Attraction of Schistosoma mansoni Sambon , 1907 miracidia to Biomphalaria glabrata ( Say , 1818 ) in the presence of Angiostrongylus costaricensis

The attraction exerted by Biomphalaria glabrata infected with Angiostrongylus costaricensis on Schistosoma mansoni miracidia of the BH strain was tested, using a glass apparatus composed by two circular chambers connected to a channel. One mollusk or a sample of a snail conditioning water (SCW) was placed in one of the chambers, randomly selected, and the remaining of the apparatus was filled with chlorine-free water. Ten miracidia were placed in the centre of the channel and their behaviour was observed for 15 minutes. Ten replicates were made for each treatment (snail or SCW), using different specimens of mollusks or different samples of SCW. Mollusks infected with A. costaricensis attracted significantly less S. mansoni miracidia than non-infected B. glabrata. In addition, miracidia were also significantly more attracted to SCW from mollusks infected with A. costaricensis.

The evidence of attraction exerted by vector mollusks to Schistosoma mansoni miracidia was suggested by Kloetzel (1958) when observing the behaviour of the trematode in the presence of B. glabrata.Apparently, the attraction exerted by the intermediate hosts to the miracidia is caused by substances released in the water by the mollusk, which are denominated as SCW (snail-conditioned water, Chernin, 1972).Analyses of the hemolymph and the SCW of B. glabrata and B. tenagophila (Brasio et al., 1985b) show some cations and amino acids (Na, K, Mg, Ca, NH, alanine, glycine, valine, treonine, serine, leucine, proline, metionine, acid aspartic, fenilalanine, glutamic acid, lisine, tyrosine, arginine) as responsible to the miracidia attraction.Paraense and Corrêa (1963), when studying the BH and SJ strains of S. mansoni, described that the intensity of miracidia attraction is dependent on the trematode strain and of the planorbid species.Brasio et al. (1985a) verified that, in addition to SCW, the mucus and the feces of the mollusks also exerted strong attraction to the miracidia.Machado et al. (1988) and Balan et al. (1993) showed that the infection by S. mansoni did not develop in B. tenagophila previously infected by furcicercariae either with or without ocellli.The presence of primary or secondary sporocysts of S. mansoni in B. glabrata (Seta et al., 1993;Magalhães et al., 1997) induced the loss of the attractiveness to miracidia, which results in an advantage for the trematode, for it is more attracted to non-parasited mollusks.
In this work we verified the attractiveness of B. glabrata infected with A. costaricensis to S. mansoni miracidia.

Material and Methods
Melanic specimens of B. glabrata from Belo Horizonte (MG, Brazil) and the sympatric S. mansoni strain maintained in mice Swiss, SPF, were used.The Crisciumal (RS, Brazil) strain of A. costaricensis was used, and maintained in B. glabrata and Sigmodon hispidus (cotton-rat).Larvae (L1) of A. costaricensis were obtained from the feces of S. hispidus, using the method of Rugai et al. (1954).For infection of B. glabrata, 120 larvae (L1) of A. costaricensis were used.The exposure period of the mollusks to the larvae was 12 hours and the infected mollusks were used in the experiments after 20 hours.Samples of the snail-conditioning wa-ter (SCW) prepared according to Chernin (1972) were also tested.The samples of SCW were prepared with non-infected mollusks and with mollusks infected with A. costaricensis.
A glass apparatus composed of two circular chambers (A and B), 30 mm in diameter and 20 mm in depth, attached to a 40 mm × 11 mm channel (C), as described by Brasio et al. (1985a) were used in the experiments.Four groups were used in the experiments: Group I -10 specimens of non-infected B. glabrata; Group II -10 specimens of B. glabrata infected with A. costaricensis; Group III -10 samples of SCW of non-infected B. glabrata; Group IV -10 samples of SCW of B. glabrata infected with A. costaricensis.The glass apparatus have been filled with chlorine-free water and the mollusks or the samples of SCW were randomly placed in one of the chambers (A).Ten S. mansoni miracidia of the BH strain were deposited with a pipette in the centre of channel "C".The behaviour of the miracidia was then observed for fifteen minutes using a stereoscopic microscope (×0.5) and with abundant and homogenously distributed illumination.After fifteen minutes, the number of miracidia which migrated to each chamber was recorded.The experiments were repeated ten times and in each replication different specimens of mollusks or samples of SCW were tested.After each observation, the apparatus was carefully washed to remove any mucus tracks and feces that could interfere with the observations (Brasio et al., 1985a).
The attractiveness to the miracidia was statistically analysed using the PROC GLM (General Linear Model), procedure of the SAS ® software (Statistical Analysis System, SAS Institute, 2006).Differences among the means were tested by the Duncan multiple comparisons test.

Results
The results are presented in Table 1.There was a significant difference in the attractiveness exerted by the infected mollusks to the miracidia when compared to the mollusks non-infected with A. costaricensis (F = 4.47; P = 0.0487).The miracidia attraction exerted by SCW to the infected mollusks and by the SCW from non-infected mollusks were not significantly different (F = 1.17;P = 0.2945) (Table 2).The Duncan test did not detect any significant differences in the attractiveness exerted to the miracidia by the SCW from infected and not-infected snails (F = 0.13; P = 0.7183) (Table 3).In addition, the SCW from infected snails attracted significantly more miracidia than the infected snails (F = 7.71; P = 0.0124) (Table 4).

Discussion
The success of parasitism can be attributed partly to the parasite's finding of the host, that can be nearby or far.Therefore, the existence of substances that attract the parasites in the vicinity of the host constitutes an important strategy for the success of parasitism.Chernin (1972) and later Brasio et al. (1985b), evidenced amino acids and cations present in the conditioning water of the mollusks and in the hemolymph that attracted S. mansoni miracidia.
In our experiment, B. glabrata without infection with A. costaricensis, (group I) attracted 86% of S. mansoni miracidia, and B. glabrata infected with A. costaricensis attracted 71%.Although we have verified significant reduction in the attraction ability by the mollusk infected with A. costaricensis, nevertheless, we noted that B. glabrata infected with this nematode continues to exercise strong attraction to miracidia.Using B. tenagophila previously infected with S. mansoni, Seta (1996) obtained 70% of attraction with miracidia of the SJ strain, results that are similar to ours.In contrast, when using B. glabrata previously infected with S. mansoni, the author obtained only 17% of miracidia attraction.B. glabrata infected with S. mansoni, containing primary sporocysts (Seta et al., 1993) or secondary sporocysts (Magalhães et al., 1997) lost the ability to attract miracidia, the same occurring with SCW.
When we submitted BH miracidia to the SCW of non-infected B. glabrata, (group III), we obtained 84% of miracidia attraction.Seta (1996) obtained 100% of miraxonal attraction when exposing BH miracidia to SCW of non-infected B. glabrata.Studies done by Machado et al. (1988) in concomitant infections by different species of trematode (Digenea) in B. tenagophila showed that the development of natural infection of mollusks was frequently monospecific.Mollusks naturally infected by cercariae of species of Echinostomatidae and by aculeate cercariae of Distomata (Digenea) presented partial resistance to the development of experimental superinfection with S. mansoni, with resistance rates of 73 and 87%, respectively.According to Balan et al. (1993), samples of B. tenagophila naturally parasitised by trematodes and experimentally superinfected with S. mansoni, showed resistance to the development of sporocysts of S. mansoni.However, Yousif and Lämmler (1977) verified that previous infection of B. glabrata with A. cantonensis did not inhibit nor delay the subsequent infection of the mollusks with S. mansoni.
Biochemical alterations in the mollusks infected with S. mansoni were verified by several authors (Target, 1962;Gilbertson et al., 1967;Michelson and Dubois, 1973;Brasio et al., 1985b;Seta, 1996).Studies done by Stewart et al. (1985) showed an elevation in the levels of glucose and enzymes in the hemolymph of B. glabrata infected with A. costaricensis, as a consequence of the alterations in the parasitised tissue, characterised by the intense haemocytic reaction around the larvae (Zanotti-Magalhães et al., 2007).Brasio et al. (1985b) and Seta (1996) reported that the mollusk SCW exerted greater attraction than that exerted by the mollusk alone.Alterations in the chemical constitution of the conditioning water (SCW) from mollusks infected with   A. costaricensis that were kept in this SCW water for 24 hours, could explain the greater attraction exerted by the SCW water compared with the attractions exerted by the mollusk alone, when it was kept for only 15 minutes in the chamber of the miraxonal attraction apparatus.Seta (1996) observed that B. glabrata (or its SCW) infected with S. mansoni repelled miracidia of the trematode, whereas B. tenagophila (or its SCW) infected with S. mansoni exerted a more intense attraction to the miracidia of this trematode than the non-infected mollusks.An alteration in the composition and in the concentration of the aminoacids was found.
We concluded that B. glabrata infected by A. costaricensis showed a low intensity attraction to S. mansoni miracidia of the BH strain.The conditioning water of B. glabrata infected by A. costaricensis attracted more BH miracidia than the mollusks infected with the nematode.

Table 1 .
Mean number of miracidia of S. mansoni attracted for B. glabrata not infected (NI) or infected (I) by A. costaricensis.Mean values with the same letter are not significantly different (overall error rate (α) = 0.05).

Table 4 .
Mean number of miracidia of S. mansoni attracted for B. glabrata infected (I) by A. costaricensis or for its SCW (SCWI).Mean values with the same letter are not significantly different (overall error rate (α) = 0.05).

Table 2 .
Mean number of miracidia of S. mansoni attracted for SCW of B. glabrata not infected (SCWNI) or infected (SCWI) by A. costaricensis.Mean values with the same letter are not significantly different (overall error rate (α) = 0.05).