Discussion

Changes in morphometric features of S. mansoni male worms were observed among groups, depending on variables such as the host (AKR/J or Swiss mice, hamsters), the time of infection (day 45 or day 60 post-infection) and the location of parasite development (peritoneal cavity or portal system), suggesting the occurrence of developmental plasticity. Differences have been verified in the biology and morphology of these schistosomes due to distinct types of hosts and times of infection (Stirewalt et al., Reference Stirewalt, Kuntz and Evans1951; Kuntz, Reference Kuntz1952; Amorim, Reference Amorim1953; Saoud, Reference Saoud1965, Reference Saoud1966; Coles & Thurston, Reference Coles and Thurston1970; Kastner et al., Reference Kastner, Kohn, Teixeira and Pitanga1975; Machado-Silva et al., Reference Machado-Silva, Galvão, Presgrave, Rey and Gomes1994, Reference Machado-Silva, Galvão, Oliveira, Presgrave and Gomes1995; Martinez et al., Reference Martinez, Neves, Oliveira, Machado-Silva and Rey2003; Neves et al., Reference Neves, Costa-Silva, Martinez, Branquinho, Oliveira, Lenzi, Gomes and Machado-Silva2004). However, the influence of the location of development of S. mansoni, even though from an essentially experimental point of view, had not been evaluated in detail. Previously, experiments on the development of this trematode in the peritoneal cavity of different models focused on the number of parasites recovered and total size (Moore & Meleney, Reference Moore and Meleney1955; Pereira et al., Reference Pereira, Coelho, Fonseca, Bredt and Pellegrino1972; Bicalho et al., Reference Bicalho, Melo and Pereira1993).

In the present study, schistosomes recovered from the peritoneal cavity of Swiss mice at day 45 post-infection showed lower mean values in all characteristics evaluated in comparison with those of worms obtained from the peritoneum of AKR/J mice. This indicates that this inbred strain presumably favours the development of the parasite in the peritoneal cavity. However, no statistical difference was observed between worms recovered from the peritoneum of mice of the AKR/J and Swiss strains at day 60 post-infection. On the other hand, parasites recovered at day 45 post-infection from the portal system of Swiss mice were more developed in terms of higher average values of total body length, extension of testicular mass and number of testes than those removed from the corresponding site in AKR/J mice. This indicates that schistosomes appear to display opposite patterns of development in the peritoneal cavity and portal system in the two mouse strains.

Despite worms from the peritoneal cavity of the AKR/J strain being more developed than parasites of the Swiss lineage at day 45 post-infection and those of AKR/J mice at day 60 post-infection in terms of significant differences in total body length and number of testes, the results suggest that the development of the parasite in the portal system of this strain was lower and/or slower than that observed in Swiss mice. Parasites recovered from the portal system of AKR/J mice at day 60 post-infection were indeed more developed compared with those schistosomes recovered at day 45 post-infection from the same site and lineage of vertebrate host. This suggests the continuation of their development process. However, among parasites from the portal system recovered at day 60 post-infection, the average total body length was higher in Swiss mice, although morphometric values of the distance between the oral and ventral suckers and the testes number did not vary between the two lineages and extension of testicular mass was higher in AKR/J mice. The values of reproductive characteristics of worms obtained from the portal system of Swiss mice were lower in the second than in the first period evaluated, i.e. significant reductions in the extension of testicular mass and the number of testes. This finding perhaps indicates the occurrence of an involution that is similar to that verified in worms from the peritoneal cavity of AKR/J mice. However, in this case, this probable involution observed in parasites is more restricted, i.e. a significant reduction only in the number of testes. In fact, parasites recovered from the peritoneal cavity of AKR/J mice had a more developed total body length and higher number of testes at day 45 post-infection than at day 60 post-infection. However, in the parasites from the peritoneal cavity of the Swiss mice the majority of the parameters, i.e. total body length, distance between oral and ventral suckers and extension of testicular mass, were higher at day 60 post-infection.

It is likely that some factor associated with AKR/J mice stimulated or inhibited worm development. These mice are a sublineage obtained from the AKR strain, which is characterized by a C5 component deficiency in the complement pathway (Ben-Efraim & Cinader, Reference Ben-Efraim and Cinader1964; Cinader & Dubiski, Reference Cinader and Dubiski1964) and a polarization for the T-helper (Th)-1 response (Curry et al., Reference Curry, Else, Jones, Bancroft, Grencis and Dunne1995; Guerassimov et al., Reference Guerassimov, Hoshino, Takubo, Turcotte, Yamamoto, Ghezzo, Triantafillopoulos, Whittaker, Hoidal and Cosio2004). In experimental schistosomiasis, AKR/J mice have been used mainly due to their immunological peculiarities, which have been correlated with the complete development of S. mansoni observed in the peritoneal cavity of these mice (Bicalho et al., Reference Bicalho, Melo and Pereira1993; Tristão, Reference Tristão2001; Mati, Reference Mati2009; Mati & Melo, Reference Mati and Melo2013). Similarly to conventional mice, the production of interleukin-4 and interleukin-5 associated with the Th-2 response, and the ability to decrease interferon production and increase total immunoglobulin (Ig)-E levels in response to S. mansoni egg antigens have been reported for the AKR/J strain (Yamaya et al., Reference Yamaya, Basaki, Togawa, Kojima, Kiniwa and Matsuura1995). However, a deficiency in modulation of immune response may explain why hepatic granulomata of AKR/J mice are larger and have greater cellularity than granulomatous lesions of Swiss mice in the initial stage of the post-postural acute phase of the disease (Tristão, Reference Tristão2001; Mati, Reference Mati2009; Mati & Melo, Reference Mati and Melo2013). This momentary status of AKR/J mice, hardily responding to antigens of mature eggs of the parasite, may be correlated with a more vigorous response to adult male worms, leading to a lowering of their development. This has been observed in schistosomes obtained from the portal system of this isogenic strain of mice in comparison with the parasites of Swiss mice, perhaps by accelerating and increasing energy expenditure in tegument turnover. A similar approach may be used to explain the reduction of sexual organs in parasites from the portal system of Swiss mice observed at day 60 post-infection, considering that granulomatous lesions reach their largest size in the liver of this host close to this time (Silva et al., Reference Silva, Fernandes, Barbosa, Oliveira and Andrade2000; Mati et al., Reference Mati, Freitas and Melo2010). However, further studies are still needed to clarify these aspects.

The site of development was substantially important in determining morphometric changes observed in male parasites. The intraperitoneal development of S. mansoni did not follow the pattern observed for worms recovered from the portal system. Furthermore, parasite development was not only slower in the peritoneal cavity of Swiss mice compared with the AKR/J strain, but often incomplete. It is known that female S. mansoni obtained from the peritoneal cavity of Swiss mice usually do not have eggs in their ootypes. However, the presence of eggs was verified in a proportion of the total of female worms recovered from the peritoneal cavity of AKR/J mice. In these rodents, despite the atypical site of development, schistosomes had rapid somatic growth and sexual maturation in comparison with those recovered from the peritoneal cavity of Swiss mice (Bicalho et al., Reference Bicalho, Melo and Pereira1993). The development of S. mansoni in the peritoneal cavity of AKR/J mice and after its involution is still not completely explained. However, in addition to the immunological aspects of this lineage already mentioned, particularly the deficiency in the complement system, AKR mice show low levels of interferon production by macrophages in the peritoneal cavity (Jiang et al., Reference Jiang, Rummage, Zhou, Chen, Herriot, Stewart, Kolosov and Leu1996), which may provide an area with a certain degree of immune privilege for the parasite that would be reversed after the start of oviposition and formation of schistosomal granulomata in situ, when immune cells are attracted to the peritoneal cavity.

Therefore, complementary studies with the evaluation of immunological parameters and longer periods of infection are desirable for improved understanding of this trend of downsizing and involution, even partial and perhaps exclusive of the acute phase, of the total body length and sexual organs of worms recovered from the portal system and peritoneal cavity of Swiss and AKR/J mice, respectively. Interestingly, mature worms of S. mansoni with a tendency to reduce their size with ageing have already been shown in experimental schistosomiasis (Fan & Pao, Reference Fan and Pao2006).

Conversely, maybe also depending on the rodent strain, a possible effect of already developed peritoneal male worms in AKR/J mice on the maturation of the females and the production of eggs cannot be ruled out. In the portal system, this influence of male schistosomes on female schistosomes has been demonstrated. Apparently, for the development and sexual maturation of parasites, interrelationships among specimens of male worms and the vertebrate host's immune system, at first, as well as subsequently the interaction among parasites of both sexes, are fundamental. Male worms, unlike the females of S. mansoni, were able to transduce signals from stimuli of the host immune system that seem necessary for their complete sexual development. Later, these already developed worms could stimulate the development of female parasites (Ribeiro-Paes & Rodrigues, Reference Ribeiro-Paes and Rodrigues1997; Kunz, Reference Kunz2001; Hernandez et al., Reference Hernandez, Lim, McKerrow and Davies2004; Loverde et al., Reference Loverde, Niles, Osman and Wu2004; Ting-An & Hong-Xiang, Reference Ting-An and Hong-Xiang2009). Adult males of S. mansoni supply a signal that is required for preventing the apoptosis of vitellocytes in female parasites, and the transcription of genes, including those encoding eggshell precursor proteins within vitelline cells, seems to be dependent on pairing (Galanti et al., Reference Galanti, Huang and Pearce2012). In addition, epigenetic regulatory mechanisms have already been demonstrated in S. mansoni and the cytosine methylation machinery appears to be linked to the process of oviposition (Geyer et al., Reference Geyer, Rodríguez-López, Chalmers, Munshi, Truscott, Heald, Wilkinson and Hoffmann2011).

The physiology of schistosomes, parasite burden and the host immunological system may also influence the pathology (Cheever et al., Reference Cheever, Lenzi, Lenzi and Andrade2002; Pyrrho et al., Reference Pyrrho, Ramos, Neto, Silva, Lenzi, Takiya and Gattass2002; Mati et al., Reference Mati, Freitas and Melo2010) and phenotype of adult worms, such as morphological aspects (Senft et al., Reference Senft, Gibler and Knopf1978; Kruger et al., Reference Kruger, Hamilton-Attwell, Tiedt, Visser and Joubert1988; Bicalho et al., Reference Bicalho, Melo and Pereira1993; Machado-Silva et al., Reference Machado-Silva, Galvão, Presgrave, Rey and Gomes1994; Neves et al., Reference Neves, Costa-Silva, Martinez, Branquinho, Oliveira, Lenzi, Gomes and Machado-Silva2004; Martinez et al., Reference Martinez, Costa-Silva, Neves, Oliveira and Machado-Silva2008). Indeed, the strain of the definitive host contributed to the determination of characteristics of the parasitism process, including the worm phenotype, and peculiar aspects related to AKR/J mice may be expected in the host–parasite relationship when this experimental model is employed. In fact, as recently described, AKR/J mice also provide a more favourable environment for the development of the intestinal trematode Centrocestus formosanus than Swiss mice, with a higher percentage of parasite recovery and a greater fecundity of these flukes in inbred mice (Mati et al., Reference Mati, Pinto and Melo2013).

When considering morphometric data obtained from worms recovered from the portal system of hamsters, the rodent species in which the LE strain of the parasite is maintained and presumably more adapted, phenotypic differences in male S. mansoni were even more evident. It is interesting that schistosomes of this strain on the first passage in mice showed morphological differences in relation to parasite morphology seen in hamsters. In accordance with this observation, a strain of this trematode species isolated from a natural host undergoes morphological alterations after its passage in mice (Machado-Silva et al., Reference Machado-Silva, Galvão, Presgrave, Rey and Gomes1994). Furthermore, if we consider the site of the development of the parasite in mice belonging to a single lineage and in the same period of infection, although parasites have a common origin and possibly a lower genetic variation, significant differences in morphometric parameters evaluated were found among worms from the portal system and peritoneal cavity. This finding suggests that the phenomenon of phenotypic plasticity occurred in this case. It is likely that the parasite has, over the course of its development, adapted to the conditions of its microenvironment in the host, favouring or inhibiting the development of certain structures. The mean values of the distance between the oral and ventral suckers and the number of testes were the closest to those of worms recovered from the portal system or peritoneal cavity, while the values of total body length were the most different among groups. In fact, the distance between suckers was the most conserved, at least in the present study using the S. mansoni mouse model, with no variation in this characteristic between S. mansoni male worms from the portal system of hamsters and Swiss mice. Interestingly, comparative analysis of adult populations of Fasciola hepatica from sheep, cattle, pigs and experimentally infected rats shows that many parameters studied, such as the body area of the fluke, were significantly different; however, the distance between suckers did not change due to the host (Valero et al., Reference Valero, Darce, Panova and Mas-Coma2001). Information is lacking that would enable us to understand better the extent of these variations, which do not seem to be determined solely or primarily by genetic factors.

Another factor that could influence worm development is the nutritional state of the host. Sudati et al. (Reference Sudati, Rivas and Fried1997) reported that a high-protein diet in mice altered worm growth in terms of the dry weight, body area and the uterine egg counts of Echinostoma caproni. This might be explained by the increased cellular and/or humoral immunity in the gastrointestinal tract of mice, making the immediate host mucosal environment less conducive to worm development. It is also known that the lack of nutritional factors, with consequent malnutrition of the host, affects unfavourably the development of some morphological parameters of S. mansoni male worms, and that a cholesterol-rich diet can also interfere with their development in mice (Neves et al., Reference Neves, Oliveira, Machado-Silva, Coutinho and Gomes2002, Reference Neves, Alencar, Aguila, Mandarim-Lacerda, Machado-Silva and Gomes2007). However, the AKR/J strain was larger and had greater carcass fat content compared with the SWR/J strain when mice were fed with a high-fat or high-protein diet (West et al., Reference West, Waguespack and McCollister1995). Whether the AKR/J strain is different from Swiss mice or not, and is more or less metabolically efficient, remains unknown, but a relative shortage of nutrients in the peritoneal cavity could be inferred from this difference.

The existence of differences among strains of S. mansoni has been sufficiently demonstrated, including those from the morphological point of view (Saoud, Reference Saoud1965, Reference Saoud1966; Coles & Thurston, Reference Coles and Thurston1970; Machado-Silva et al., Reference Machado-Silva, Galvão, Oliveira, Presgrave and Gomes1995; Bogéa, Reference Bogéa2005). However, as in the present study, a substantial influence of the host on a specific strain of the trematode has been verified, and attention should be given to the vertebrate host in the characterization of intra-specific variations of S. mansoni by morphological studies, since there are instances in which morphological analysis alone cannot be sufficient to characterize the parasite lineage or species. In fact, in the case of Monogenea, for example, differences in the development and morphometric aspects of these parasites due to changes in their habitat have even led to taxonomic confusion in some species (Matejusová et al., Reference Matejusová, Koubková, Gelnar and Cunningham2002; Olstad et al., Reference Olstad, Bachmann and Bakke2009).

Although morphological differences in S. mansoni from different hosts are relatively well documented, the term ‘phenotypic plasticity’ is not always used. In relation to schistosomes, Kruger et al. (Reference Kruger, Hamilton-Attwell, Tiedt, Visser and Joubert1988) were the first to use it, by stating that the protrusion of tubercular spines in S. margrebowiei and S. mattheei is correlated with plastic phenomena controlled by factors such as the type of the vertebrate host and whether there is parasite pairing. Until then, before the scanning electron microscopic analysis performed by these authors, the question concerning the presence or absence of tubercles on the tegument of these bovid schistosomes yielded conflicting interpretations. Nevertheless, changes in the tegument of the species S. mansoni have also been suggested to be influenced by the vertebrate host, and it was indeed shown that in non-permissive hosts tegument maturation is later and incomplete, and the tubercles are aspinous (Senft et al., Reference Senft, Gibler and Knopf1978).

It is probable that S. mansoni routinely encounters environmental stressors and uses phenotypic plasticity to compensate for the costs of these disturbances. Plastic developmental strategies may have been selected to increase the probability of transmission between hosts during the evolutionary history of trematodes (Davies & McKerrow, Reference Davies and McKerrow2003; Poulin, Reference Poulin2003), and traces of this ability might persist in schistosomes. A high potential for the occurrence of this phenomenon in certain characteristics with substantial importance to the survival of the organism may represent adaptive advantages for the species. In fact, among the non-blood flukes, adopting a progenetic cycle seems to be a conditional feature in response to the difficulty of passage from one intermediate host to the definitive host (Lefebvre & Poulin, Reference Lefebvre and Poulin2005; Herrmann & Poulin, Reference Herrmann and Poulin2012).

Taking into account the same species and strain of vertebrate host, plastic phenomena were observed for the first time in S. mansoni specimens from the same host, including the same individual, but at different sites of development. On the other hand, not only aspects related to feeding, but also immunological aspects are probably involved in the phenotypic plasticity observed in this schistosome, since in the peritoneal cavity, where it is assumed that there are dietary restrictions for the worms, mainly the absence of blood, there were also differences in development between parasites of Swiss and AKR/J mice. Nevertheless, even without access to blood, worms usually reach maturity and lay eggs in the peritoneal cavity of AKR/J mice, as already mentioned.

The ability of worms to adapt to their habitat might also be related to the data shown in the present study, regarding the differences between the development of the parasite in the peritoneal cavity and portal system in both AKR/J and Swiss mice. What favours the parasite in the portal system of Swiss mice and in the peritoneal cavity of AKR/J mice remains unknown. Further studies are still necessary to elucidate why, in the peritoneal cavity, worm development is different in comparison with that customarily observed in the portal system, considering the different host strains. Furthermore, data obtained confirm that mice of the AKR/J strain experimentally infected with S. mansoni are, especially as regards the development of peritoneal parasites, an interesting model for the study of schistosomiasis.

Developmental plasticity has been suggested in parasites as an outcome of epigenetic factors and the present results reinforce the idea of the high plastic potential and adaptive capacity of S. mansoni. The morphology of the worm depends on the balance of the host–parasite relationship and, to understand the biology of this parasite, efforts to characterize its relationship with its invertebrate and vertebrate hosts must be continued.