The susceptibility of Biomphalaria tenagophila from Espírito Santo (ES), Brazil was evaluated. Specimens F1 of B. tenagophila (ES) and B. glabrata (MG) control, were individually exposed to miracidia of the LE, SJ and AL strains. The snails were examined 30 days after the exposure and weekly thereafter for a period of 80 days. The snails that died during the experiment were subjected to the low-stringency polymerase chain reaction (LS-PCR) technique to detect Schistosoma mansoni DNA. The infection rate for B. tenagophila (ES) was 0% to all strains used, whereas the rates for B. glabrata were 81, 90 and 94.3% for the SJ, AL and LE strains, respectively. B. tenagophila snails that died during the experiments were not positive for S. mansoni, while all of the B. glabrata were positive for S. mansoni. In addition, to verify if the miracidia had penetrated in the molluscs and how long it was eliminated, B. tenagophila specimens were exposed to miracidia and ten snails were sacrificed at different times and subjected to LS-PCR. Forty-eight hours after the exposure, S. mansoni was not detected in B. tenagophila. We concluded that this B. tenagophila population is resistant to infections of S. mansoni (100 miracidia/snail).

Caldeira RL, Jannotti-Passos LK, Teodoro TM, dos Santos Carvalho O (2018) Resistance of Biomphalaria Tenagophila (Mollusca: Gastropoda) From The State of Espirito Santo, Brazil to Schistosoma Mansoni (Platyhelminthes: Trematoda) Infection. Ann Clin Pathol 6(2): 1136.

•    Biomphalaria tenagophila
•    Resistance
•    Schistosoma mansoni
•    LS-PCR 

ES: Espírito Santos; LE: Luis Evangelista; SJ: São José dos Campos; AL: Alagoas; CMM-Fiocruz: Medical Malacology collection from Fundação Oswaldo Cruz; LS-PCR: low strincengy polymerase chain reaction; PCR-RFLP: polymerase chain reaction and analyses of the restriction fragment-length polymorphism; ITS: internal transcribed spacer; DNA: deoxyribonucleic acid; rDNA: ribosomal deoxyribonucleic acid.

Biomphalaria glabrata (Say, 1818), B. tenagophila (Orbigny, 1835), B. straminea (Dunker, 1848) are found naturally infected by the trematode Schistosoma mansoni (Sambon, 1907), the agent of schistosomiasis. Biomphalaria peregrina, B. amazonica and B. cousini were reported as potential hosts of the parasite since they had been infected experimentally [1-4].

Schistosoma - has a complex life cycle comprising sexual reproductive stage in the definitive mammalian host and asexual reproductive stage in the snail intermediate host.

Studies of the host-parasite interaction using the relationship between B. glabrata and S. mansoni as a model have demonstrated this species shows a varied degree of susceptibility to this trematode infection [5-6]. This susceptibility of the planorbid snails to S. mansoni infection is a trait genetically controlled and inherited over generations [7], being controlled both by parasite and snail genes [8].

The snail B. tenagophila exhibits populations that are susceptible to S. mansoni infection [5] and a population that is resistant from the Taim Ecological Reserve, Rio Grande do Sul - RS. In the laboratory this population has been exposed by different strains of S. mansoni with a variable number of miracidia and was always resistant to infection [9-13].

A range of studies has already been conducted to improve our understanding of the resistance of snails to trematode infections [14-17]. The snail internal defense system comprises hemocytes and soluble factors in the hemolymph. These two components act together in the snail defense, thus defining those molluscs susceptible and/or resistant to pathogens [18]. Few studies have defined whether uninfected snails are resistant to S. mansoni infection. Souza & Jannotti-Passos [19] showed that the parasite was destroyed at the first week after its penetration in the B. occidentalis by low strincengy polymerase chain reaction (LSPCR). Pimenta-Nacif et al. [20], focusing on the initial phase (from 1h to 10h after exposure) of the interaction process between the snails and the S. mansoni sporocysts, compared by histology, susceptible versus resistant B. tenagophila populations. They showed that even at the earliest time point (1h a.e), fibrous host cells of both snail populations were arranged as a thin layer around the sporocysts.

The diagnosis of infection in snails is also an important aspect since it is not viable to detect infection in the prepatent period and in dead snails using traditional methods, and also it is not possible to differentiate S. mansoni from other trematodes. Molecular techniques have been used to aid in the diagnosis regarding the infection of snails [21-23].

The aim of this study is to analyze B. tenagophila population from the state of Espírito Santo (Brazil) exposed to different S. mansoni strains and elucidate whether the population is naturally resistant to this trematode.

Snails

Specimens from the F1 generation of B. tenagophila collected in the Mãe Bá Lagoon (geographical coordinates: 20º45’19”W, 46º34’29”S) were used. As an experimental control of infection and mortality, B. glabrata originating from Belo Horizonte, Minas Gerais (MG), Brazil, was used. For species identification, one of the tentacles of the snails (the snails were not sacrificed at this stage) was removed for DNA extraction using the Wizard Genomic DNA Purification kit (Promega) and subjected to polymerase chain reaction and analyses of the restriction fragment-length polymorphism (PCR-RFLP) using the rDNA internal transcribed spacer (ITS) with the enzyme DdeI [24,4]. The profiles were compared to the standard profiles of the DNA extracted from snails tissue from the Medical Malacology Collection (CMM-Fiocruz). These snails were maintained and raised in the “Lobato Paraense” Mollusc Rearing of René Rachou Institute - IRR/FIOCRUZ, in Belo Horizonte, MG, Brazil according to Jannotti-Passos et al. [25].

Experimental research on vertebrates and invertebrates have been approved by an appropriate ethics committee CEUA/ Fiocruz LW30/13.

Parasites

The LE, SJ and AL strains of S. mansoni were used. The LE strain was isolated from a patient residing in Belo Horizonte, MG, and maintained in the “Lobato Paraense” Mollusc Rearing since 1968. The SJ strain was isolated from naturally infected snails from the region of São José dos Campos, São Paulo state (Brazil), maintained in the “Lobato Paraense” Mollusc Rearing since 1975. The AL strain was isolated in 1980 from B. glabrata from Alagoas state (Brazil). The maintenance of the cycles of S. mansoni strains was performed through successive passages in hamsters (Mesocricetus auratus) and B. glabrata, according to the technique described by Pellegrino and Katz [26] and modified by Jannotti-Passos et al. [25].

Susceptibility experiments

A. To verify the susceptibility status of the snails, 100 B. tenagophila and 100 B. glabrata (infection control) were individually exposed to 100 and 10 miracidia/snail, respectively, of the LE/SJ/AL strains according to Jannotti-Passos et al. [25]. The snail diameters were 4-6mm for B. tenagophila and 6-8mm for B. glabrata, the same diameter standardized in the Mollusc Rearing to S. mansoni routine infections. A total of 25 snails from each species and with the same diameters, were used as a mortality control and not exposed to miracidia.

C. To verify if the miracidia had penetrated in the molluscs and how long it was eliminated 60 B. tenagophila and 60 B. glabrata (control) were individually exposed to 100 and 10 miracidia SJ (strains), respectively. At intervals of 1, 5, 10, 24, 36 and 48 hr after exposure, 10 snails from each species were sacrificed and subjected for molecular studies.

Examination of the snails

The snails were individually introduced into recipient dishes with 5mL of unchlorinated water, exposed to artificial light for 30 min and then taken to the stereoscope for observation of possible S. mansoni cercariae (experiment “A”). The first examination was performed 30 days after the exposure of the snails to the miracidia, and the other examinations were performed weekly for a total period of 80 days after exposure [25]. At the end of this period, the snails that survived were examined by crushing them between glass plates. The snails that died during the experiment and experiment “B” snails were subjected to DNA extraction using the Wizard Genomic DNA Purification kit (Promega) according to the manufacturer’s instructions, and subjected to the LS-PCR technique to verify the presence of S. mansoni DNA [22]. The pair of primers used in these reactions was designed to amplify across adjacent in tandem minisatellite units from S. mansoni mtDNA [22]. This procedure was repeated for all strains used and the infection and mortality rates were calculated for each experiment.

The snail B. tenagophila is an important intermediate host of S. mansoni in southern Brazil [27], and a population of this snail from Taim reserve (RS) has been shown to be resistant to S. mansoni infection [9-13]. In this study, descendants of B. tenagophila from Espírito Santo state, were exposed to miracidia of the three S. mansoni strains. Infection was not observed in any of the exposured specimens. On the other hand, the infection rates for B. glabrata (control) were 81, 90 and 94% for the SJ, AL and LE strains, respectively. The mortality rates of B. glabrata were 8, 16 and 11% for the AL, SJ and LE strains, respectively, and for B. tenagophila, were 25, 21 and 20% for the AL, SJ and LE strains, respectively. The specimens of B. tenagophila that died during the experiments were examined by LS-PCR and showed no profile of S. mansoni (Figure 1, lane 3-11). In figure 1, the pair of primers seemed to be highly specific for this parasite, as the DNA derived from uninfected snails did not show the typical pattern obtained with mtDNA. While, the B. glabrata specimens that died during the experiments exhibited the presence of a ladder-type arrangement of the bands, corresponding to the amplification of the tandem repeated region of the 62-bp mtDNA fragment, the characteristic profile of S. mansoni (Figure 3 lane 9). To date, the study of snails susceptible to S. mansoni infection has been performed through the exposure of snails to miracidia and examination by light exposure or crushing. However, these techniques do not detect infection in the prepatent period or died snails, making it impossible to know if the miracidia penetrated the snails. LS-PCR is an important technique in susceptibility studies because it is capable of detecting the DNA of this trematode in the prepatent period [22]. Souza et al. [19], used the LS-PCR technique and showed that there was no S. mansoni DNA in B. occidentalis after 7 days of exposure. That is, the miracidia penetrated and were destroyed by the defense system of the snail, which was therefore considered resistant to S. mansoni infection. In our study, using the LS-PCR technique, it was possible to observe the presence of the a ladder-type arrangement of the bands in B. tenagophila exposed to S. mansoni (SJ strain) after 1, 5, 10, 24 and 36 hr only (Figure 2, lanes 3-7) as well as in adult S. mansoni worm (Figure 2, lane 2). Moreover, it was possible to observe the presence of this profile in all B. glabrata, in all intervals of sacrifice after exposure tested (Figure 3 lanes 3-8). It not was possible to observe the presence of S. mansoni in negative B. glabrata (Figure 3, lane 10), in the B. tenagophila exposed to S. mansoni after 48 hr (Figure 2, lane 8) and negative B. tenagophila (Figure 2, lane 9).

The host-parasite relationship is complex and there are still gaps in the knowledge about the susceptibility and resistance of snails to trematode infection and the capacity of development of this trematode in the intra-snail stage. It is known that in B. glabrata, 30% of the miracidia are capable of penetrating and transforming to sporocysts, 30% penetrate but do not transform, and 40% are incapable of even penetrating the snail [28]. In resistant B. glabrata and B. tenagophila, the miracidia penetrate and are recognized as foreign bodies and destroyed by the immune defense system, composed of hemocytes and soluble factors present in the hemolymph, in the first hours following penetration [16-17,20,28-30]. In refractory snails, the miracidia do not penetrate. In susceptible snails, the miracidia penetrate and develop within the snails, producing cercariae that are released to the external environment. According to Lewis et al. [8], parasite-host interactions are influenced by the snail genes that control susceptibility and the parasite genes that determine infectivity.

Biomphalaria tenagophila (ES) snails used in this study did not release cercariae after the exposure with S. mansoni miracidia and examination by light stimulation. Furthermore, the LS-PCR technique used detects the presence of S. mansoni DNA up to 36 hr after the exposure, demonstrating that the studied B. tenagophila population is resistant to strains used.

We would like to thank Fiocruz-MG, Fapemig (APQ-01766- 15) and CNPq (308869/2017-6) for financial support for this study.

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