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. Author manuscript; available in PMC: 2021 May 7.
Published in final edited form as: Exp Parasitol. 2020 Mar 26;213:107887. doi: 10.1016/j.exppara.2020.107887

Biochemical and apoptotic changes in the nervous and ovotestis tissues of Biomphalaria alexandrina following infection with Schistosoma mansoni

Mohamed R Habib a,*, Samah I Ghoname a, Rasha E Ali a, Rasha M Gad El-Karim a, Alaa A Youssef a, Roger P Croll b, Mark W Miller c,d
PMCID: PMC8105080  NIHMSID: NIHMS1698529  PMID: 32224062

Abstract

Infection with trematodes produces physiological and behavioural changes in intermediate snail hosts. One response to infection is parasitic castration, in which energy required for reproduction of the host is thought to be redirected to promote development and multiplication of the parasite. This study investigated some reproductive and biochemical parameters in the nervous (CNS) and ovotestis (OT) tissues of Biomphalaria alexandrina during the course of Schistosoma mansoni infection. Antioxidant and oxidative stress parameters including catalase (CAT), nitric oxide (NO) and lipid peroxidation (MDA) were measured. Levels of steroid hormones, including testosterone, progesterone and estradiol, were also assessed. Finally, flow cytometry was used to compare measures of apoptosis between control snails and those shedding cercariae by examining mitochondrial membrane potential with the stain 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) and poly(ADP-ribose) polymerase (PARP). Infection with S. mansoni caused a 47.7% reduction in the net reproductive rate (Ro) of B. alexandrina. CAT activity was increased in the CNS at 21 days post infection (dpi) but by 28 dpi it was reduced below control values. Also, CAT activity increased significantly in the OT at 14, 21 and 28 dpi. In CNS tissues, NO levels were reduced at 7 dpi, increased at 14 and 21 dpi, and reduced again at 28 dpi. The overall level of lipid peroxidation gradually increased during the course of infection to reach its highest levels at 28 dpi. Steroid hormone measurements showed that concentrations of testosterone and estradiol were reduced in the CNS tissues at 28 dpi, while those of progesterone were slightly increased in the CNS and OT tissues. The percentage of cells that positively stained with JC-1was significantly increased in CNS and OT tissues of infected snails while the percentage of cells positively stained with PARP was decreased compared to controls. Together, these findings indicate that infection initiates diverse biochemical and hormonal changes leading to loss of cells responsible for egg laying and reproduction in B. alexandrina.

Keywords: Apoptosis, Biomphalaria alexandrina, Schistosoma mansoni, CNS, Castration

1. Introduction

Schistosomiasis is a public health challenge that impacts the lives of millions worldwide (Colley et al., 2014; Toor et al., 2018). Snails of the genus Biomphalaria act as obligatory intermediate hosts for the transmission of Schistosoma mansoni, the parasite that causes the most widespread form of intestinal schistosomiasis. Infection of Biomphalaria with schistosomes causes many physiological and behavioural changes including parasitic castration (Faro et al., 2013; Alberto-Silva et al., 2015). Infection-induced parasitic castration, or loss of fertility, has been documented in many snail-trematode systems (Baudoin, 1975; Joosse and Van Elk, 1986; Crews and Yoshino, 1990; Tunholi et al., 2011). Fecundity of infected snails may be partially or completely inhibited by parasitism (Minchella, 1985).

Many mechanisms contribute to parasitic castration, including direct and/or indirect effects of the parasite on its snail host (Sluiters et al., 1980). Infection-induced fertility loss cannot be attributed to a specific factor as it reflects many complex events that facilitate the parasite establishment within snails (Sullivan et al., 1985; De Jong-Brink, 1995). Indirect mechanisms may include actions on the snail’s nervous system leading to loss of specific signals required for reproduction (see Joosse et al., 1988). For example, levels of biogenic amines, a class of neurotransmitters that regulate reproduction, were found to be reduced in the snail nervous system and plasma during the course of infection with S. mansoni (Manger et al., 1996). It was hypothesized that miracidia-to-mother-sporocyst transformation depends on scavenging dopamine and serotonin from the surrounding tissues and nerve endings of the host, thereby leading to their depletion in the nervous and reproductive tissues (see also Vallejo et al., 2014).

Direct changes in the reproductive organs mediated by the parasite larvae are also responsible for diminished egg production in infected snails. Interference can occur through competition for nutrients (Sullivan et al., 1985). Invasion of the gonads by parasites decreases the organ volume and subsequently leads to their dysfunctionality through alteration in host’s hormonal balance (Bayne and Loker, 1987; Gerard and Theron, 1997). Crews and Yoshino (1989) attributed the inhibition of B. glabrata reproductive activity to the regulation of gonadal growth by daughter sporocysts of S. mansoni. However, it is not clear if this regulation is mediated through the parasite loads in gonads or disturbance to snail host metabolism.

The current study was designed to extend and update the existing information on parasitic castration using the B. alexandrina-S. mansoni system. Biochemical and apoptotic parameters were measured in the CNS and ovotestis (OT) of infected snails and compared to unexposed specimens.

2. Materials and methods

2.1. Biomphalaria alexandrina infection with Schistosoma mansoni and fecundity

Sixty mature, laboratory-bred B. alexandrina (> 10 mm shell diameter), originating from field populations collected from Giza Governorate, Egypt and maintained for several generations in the Medical Malacology Laboratory, were divided into two groups (each of 30 snails). Maturity of snails was confirmed by maintaining them for one week in dechlorinated water before starting the experiment. Specimens that laid eggs were considered sexually mature. Snails in the experimental group (triplicate groups of 10) were individually exposed to 10 freshly hatched S. mansoni miracidia obtained from the Schistosome Biological Supply Center (SBSC), Theodor Bilharz Research Institute, Egypt (originally an Egyptian strain obtained from Giza Governorate) for 3 h at 25 °C in 2 ml vials containing dechlorinated tap water. The control snails (triplicate groups of 10) were placed individually in 2 ml vials without exposure to miracidia. Following exposure to miracidia, the snails were transferred to plastic aquaria (10 snails/aquarium; 16 × 23 × 10 cm) containing dechlorinated water. The water was changed two times per week and lettuce leaves (Lactuca sativa) were added as food source. Pieces (5 × 5 cm with 0.5 cm thickness) of styrofoam sheets floating on the water surface were used as substrates for oviposition. Egg masses were collected weekly for four successive weeks. The survivorship of snails (Lx) and the total number of eggs laid per snail (Mx) were recorded weekly for each aquarium. The reproductive rate (Ro) was calculated at the end of the experiment (Costa et al., 2004; Ragheb et al., 2018).

For the estimation of biochemical parameters and apoptosis, 700 adult snails with average shell diameter from 10 to 12 mm were infected with 10 freshly hatched S. mansoni miracidia and monitored for four successive weeks as described in the previous section. Each week, a cohort of snails was carefully crushed between two glass slides, the shells were removed, and the soft parts were pinned and dissected in Sylgard-lined (Dow Chemical) Petri dishes containing snail saline (see Habib et al., 2015). CNS and OT tissues were collected weekly and stored at −20 °C until used for analysis.

2.2. Oxidative stress and antioxidant parameters

CNS and OT tissues from infected and control snails were separately collected and pooled into triplicates (10 each for ovotestis and 20 each for nervous tissues) at 7, 14, 21 and 28 days post infection (dpi, shedding snails). Tissue samples were washed in distilled water, weighed and homogenized in PBS (phosphate buffer solution; 50 mM potassium phosphate, 1 mM EDTA, pH 7.5) in a 1:10 wt to volume ratio using a glass homogenizer. Tissue homogenates were centrifuged at 1,700 g for 15 min at 4 °C. The resultant supernatant was used to calculate the concentration of malondialdehyde (MDA) based on Kei (1978) using a lipid peroxide kit (Biodiagnostic Company, Dokki, Giza, Egypt; Cat. No. MD 2529). Measurement of catalase (CAT; E.C. 1.11.1.6) activity was based on the method of Aebi (1984) using a catalase assay kit (Biodiagnostic Company, Dokki, Giza, Egypt; Cat. No. CA 2517). Nitric oxide (NO) concentration was determined with a colorimetric NO kit (Biodiagnostic Company, Dokki, Giza, Egypt; Cat. No. GR 2511) based on Montgomery and Dymock (1962).

2.3. Steroid hormones

CNS and OT tissues were collected in triplicates as previously described for enzyme measurements. Tissue samples were weighted and homogenized in PBS (1:10 wt to volume). Following centrifugation at 950 g for 45 min at 4 °C, supernatants aliquots were used to calculate steroid hormone concentrations using commercial enzyme immunoassay kits available from DiaMetra (Via Pozzuolo, Spello, Italy) for progesterone (Cat. No. DCM006–9), testosterone (Cat. No. DCM002–12) and estradiol (Cat. No. DCM003–11). Absorbance of the calibrators, controls, and test samples was measured with an Immunospec micro-plate reader (Cat. No. INS28-CA-2000, Immunospec Corp., USA). The standard best-fit curve for each hormone was drawn through the plotted points following kit instructions. The sensitivities were as follows: 0.05 ng/ml for progesterone with an intra-assay variability less than 4% and an inter-assay variability less than 9.3%; 0.10 ng/ml for testosterone with an intra-assay variability less than 7% and an inter-assay variability less than 8.3%; and 8.86 pg/ml for estradiol (17β-estradiol) with an intra-assay variability less than 9% and an inter-assay variability less than 10%.

2.4. Measurement of mitochondrial transmembrane potential by flow cytometry

Mitochondrial membrane potential, an indicator of apoptosis, was evaluated using the lipophilic fluorochrome JC-1 and BD™ MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit (Catalog No. 551302). Snail CNS and OT tissues were washed with isotonic tris EDTA buffer and homogenized in distilled water pH 7.5. Aliquots (100 μl) of each cell suspension were transferred into a sterile 15 ml polystyrene centrifuge tube. The samples were centrifuged at 350 g for 10 min, the supernatant was removed, and 500 μl JC-1 working solution was added to each cell pellet. Samples were incubated for 10–15 min at 37 °C in a CO2 incubator. Cells were then washed three times using 1x assay buffer obtained with the kit (2 ml, 1 ml and 0.5 ml, respectively) for 5 min and immediately analyzed on an Accuri C6 flow cytometer (Becton Dickinson, Sunnyvale, CA, USA). JC-1 accumulates in the mitochondria in a potential-dependent manner and its increase forms JC-1 aggregates. Staining values were calculated as the percentage of the total number of cells counted. All experiments were repeated three times.

2.5. Measurement of poly (ADP-ribose) polymerase-1 (PARP) by flow cytometry

PARP was measured in CNS and OT tissue homogenates using the BD Pharmingen™ PARP protocol. A cell suspension (100 μl) was transferred to a test tube and 10 μl of purified mouse monoclonal antibody against PARP conjugated with FITC fluorochrome (Catalog No. 551024; San Jose, CA, USA) was added at the recommended dilution for each sample. The mixture was incubated at room temperature for 30 min. The cells were then washed with 2 ml of phosphate buffer saline (8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4 and 0.2 g KH2PO4 dissolved in 1L distilled water, pH 7.4)/bovine serum albumin (BSA) and centrifuged at 240 g for 5 min. The supernatant was discarded and the cells were re-suspended in 0.2 ml of PBS/BSA or in 0.2 ml of 0.4% paraformaldehyde in PBS/BSA. Finally, PARP values were measured in the green (FL 1-H) channel on the flow cytometer.

2.6. Statistical analysis

One-way analyses of variance (ANOVA) followed by the post hoc Dunnett’s t-test were used to assess significant differences in enzyme and hormone concentrations. Calculations were performed using the Statistical Package of Social Science (SPSS) software (version 20.1, Chicago, IL, USA). Data are presented as mean values ± standard deviation or standard error of the mean. The limit for statistical significance was set at p < 0.05 (i.e. a confidence level of 95%).

3. Results

3.1. Effect of infection on snail fecundity

The prevalence of infection in B. alexandrina at 28 dpi was detected by cercarial shedding from living snails and examination of snails that died during the pre-patent period. Infection was present in 75% of the total specimens exposed. The survival rate of infected snails at this first shedding was 93% compared to 100% in control snails. The survivorship (LX) of infected B. alexandrina was reduced to 87% over the period of infection at 28 dpi compared to 96% in control snails.

Infection also led to a marked reduction in the number of eggs laid/snail/week (MX) compared to non-infected snails. The pattern of reduction in MX was similar in both infected and control snails during the pre-patent and early patent periods (7–21 dpi). However, at 28 dpi the infected group showed a marked (p < 0.001) reduction in the number of eggs laid/snail (5.5 ± 3.3) compared to controls (15.0 ± 5.2). The net reproductive rate (Ro) was significantly reduced (p < 0.001) in infected snails to 47.7% of its value in controls (Table 1).

Table 1.

Effect of infection with Schistosoma mansoni on reproductive parameters of Biomphalaria alexandrina snails during 4 weeks of exposure.

Exposure period (weeks) Control Infected
LX MX LXMX Lx MX LXMX
1 1.00 19.1 ± 8.3 1 1.00 19.1 ± 8.3 1
2 1.00 9.2 ± 5.4 2 1.00 9.2 ± 5.4 2
3 1.00 5.1 ± 2.6 3 1.00 5.1 ± 2.6 3
4 0.96 15 ± 5.2 4 0.96 15 ± 5.2 4
Ro Σ LXMX 47.8 25.02
Reduction % 47.7 ***
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Data presented as Mean ± Standard Error.

***

significant compared to control at p < 0.001, LX = Survivorship as a proportion (1.0 = 100%), MX = The mean number of eggs/snail/week, Ro = The net reproductive rate (Σ LXMX) of snails throughout the experimental period.

3.2. Oxidative stress parameters

Schistosoma mansoni infection altered the levels of CAT, NO, MDA in B. alexandrina. CAT activity was higher in the CNS than in the OT at all times. CAT remained relatively stable in the OT for the first 1–2 weeks, increased slightly in the subsequent two weeks, and then fell by week 4 post infection. CAT in the CNS also remained stable for the first 1–2 weeks, but then rose to a peak on week 3, and then fell precipitously by week 4 (Fig. 1A). Nitric oxide concentrations in the OT showed a pattern of significant reduction (p < 0.05) during the period from 1 to 3 weeks before increasing again by week 4. In the CNS, NO showed a peak of increment after 2 weeks post infection before a marked reduction (p < 0.05) at the 4th week where its value was 1.50 ± 0.10 μmol/g compared to 4.50 ± 0.30 μmol/g in controls (Fig. 1B).

Fig. 1.

Fig. 1.

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Antioxidant and oxidative stress parameters in the nervous (CNS) and ovotestis (OT) of Biomphalaria alexandrina following infection with Schistosoma mansoni. A: Catalase activity. B: Nitric oxide concentration. C: Malondialdehyde concentration. Data presented as Mean ± Standard Error. * significant at p < 0.05.

Lipid peroxidation was detected by measuring the concentration of malondialdehyde (MDA). In the CNS and OT of infected snails, MDA increased over the period of infection compared to its value in control snails. The highest MDA concentrations in the CNS and OT were measured at week 3, MDA values were 72.97 ± 4.97 nmol/g and 34.53 ± 1.03 compared to 11.81 ± 1.01 and 23.50 ± 3.50 nmol/g in controls, respectively, for CNS and OT (Fig. 1C).

3.3. Steroid hormones

While infection did not induce significant changes in progesterone at any time interval following infection (Fig. 2A), it did induce significant decreases in the concentration of testosterone at 14 and 28 dpi in the CNS and 7 and 14 dpi in the OT of infected snails compared to non-infected controls (Fig. 2B). Estradiol, the most affected hormone, decreased in all time intervals following infection. The lowest concentration of estradiol in the CNS was recorded at week 4 post infection. In the OT, it was the lowest at 1 and 2 weeks post-infection compared to its concentration in non-infected controls (p < 0.05; Fig. 2C).

Fig. 2.

Fig. 2.

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Concentration of steroid hormones in the nervous (CNS) and ovotestis (OT) of Biomphalaria alexandrina following infection with Schistosoma mansoni. A: Testosterone. B: Progesterone. C: Estradiol. Data presented as Mean ± Standard Error. * significant at p < 0.05.

3.4. Flow cytometry

Measurement in the CNS and OT of B. alexandrina indicated an increase of apoptosis in the tissues of infected snails compared to non-infected controls. A significant increase (p < 0.001) in the percent of cells stained with the mitochondrial apoptotic marker, JC-1 was observed in the CNS and OT of infected snails compared to controls. The increase of apoptosis in infected snails was coincident with a marked decrease (p < 0.001) in the percent of cells stained with PARP, the enzyme responsible for DNA repairs and reversing apoptosis, in the CNS and OT. In infected snails, the percentage of cells in which PARP was detected was decreased (p < 0.001) in the CNS and OT to 1/3 its initial value and less than half of its value in controls, respectively (Fig. 3).

Fig. 3.

Fig. 3.

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Percent of Mitochondrial transmembrane potential (JC-1) and Poly (ADP-ribose) polymerase (PARP) markers in the nervous (CNS) and ovotestis (OT) of control and Schistosoma mansoni-infected Biomphalaria alexandrina. Data presented as Mean ± Standard Deviation. *** significant at p < 0.001(sample t-test).

As in Figure (4), the percentage of cells positively stained with JC-1 (M6) is much higher than that of cells with negative stain (M5). However, the percentage of cells positively stained with PARP (M2) was much lower than those negatively stained (M1) (Fig. 5).

Fig. 4.

Fig. 4.

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Flow cytometric analysis of Mitochondrial transmembrane potential in CNS and OT tissues of control and infected Biomphalaria alexandrina snails. A and B: CNS from control and infected snails, respectively. C and D: OT of control and infected snails, respectively. M5 indicates negatively stained population of cells while M6 indicates positively stained populations.

Fig. 5.

Fig. 5.

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Flow cytometric analysis of Poly (ADP-ribose) polymerase (PARP) in CNS and OT tissues of control and infected Biomphalaria alexandrina snails. A and B: CNS from control and infected snails, respectively. C and D: OT of control and infected snails, respectively. M1 indicates negatively stained population of cells while M2 indicates positively stained populations.

4. Discussion

4.1. Effect of infection on reproduction

Trematode infection usually causes castration in snails by reducing or completely inhibiting their ability to lay eggs. While it is clear that parasitic castration is mediated through an array of events initiated following parasite penetration into their intermediate hosts, more studies are needed to disclose mechanisms that underlie this phenomenon. The present study contributes to our understanding of castration by exploring some biochemical and apoptotic markers in B. alexandrina during the course of S. mansoni infection.

Infection of B. alexandrina with S. mansoni caused a 47% reduction in the net reproductive rate accompanied by a gradual decrease in the survival rate of snails. Similar observations were reported by Thornhill et al. (1986), who found significant alterations in the reproductive parameters of exposed and control B. glabrata during the patent period. Moreover, Alberto-Silva et al. (2015) demonstrated that 15.2% of B. glabrata with positive S. mansoni infection failed to lay egg masses. Reduction in fecundity was also recorded in three Bulinus species. Infection with S. haematobium caused 34%, 63% and 90% reduction in the fecundity of Bulinus senegalensis, B. truncatus and B. globosus, respectively. This inhibition of reproductive output of infected snails occurred during the early weeks, with the greatest reduction observed during the patency period from weeks 10–19 post infection (Fryer et al., 1990). In our study, the mean number of eggs/snail was significantly reduced in infected snails during the first three weeks of infection. The difference was highest between infected and non-infected snails at week 4 where it was one-third its control value. This is in accordance with Crews and Yoshino (1989) who reported a reduction in the mean number of eggs laid per B. glabrata between the first and third week of infection with S. mansoni. These authors related this reduction to the movement of sporocysts to digestive glands and gonads. Moreover, Meier and Meier-Brook (1981) reported a drop in the number of eggs laid by B. glabrata by the second week after exposure to S. mansoni. Similarly, inhibition of egg production in Lymnaea truncatula occurred between week 1 and week 3 post infection with Fasciola hepatica as a result of ovotestis consumption by the parasite rediae (Wilson and Denison, 1980). However, Minchella and Loverde (1981) documented a significant increase in the reproduction of B. glabrata during the first two weeks of infection. The authors suggested that this is a mechanism of fecundity compensation in which the snails increase their reproductive output immediately following challenge with miracidia. In the present results, infection with S. mansoni decreased the reproductive parameters of B. alexandrina during 4 weeks of observation and no fecundity compensation was observed at any time point post infection.

A comparison between B. glabrata that had been exposed and those not exposed to S. mansoni showed a significant difference in the number of egg masses/snail and eggs/snail in the period from 42 to 62 dpi. B. glabrata shedding cercariae showed a reduction in fecundity and fertility (Faro et al., 2013). This difference between exposed and unexposed snails, particularly during the patent period of infection, suggests a reduction in the energy allocated for reproduction, and a consequent interruption of oviposition. The reduced energy might be also redirected towards increasing the animal growth (gigantism) as usually observed with infected snails (Mangal et al., 2010).

4.2. Oxidative stress parameters

Trematode infection changed the levels of CAT, NO, and MDA in both the CNS and OT of the snails. The overall trend of CAT activity indicated that this enzyme was inhibited during the first week of infection and followed with a slight increase at week 2 during the pre-patent period of infection. The marked elevation in catalase activity was observed at week 3 (21 dpi). However, this increase did not reduce the lipid peroxidation that was increasing in the CNS and OT from the first week of infection and afterward. The oxidative metabolism in nervous tissue is increased during S. mansoni infection, but the catalase activity did not change significantly to attenuate the reactive oxygen intermediates formed.

NO was significantly decreased in the nervous tissue at week 1 and 4 post infection. This decrease may be due to that developing schistosome larvae scavenge nutrients from the snail’s hemolymph, resulting in a reduction in the amount of nutrients circulating to the nervous system at these periods. Insufficient nourishment will cause a decline in the nervous system activity. In snails, there is an association between the internal defense system and the neuroendocrine system and both systems are vulnerable to schistosome infection (De Jong-Brink et al., 1997). Thus, the reduction in CNS activity may culminate in a deficiency in the function of the defense system of snails. These events occur at day 7 dpi when the intense asexual reproduction of schistosome larvae causes significant tissue lesion and at 28 dpi when the cercariae must evade the immune system to migrate and emerge to the external environment. These fluctuations in the oxidative and antioxidant parameters of infected snails are proposed to reflect an attempt of snails to overcome the stress caused by invading larvae. These changes were also observed in the OT of infected snails where they may reflect direct damage induced by the parasitic larvae to the surrounding tissues.

The effects of infection on the antioxidant system of Biomphalaria were also reported by Mossalem et al. (2018) who found a significant decrease in CAT and GSH and an increase of MDA in the tissues and hemolymph of B. alexandrina following infection with S. mansoni. These changes in the antioxidant system of snails were restored to near control values when the snails were exposed to antioxidants extracted from the plant, Eucalyptus camaldulensis. Moreover, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase, CAT, GSH, and glutathione reductase levels were decreased while malondialdehyde, protein carbonyl, total protein, albumin, globulin, cholesterol, low density lipoprotein and triglycerides levels were increased in Schistosoma infected snail tissues. Apoptotic changes were also reported in infected snail tissue homogenates (Koriem et al., 2016). The above-mentioned biochemical parameters were restored to their values in control uninfected snails upon treatment with sodium fluoride, suggesting its ability to inhibit oxidative stress and apoptosis produced in Schistosoma infected snails (Koriem et al., 2016).

Flow cytometric analysis of CNS and OT cell suspensions during the patent period of infection indicated a significant increase of apoptosis in infected snails compared to controls. Regulation of apoptosis is a key determinant of host-parasite interactions (Barcinski and DosReis, 1999). In the present study, we observed an increase in JC-1 percentages, indicating an effect on the function of mitochondria. The subsequent induction of apoptosis during PARP inhibition is indicative of apoptosis and the inability of cells to repair damaged DNA following infection induced apoptosis.

Mitochondria play an important role in apoptosis by releasing cytochrome c and other proteins that activate apoptotic enzymes including caspase 3, which breaks down proteins and leads to cell death (Fischer et al., 2003). Decreased mitochondrial outer membrane permeabilization triggers release of cytochrome c. The mechanism of this release is suggested to occur due to collapse of the mitochondrial membrane potential (Schultz and Harrington, 2003; Yuan et al., 2003). This intrinsic pathway of apoptosis that includes permeablization of mitochondrial membrane, cytochrome c release and DNA damage is also present in molluscs (Pirger et al., 2009). JC-1 is a membrane-permeable lipophilic fluorochrome that is used as a probe of the status of mitochondrial membrane potential (Gravance et al., 2000). PARP inhibition or over-activation has been implicated in several neurological diseases (Bhaskara et al., 2005). Both caspase 3 and PARP are activated in neurons involved in long-term synaptic plasticity in molluscs (Bravarenko et al., 2006; Cohen-Armon, 2008; De Lisa et al., 2012).

Analysis of the B. glabrata genome revealed the presence of an extensive gene set for apoptosis with a potential regulatory role in apoptosis and innate immunity (Adema et al., 2017). Wang et al. (2017) observed a reduction of the oxysterol-binding protein-related protein 9 within the nervous system of infected B. glabrata. This protein was found to interact with S. mansoni p53 apoptosis stimulating protein that facilitates survival and development of Schistosoma (Han et al., 2013). Indeed, Ghoname et al. (2017) recorded a down-regulation of p53 levels in the tissues of B. alexandrina that failed to produce S. mansoni infection compared to susceptible snails that showed an up-regulation of p53 levels. The antioxidant system of B. alexandrina is also suggested to contribute to infection-induced apoptosis. The generation of reactive oxygen species during infection account for the loss of mitochondrial membrane potential and for phosphatydylserine exposure at the cell surface membrane suggesting the execution of an intrinsic apoptotic pathway (Russo and Madec, 2007). Moreover, oxidative stress breaks DNA and activates PARP which leads to inflammation, apoptotic and necrotic cell death (Langelier and Pascal, 2013).

A possible mechanism by which infection-induced apoptosis contributes to parasitic castration is through a reduction of neurons responsible for the production of neurotransmitters and neuropeptides important for egg laying and other aspects of reproduction in B. alexandrina. Rizk et al. (2018) showed that apoptosis in the digestive glands of Pirenella conica infected with Heterophyes spp. can contribute to dopamine and serotonin reduction during infection. Biogenic monoamines control many physiological functions in molluscs including reproduction. Serotonin and dopamine are abundant in the nervous and peripheral tissues of Biomphalaria and their distributions suggest their participation in the Biomphalaria-Schistosoma interaction (Delgado et al., 2012; Vallejo et al., 2014). Serotonin and dopamine, and other catecholamines were found in large quantities in the nervous system and albumen gland of B. glabrata, whilst serotonin and its metabolites were predominantly abundant in ovotestis (Santhanagopalan and Yoshino, 2000). In vitro assessment of the possible role of serotonin and dopamine on the function of albumen glands, the organ responsible for perivitelline fluid and galactogen synthesis, showed their ability to stimulate the discharge of secretory proteins. This stimulation was inhibited when a dopamine receptor antagonist (chlorpromazine) was added to the culture. The same protein secretion was stimulated upon exposure to exogenous serotonin suggesting that these amines play essential roles in regulating reproduction in Biomphalaria spp. (Boyle and Yoshino, 2002).

Induction of apoptosis in the nervous and ovotestis tissues of B. alexandrina following infection with S. mansoni may be responsible for loss of neurons responsible for the production of these important transmitters leading to deterioration of the reproductive capacity of the snails. Indeed, both serotonin and dopamine were significantly reduced in the nervous tissues of S. mansoni-infected B. glabrata at 14 dpi and L-dopa was decreased at 21 dpi (Manger et al., 1996). The role of serotonin in reproduction was confirmed when B. glabrata, infected and uninfected, were exposed to exogenous source of serotonin. Repeated treatment initiated ovulation and oviposition. Uninfected-treated snails laid more eggs than uninfected, untreated ones. Moreover, infected treated snails showed egg-laying rates comparable to both serotonin-treated and untreated, uninfected snail groups, thus reversing the castrating effects of larval infection. Thus, depletion of serotonin, and possibly other biogenic amines, may be involved in parasitic castration in Biomphalaria (Manger et al., 1996).

In B. alexandrina, numerous neuropeptides that belong to the neuroendocrine system of other pulmonates were localized in the cerebral ganglia. These include egg laying hormone, APGWamide and FMRFamide, all of which are involved in the reproduction of snails (Acker et al., 2019). The above-mentioned peptides were significantly reduced in the nervous system of B. glabrata during pre-patent infection with S. mansoni suggesting their role in parasite-induced reproductive castration (Wang et al., 2017).

4.3. Steroid hormones

The concentrations of steroid hormones, testosterone and estradiol in the CNS and OT of Biomphalaria were reduced during the course of infection. Steroid hormones were previously reported in numerous molluscs including B. alexandrina (Oehlmann and Schulte-Oehlmann, 2003; Croll and Wang, 2007; Omran, 2012; Ragheb et al., 2018). Changes in testosterone and estradiol were reported in the clam Sinonovacula constricta during its reproductive cycle (Yan et al., 2011). The hormonal reduction observed in the present study may contribute to fecundity loss in infected snails. For example, testosterone administration prompted the production of male secondary sex characteristics in the castrated slugs Euhadra peliomphala (Takeda, 1980). In other molluscan species, administration of testosterone, estradiol, and progesterone stimulated spermatogenesis and oogenesis in the gonads (Sakr et al., 1992; Wang and Croll, 2004).

Numerous authors related parasitic castration to chemical manipulations of the host’s neuroendocrine system by parasite larvae Joosse and Van Elk, 1986; Joosse et al., 1988; Hurd, 1990; De Jong-Brink, 1995). Developing larvae reduce the gonad volumes and retard its growth leading to an alteration in the hormonal homeostasis and subsequent inhibition of egg production (Bayne and Loker, 1987). Schistosomin, a peptide produced by the nervous system of snails following schistosome infection, interferes with the host’s neuroendocrine systems impacting the synthetic activity of albumen glands by inhibiting the action of one of the reproductive hormones (De Jong-Brink, 1995).

5. Conclusion

The results of the present study indicate that the reduction in egg production of S. mansoni-exposed B. alexandrina may occur as a result of many interrelated factors that include a series of biochemical changes. For example, infection-induced apoptosis in the CNS and OT may be responsible for reduction in hormones and enzymes in infected B. alexandrina. Likewise, deterioration of antioxidant systems and increased lipid peroxidation may lead to cell death. Castration may also be caused by a reduction in the number of cells responsible for producing important monoamines involved in reproduction.

Acknowledgement

This work received funding from the National Academy of Sciences (NAS; USA) - Science and Technology Development Fund (STDF, Egypt) joint fund: 2000007152 (USA) & USC17-188 (Egypt) and National Science Foundation (NSF; USA): OISE 1545803.

Footnotes

Compliance with ethical standards

All the institutional guidelines were considered during conducting the current study. In addition, the study did not include any human subjects.

Declaration of competing interest

The authors declare that they have no competing interests.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.exppara.2020.107887.

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