Interaction of Schistosoma mansoni Sporocysts and Hemocytes of Biomphalaria

Human infection by Schistosoma mansoni affects more than 100 million people worldwide, most often in populations of developing countries of Africa, Asia, and Latin America. The transmission of S. mansoni in human populations depends on the presence of some species of Biomphalaria that act as an intermediate host. The compatibility between S. mansoni and its intermediate host is influenced by behavioral, physiological, and genetical factors of the mollusc and the parasite. The susceptibility level of the mollusc has been attributed to the capacity of internal defense system (IDS)—hemocytes and soluble components of the hemolymph—to recognize and destroy the parasite, and this will be the center of interest of this paper. The schistosome-resistant Biomphalaria can be an alternative strategy for the control of schistosomiasis.


Introduction
Schistosomiasis is an important health problem that affects over 200 million people worldwide. Among the schistosome species that infect humans, Schistosoma mansoni is the most prevalent species causing intestinal and hepatic schistosomiasis in more than 100 million people living mainly in sub-Saharan Africa, the Caribbean, and South America, including Brazil [1,2]. Although campaigns for schistosomiasis control based on chemotherapy have reduced the morbidity and prevalence of this disease, transmission continues in almost all the areas in which interventions has been attempted. The transmission of S. mansoni in human populations has been associated with environmental and socioeconomic conditions, but the presence of susceptible intermediate hosts, consisting of some species of Biomphalaria, is obligatory. In Brazil, out of the eleven species Biomphalaria [3], only three were found naturally infected by S. mansoni: B. glabrata, B. tenagophila, and B. straminea [4].
The development of S. mansoni inside the intermediate host starts immediately after the active penetration of the snail by the miracidium, a swimming ciliated larva, through the exposed snail tegument. After penetration, the parasite undergoes morphological and physiological changes, being transformed into primary sporocyst (or mother sporocyst) that remains in the fibromuscular tissue of the host's cephalopodal region, near the penetration site. After 2-3 weeks, primary sporocysts generate secondary ones (or daughter sporocysts), which migrate from the cephalopodal musculature to the digestive glands or hepatopancreas of the mollusc, where their germinative cells can generate the cercariae [5][6][7]. The susceptibility level of different Biomphalaria species or strains to infection with the same lineage of S. mansoni can be very diverse, and it is a determinant of vectorial competence.
The compatibility between S. mansoni and its intermediate host is influenced by behavioral and physiological factors of the mollusc. However, the susceptibility level of Biomphalaria to S. mansoni is also determined by the genetic differences of the molluscs, as well as by the genetic constitution of Schistosoma [8,9]. Newton [10,11] demonstrated that the susceptibility of B. glabrata snail to S. mansoni depends largely upon genetic factors. Later, these results were corroborated by Richards [12], who demonstrated that the resistance character, acquired at the maturity phase, is determined by a single dominant gene with mendelian inheritance. The genetic dominance of the resistance character was also confirmed in crossbreeding with the susceptible and resistant strain of B. tenagophila [13,14]. One of the factors that influence susceptibility, and that may be genetically determined, is the activity of the snail internal defense system (IDS).
The Biomphalaria IDS is composed of soluble components of hemolymph and circulating cells, termed hemocytes, which work in association during the snail responses against infectious agents [15]. In snails, circulating hemocytes, especially the phagocytic cell population, are the principal line of cellular defense involved in destruction of S. mansoni larvae inside the intermediate host [16][17][18][19][20][21][22][23]. However, there is experimental evidence that soluble elements of the hemolymph participate in the protective mechanism against pathogens in many invertebrates [24][25][26][27]. Soluble components of the hemolymph can interact directly with pathogenic agents producing toxic substances or lytic peptides, or indirectly through mediator molecules for recognition of the pathogen or hemocyte activators [22,[28][29][30][31][32]. In Biomphalaria, hemocytes circulating in hemolymph or fixed in tissues are mainly produced by a well-defined region located between the pericardium and the posterior epithelium of the mantle cavity, called the amebocyte producing organ (APO) [33]. However, there is some evidence [34][35][36][37] that B. glabrata hemocytes may have multicentric origin and sites with proliferation of hemocytes were detected also at the saccular portion of the renal tubules and in the ventricular cavity of the heart.
The existence of a cellular defense mechanism deployed by molluscs against trematode infection was initially suggested by the finding of histological reactions around parasite sporocysts [10]. Histopathological analysis of S. mansoniinfected Biomphalaria showed that hemocyte infiltration around parasite larvae was faster and stronger in snail strains that are more resistant to parasite infection [23,38]. The confirmation of hemocyte participation in S. mansoni-sporocyst control was provided by experiments that transferred the APO from resistant to susceptible snail strains. In these experiments the APO recipient snails were able to control S. mansoni infection better than the respective controls [39,40].
The effector mechanisms by which hemocytes are able to kill trematode larvae are partially dependent on the capability of these cells to recognize sporocyst tegument molecules, leading to parasite encapsulation and cellular activation, that result in production of highly toxic metabolites of oxygen and nitrogen associated with parasite killing [41][42][43][44][45]. In this context, a better knowledge of the interactions between the parasite tegument and snail hemocytes is essential for understanding the snail susceptibility to S. mansoni infection. This is needed in order to propose new strategies for parasite transmission control. During the last few years, our research group has used the experimental model of S. mansoni infection in B. tenagophila of Taim strain to explore this interaction and the results are discussed below.  [40]. The process of destruction of S. mansoni larvae by hemocytes starts with the recognition and encapsulation of the newly penetrated sporocyst. The tegument of S. mansoni transforming miracidium is an important interface for molecular communication between the parasite and Biomphalaria [50]. In this context, the first step in the activation of this defense mechanism is the recognition of the parasite presence by hemocytes. The tegument of S. mansoni sporocyst is composed of highly glycosylated [50][51][52] molecules that bind to soluble proteins of B. glabrata hemolymph in a carbohydrate-dependent manner [50]. Furthermore, it was demonstrated that excretorysecretory glycoproteins from S. mansoni sporocysts also bind to hemocytes via carbohydrate binding receptors [53]. Therefore, lectin-carbohydrate binding could mediate the association of hemocytes with the trematode tegument [15,44,53], and consequently it could be a determinant factor of Biomphalaria susceptibility to S. mansoni infection.

Schistosoma mansoni Infection in Biomphalaria tenagophila of Taim Strain
To better understand the interaction of hemocytes with parasite larvae, we used the in vitro assay first developed by Bayne et al. [16]. Using this procedure we tested the effect of purified circulating hemocytes plus soluble hemolymph from different Biomphalaria species or strain on the axenically transformed primary or secondary sporocysts of S. mansoni.
The data clearly showed that addition of purified hemocytes from resistant snail strains, such as B. tenagophila Taim, into culture with primary sporocysts resulted in higher levels of parasite mortality compared to sporocysts cultured with hemocytes from susceptible snail strains, such as B. tenagophila Cabo Frio [54]. Moreover, in primary sporocyst cultures containing hemocytes from B. tenagophila Cabo Frio, the addition of cell-free hemolymph from B. tenagophila Taim resulted in increase of hemocyte binding to parasite tegument and higher mortality rates [54]. Therefore, we demonstrated that high levels of sporocyst mortality were associated with higher number of hemocytes bound to parasite tegument leading to parasite encapsulation [32,54], experimentally confirming that the ability of hemocytes to recognize the primary sporocyst is related to the resistance of B. tenagophila Taim.
Finally, we investigated if lectin-carbohydrate binding could mediate the association of hemocytes from B. tenagophila Taim with S. mansoni primary sporocysts. Previous work [31] with S. mansoni infection in B. tenagophila Taim showed that most of the circulating hemocytes recovered from B. tenagophila Taim, but not from B. glabrata BH or B. tenagophila Cabo Frio that are susceptible to S. mansoni infection, were intensively labeled by FITC-conjugated PNA and WGA lectins, and these labeled cells almost disappeared from the circulation during the first few hours after S. mansoni infection. Based on these data we tested, in vitro, the participation of N-acetyl-D-glucosamine carbohydrate moieties on the adhesion and destruction of S. mansoni sporocysts by hemocytes of B. tenagophila Taim. Similarly to the previous data, cultures containing hemocytes plus hemolymph from B. tenagophila Taim encapsulated and destroyed over 30% of the S. mansoni sporocysts in culture. Interestingly, the addition of N-acetyl-D-glucosamine to culture medium, but not mannose, resulted in significant inhibition of cellular adhesion to the parasite tegument and reduction of parasite mortality to 5% [32]. In conclusion, the data indicate that N-acetyl-D-glucosamine moieties influence the recognition of schistosome primary sporocysts by hemocytes of B. tenagophila Taim and implies the mechanism is a determinant of snail resistance against S. mansoni infection.
According to Lodes and Yoshino [55]; the general pattern of synthesis and release of protein by primary and secondary sporocysts in culture is quite different, showing that the two sporocyst stages are metabolically different. The study of gene expression profiles of S. mansoni daughter sporocysts identified different stage-specific genes, several of which are related to adaptation and development of the parasite in the host [56,57]. The in vitro interaction of axenically transformed S. mansoni primary sporocysts or secondary sporocysts obtained from infected snails with IDS components of B. glabrata (susceptible) and B. tenagophila Taim (resistant) revealed that the secondary sporocysts are less affected by the IDS, mainly of the resistant snail. Secondary sporocysts had fewer cells adhered to the surface, lower mortality, and less surface damage. These results suggest higher resistance of secondary sporocysts to the effector mechanisms of Biomphalaria when compared to the primary sporocyst. However, the secondary sporocysts were unable to grow when inoculated into B. tenagophila Taim but were able to develop into B. glabrata [58].
Many authors have found that sporocysts can interfere with snail host reproductive physiology and alter other aspects of the parasite-host interaction, secreting molecules (excretory/secretory products (ESP)), adsorbing Biomphalaria antigens when cultivated in the presence of snail molecules, and synthesizing molecules similar to host molecules even in the absence of Biomphalaria components [55,[59][60][61][62][63][64][65]. Recently, experiments with molecular and biochemistry approaches using ESP or sporocysts and hemocytes from schistosome-susceptible and schistosomeresistant B. glabrata demonstrated that the parasite is able to interfere with extracellular signal-regulated kinase (ERK) pathway in susceptible B. glabrata [66][67][68]. Moreover, resistant B. glabrata presented differential expression of genes potentially associated with the snail IDS after infection with S. mansoni when compared with susceptible strain [69].
The hemocytes from resistant Biomphalaria species can recognize, encapsulate, and destroy the sporocysts soon after S. mansoni invasion. On the other hand, the ability of the parasite to avoid or disrupt the immune response of the host is fundamental to the establishment of parasite-host compatibility [70]. Similar molecules have been found in S. mansoni and Biomphalaria, suggesting an evolutionary convergence of molecular expression between parasite and snail host [8,59,[70][71][72]. This similarity is important for the escape process of the parasite: molecular mimicry [72][73][74]. According to Salzet et al. [75], this mechanism can prevent the recognition of the parasite by the host IDS.
These data help us to understand why the snails defense in particular its destruction of primary sporocysts, occurs in the first hours after miracidia penetration. Furthermore, van Die and Cummings [71] and Lehr et al. [72] have suggested that glycans play a role in the parasite molecular mimicry process. Although the evolutionary advantages of this adaptive process for the parasite are well understood, it is not known how this process interferes with schistosomiasis [75] or whether this mechanism could interfere with the snail's resistance mechanisms. Thus, more experiments using daughter sporocysts must be performed to clarify aspects involved with molecular mimicry in the S. mansoni/Biomphalaria (susceptible and resistant) interaction.