fig. S1) but does not discriminate each stages in the snails. Bahia et al. reported that the S. mansoni protein kinase C1 (SmPKC1) is differentially expressed during the lifecycle of this parasite (adult worm, miracidium, sporocyst, cercaria, schistosomula) [20], and Fernandes et al. found that expression patterns of the S mansoni venom allergen-like protein (SmVAL) family differed among the germ ball, cercaria, and schistosomula [21]. We consider that immunohistological analysis of SmPKC1and SmVAL expression patterns would make it possible to identify the developmental stage of larvae found at 10 and 56 DPI.
Schistosome infects only certain species of intermediate host snails, while B. glabrata is known to be the host snail for S. mansoni [6,7]. The albino B. glabrata snails (Newton's NIH Puerto Rican/Brazilian M-line strain) used in the present study have been reported to produce S. mansoni cercariae [18], which was con rmed by our ndings. Some strains of B. glabrata, such as the NMRI and M line, are also susceptible to the infection by S. mansoni [22,23], while resistant strains such as BS-90 also allow infection with S. mansoni miracidia, though it does not lead to production of cercariae [24,25]. Snails have innate immune defense systems that can specifically or non-specifically target schistosomes for destruction [26,27]. Even following successful infection, miracidia and sporocysts are exposed to humoral factors, and attacked by motile hemocytes in the hemolymph and other tissues of the snail [28], resulting in production failure of cercariae. Therefore, an immunohistological investigation of larval distribution using resistant snails such as BS-90 may provide temporal and spatial information regarding the arrest of larval development. Indeed, larval encapsulation and several factors including reactive oxygen species [29] and other resistance-associated factors [30] have been noted as possible mechanisms by which the development of larvae fails to proceed.
The present results demonstrated changes in larval locations inside snails between early and late infection, strongly suggesting that the environment favoring Schistosoma larvae differs depending on the developmental stage. Several methods for culturing Schistosoma larvae in vitro have been reported [31,32]. The Bge cell line, established from 4-to 5-day-old embryos of an albino B. glabrata snail, has been utilized to support the development and growth of Schistosoma spp [33]. larvae in vitro. According to the report by Yoshino et al., Bge cells supported the induction of sporocysts but failed to produce cercariae [34]. Furthermore, Ivanchenko et al. observed the generation of daughter sporocysts, whereas cercariae production was scarcely observed in the cultures with Bge cells [35]. Considering previously reported information about culture systems, the use of Bge cells alone seems to be inadequate to achieve complete production of sporocysts and cercariae. Identi cation of environmental factors required for the development of those larvae would make it possible to grow them in vitro.

Conclusion
The present study revealed differential localization of S. mansoni larvae at early and late infection stages in intermediate snails. Our ndings may lead to the development of novel methods for in vitro larvae cultivation using larval stage-speci c environmental factors.