New Approach for Controlling Snail Host of Schistosoma mansoni,Biomphalaria alexandrina with Cyanobacterial Strains-Derived C-Phycocyanin

Abstract

Schistosomiasis is one of the major communicable diseases of public health and socioeconomic importance in the developing world. It is a waterborne disease in which Biomphalaria alexandrina snails are known to be the intermediate molluscan host for Schistosoma mansoni: the causative agent of human intestinal schistosomiasis. Therefore, snail control is one of the cornerstones of schistosomiasis control programs. Several methods have been used to eliminate snail hosts. One of these methods is chemical molluscicides, which have undesirable effect to nontarget organisms. Consequently, the search for biologically derived molluscicides to complement the use of synthetic molluscicides is a top priority. In this concern, this study is the first to evaluate the molluscicidal potency of Cyanobacterial Phycocyanin (C-PC) as a virtually untapped source. Laboratory assessment of three freshwater Cyanobacterial strains: Anabaena oryzae SOS13, Nostoc muscorum SOS14, and Spirulina platensis SOS13-derived C-Phycocyanin as a biocontrol agent against freshwater mollusks; B. alexandrina snails were performed. Also, the safety of tested C-PC on nontarget organisms (Tilapia fish) was assessed. Results reveal that C-PC extracted from all tested Cyanobacteria strains showed a promising molluscicidal activity (the mortality rate was 100% at 100 μg/mL concentration). Out of the examined strains, A. oryzae SOS13 phycocyanin was found to be the most potent strain (LC₅₀ and LC₉₀ were 38.492 and 49.976 μg/mL, respectively). Moreover, C-PC extracts from all tested strains have been found to be safe to Tilapia fish as the survival rate was 100% at the effective molluscicidal concentrations. We can conclude that C-PC extracts are the first promising microbial biopesticides for the control of freshwater B. alexandrina snails.

Introduction

Schistosomiasis, the second-most widespread human parasitic sickness after malaria, is caused by trematodes in the genus Schistosoma (Chitsulo et al. 2000). It is endemic in Egypt and responsible for about 70% of chronic liver diseases in adults and 35% of liver diseases in children (El-Khoby et al. 2000). Schistosoma mansoni (Sambon 1907), the causative agent of human intestinal schistosomiasis, which has a complex life cycle, uses the freshwater snail Biomphalaria alexandrina (Mollusca: Planorbidae) as an intermediate host which is prominent in Upper and Lower Egypt (WHO 2002). Also, it could infect rodents (D'Andrea et al. 2000) and wild primates, particularly baboons (Müller-Graf et al. 1997, Munene et al. 1998). One of the most successful strategies for eradicating schistosomiasis is to disrupt the parasite life cycle through control of the snail host (Jolcelyn 2004). Control efforts to date have largely focused on diminishing snail populations through physical, chemical, or biological means (Bakry and Hamdi 2006, Halima et al. 2006, Radwan and El-Zemity 2007, Duval et al. 2015). Physical and chemical molluscicides have several drawbacks. Both are costly, have unintended effects on nontarget organisms, and snails are becoming more and more resistant to synthetic chemical molluscicides (Andrews et al. 1982, Clearwater et al. 2008, Oliveira-Filho et al. 2010). Consequently, the search for biologically derived molluscicides to complement the use of synthetic molluscicides is a top priority. In the interest of this, new approaches for screening compounds and extracts from plants, bacteria, fungi, and algae are needed.

Cyanobacteria are a diverse group of oxygenic photosynthetic prokaryotes that occur worldwide in marine, aquatic, and terrestrial habitats. Anabaena and Nostoc are the major genera whose members are found in plants. Cyanobacteria stand out as one of the most promising groups of organisms able to produce useful bioactive compounds. Recently, these compounds have received much attention from researchers and companies because of their potential applications in various life science fields. These secondary metabolites show a broad spectrum of biological activity, including antibacterial, antitumor, antiviral, antifungal, antioxidant, antiprotozoal, and molluscicidal effects (Yang et al. 2006, Abdel-Raouf and Ibraheem 2008, Mostafa and Gawish 2009, Guedes et al. 2011, Shanab et al. 2012, Niedermeyer 2015). Phycocyanin is a main photosynthetic accessory pigment among the phycobiliprotein pigments in cyanobacteria, and is of incredible significance due to its various biological properties. Cyanobacterial phycocyanin (C-PC) exists in many species, such as Anabaena, Nostoc, and Spirulina platensis, all of which display high C-PC contents (Eriksen 2008). Few studies have been done to screen cyanobacteria for molluscicidal activity (Falch et al. 1992, Shanab et al. 2005, Mostafa and Gawish 2009, Pereira et al. 2011). In fact, there are no previous studies on the efficacy of C-PCs as molluscicides against any type of snails. Therefore, this study is the first evaluation of the molluscicidal potency of C-PC.

We isolated and tested the molluscicidal efficacy of C-PC extracts from three species of cyanobacteria: Anabaena oryzae SOS13, Nostoc muscorum SOS14, and S. platensis SOS13, against the intermediate host of S. mansoni, B. alexandrina snails.

Materials and Methods

Snails

Ten laboratory-bred B. alexandrina snails (10–12 mm in shell diameter) were obtained from the Medical Malacology Department at Theodor Bilharz Research Institute, Al Warraq, Giza, Egypt. Snails were reared in aqueous dechlorinated medium and fed on green lettuce ad libitum and allowed to reproduce in a laboratory of Parasitology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt, for a long period of time before the experiment (Liang et al. 1987).

Cyanobacteria preparation

Cyanobacterial culture preparation

Cyanobacterial strains: A. oryzae SOS13, N. muscorum SOS14, and S. platensis SOS13, were previously isolated from rice fields in Sharkia Governorate, Egypt (Ali 2012) and identified based on morphological and physiological characteristics (Salem et al. 2011). These strains were genetically identified by sequencing fragments of the 16S ribosomal RNA gene and comparing them with sequences from the GenBank (Salama et al. 2015).

Phycocyanin extraction and purification

C-PCs were extracted from cultured Anabaena, Nostoc, and Spirulina using the modified methods of Sarada et al. (1999). Ten-day-old Anabaena, Nostoc, and Spirulina cultures were used to inoculate media in each of three 500 mL Erlenmeyer flasks (10% v/v). Anabaena and Nostoc were cultured in 250 mL of BG⁰11 medium. Spirulina was cultured in 250 mL of Zarrouk synthetic medium (Zarrouk 1966). The inoculated flasks were incubated at 26°C ± 2°C for 30 days under continuous illumination (600–800 lux) using a 36 W white fluorescent lamp. After 30 days, cyanobacterial cells were collected by centrifugation at 5000 ×g for 20 min at 20°C. Cell pellets were separated and washed with 1 M Tris–HCl buffer (pH 8.1). One volume of the washed cell mass was resuspended in five volumes of the same buffer and subjected to a freeze–thaw cycle (freezing at −50°C and thawing at 25°C). The resultant suspension was centrifuged at 14000 rpm for 10 min, and the supernatant (the crude extract) was kept in the refrigerator. Pigment absorption was measured at 615 and 652 nm in a JENWAY—England 6405 UV/VIS Spectrophotometer against 0.05 M phosphate buffer as a blank. Final purification of C-PC was then carried out using an ammonium sulfate fractionation method according to Soni et al. (2006).

Evaluation of the molluscicidal activity of C-PC extract

Five concentrations, 20, 30, 40, 50, and 100 μg/mL, were prepared from each C-PC extract. For each experimental concentration, 10 B. alexandrina snails were submerged in 1 L of phycocyanin solution for 48 h. Snails were then washed and immersed in dechlorinated water and allowed to recover for 24 h. A set of control snails was prepared using dechlorinated water and run parallel to all phycocyanin treatments. Three replicates were used for each concentration (10 snails each). The mortality percentage was calculated. Mortality rates were recorded and corrected according to Abbott (1925). Computation of the median lethal concentrations (LC₅₀) and LC₉₀ values was determined through probit analysis, within 95% confidence limits, using the Propan program.

Evaluation of C-PC extracts effect on nontarget organism

Tilapia fishes were exposed to C-PC extracts to assess the effects of these potential molluscicides on nontarget organisms. Ten Tilapia fishes were exposed to each of the aforementioned concentrations of the three C-PCs, and mortality rates were observed. The exposure period was 48 h and the recovery period was 24 h.

Results

The molluscicidal potency of C-PC extract

The preliminary screening of phycocyanins extracted from three freshwater cyanobacteria, A. oryzae SOS13, N. muscorum SOS14, and S. platensis SOS13, was done to evaluate their molluscicidal efficacy against B. alexandrina snails. All the tested C-PC extracts proved to have molluscicidal properties against target snails (the mortality rate was 100% at concentration of 100 μg/mL) with varying degrees of potency. It was evident that B. alexandrina snails were greatly affected when exposed to certain concentrations of C-PC extract (Table 1). According to the results of LC₅₀, the best results in terms of toxicity to snails were exhibited by Anabaena, followed by Nostoc and Spirulina. There is a rapid increase in the mortality rate of snails as they were exposed to increasing concentrations of A. oryzae SOS13 phycocyanin (Table 1). The LC₅₀ and LC₉₀ values of C-PC extracts against B. alexandrina snails are listed in Table 2. The median lethal values of extracts from Anabaena, Nostoc, and Spirulina (LC₅₀) against tested snails were 38.492, 49.358, and 58.278 μg/mL, respectively, whereas the respective LC₉₀ values were 49.976, 69.693, and 77.010 μg/mL. Based on the LC₅₀ and LC₉₀ values, the results clarified that C-PC extracts from three freshwater cyanobacteria stains, A. oryzae SOS13, N. muscorum SOS13, and S. platensis SOS13, have a molluscicidal activity, with Anabaena extract being the most potent.

Table 1.

Effect of Tested Cyanobacterial Phycocyanin Against Biomphalaria alexandrina Snails

C-PC concentration (μg/mL)	Anabaena oryzae SOS13 No. of dead snails (%)	Nostoc muscorum SOS14 No. of dead snails (%)	Spirulina platensis SOS13 No. of dead snails (%)
20	0 (00.00)	0 (00.00)	0 (00.00)
30	3 (10.00)	0 (00.00)	0 (00.00)
40	18 (60.00)	10 (33.33)	2 (6.67)
50	29 (96.67)	13 (43.33)	6 (20.00)
100	30 (100.00)	30 (100.00)	30 (100.00)

Total number of tested snails for each concentration is 30.

C-PC, cyanobacterial phycocyanin.

Table 2.

LC₅₀ and LC₉₀ of Tested Cyanobacterial Phycocyanin Extract Against Biomphalaria alexandrina Snails (Confidence Limits at 95%)

C-PC	LC₅₀ (μg/mL) 95% CI	LC₉₀ (μg/mL) 95% CI	Slope
Anabaena oryzae SOS13	38.492	49.976	(11.302 ± 1.209) μg/mL
Nostoc muscorum SOS14	49.358	69.693	(8.554 ± 0.903) μg/mL
Spirulina platensis SOS13	58.278	77.010	(10.588 ± 1.184) μg/mL

LC, lethal concentrations.

Effects of C-PC extract against a nontarget organism

C-PC extracts from all tested strains have been found to be safe to the nontarget organisms (Tilapia fish) as the survival rate was 100% at different concentrations (20, 30, 40, 50, and 100 μg/mL).

Discussion

Schistosomiasis remains a public health problem in many developing countries. In addition to disease prevention through chemotherapy (WHO 1993), these countries must apply efficient strategies for the control of disease transmission. Consequently, effective use of molluscicides is among the best potential strategies for mollusk control (Abou-El-Naga 2013). The undesirable effects of chemical molluscicides on the nontarget organisms and escalating levels of resistance have caused the scientists to search for a safe ecofriendly counterpart to synthetic molluscicides. Various biopesticides and biological control agents have been tested, including bacteria, fungi, plant extracts, and snail predators (Bakry 2009, Molla et al. 2013, Duval et al. 2015, Abd El-Ghany and Abd El-Ghany 2017, Younes et al. 2017). Screening for safe and effective microbial pathogens for the control of snail hosts was a major aspect of WHO recommendations for future research (WHO 1984). Few such studies have been performed (de Oliveira et al. 2004, Gamalat et al. 2011, Duval et al. 2015, Abd El-Ghany and Abd El-Ghany 2017).

Despite the recognized potential for cyanobacteria to provide a wealth of useful bioactive compounds, the assessment of cyanobacteria for molluscicidal activity has not received much attention. However, few studies have been conducted to measure their molluscicidal activity. For example, the cyanobacterium, Phormidium valderianum, was found to be toxic to B. alexandrina and Bulinus truncates snails (Shanab et al. 2005), both of which are vectors of schistosomiasis. Byproducts of S. platensis were observed to have molluscicidal effects on B. alexandrina snails (Mostafa and Gawish 2009). This study is considered the first assessment on the potential activity of C-PC extracts from three cyanobacteria strains: A. oryzae SOS13, N. muscorum SOS14, and S. platensis SOS13, as molluscicides against B. alexandrina snails. Among the tested C-PC extracts, Anabaena showed the highest molluscicidal activity against target snails, followed by Nostoc, then Spirulina. The examined Anabaena strain was previously identified as A. oryzae SOS13 (Salama et al. 2015) and its phycocyanin extract exerted antibacterial effects against B. cereus, S. aureus, Escherichia Coli, and Klebsiella pneumonia (Sitohy et al. 2015). Our results demonstrated that A. oryzae SOS13 had a highly lethal effect of 60%, 96.67%, and 100% at concentrations of 40, 50, and 100 μg/mL, respectively, against the target snails. Consequently, Anabaena phycocyanin extracts proved to be the most potent molluscicides (LC₅₀ and LC₉₀ were 38.492 and 49.976 μg/mL, respectively) examined in this study.

Furthermore, N. muscorum SOS14 phycocyanin extracts were examined for their molluscicidal impact, and the mortality rate was found to be 33.33%, 43.33%, and 100% at concentrations of 40, 50, and 100 μg/mL, respectively, and the survival rate was 100% at concentration of 20 μg/mL. Our observation is in contrast with finding by Jaki et al. (2000).

Byproducts of S. platensis have previously proven to have molluscicidal effects on B. alexandrina snails. The mortality rate against the B. alexandrina snails was 100% at 2% concentration of S. platensis culture filtrate after 24 h exposure. LC₅₀ and LC₉₀ were 0.20% and 0.23%, respectively (Mostafa and Gawish 2009). These observations were in agreement with this study, which demonstrated that the mortality rate from exposure to S. platensis SOS13 phycocyanin extract was 6.67%, 20%, and 100% at concentrations of 40, 50, and 100 μg/mL, respectively. C-PC extracts from Spirulina maxima have been tested as antibacterial agents against Streptococcus sp., Staphylococcus sp., E. coli, Bacillus sp., and Pseudomonas sp. (Muthulakshmi et al. 2012).

Previous studies have evaluated the safety of phycocyanin extract in some experimental models. They found that LD₅₀ values were greater than 3 g/kg taken orally for rats and mice, and no mortalities were observed (Romay et al. 1998). Also, Song et al. (2012) studied the chronic toxicity of phycocyanins on Sprague Dawley rats for 12 weeks. Results showed no adverse effects on routine blood serum indicators and organ/bodyweight rate. These findings were in agreement with our results, which suggested low toxicity of phycocyanin extracts in a nontarget organism (Tilapia fish) at effective molluscicidal concentrations (100 μg/mL) with no observed mortality. This suggests that the use of C-PC extracts from three fresh cyanobacteria strains: A. oryzae SOS13, N. muscorum SOS13, and S. platensis SOS13 could be a safer and ecofriendlier alternative to synthetic chemical pesticide use.

The phytochemical constituents of the S. platensis culture filtrate are alkaloids, saponins, and total phenolic compounds, all of which may have molluscicidal properties (Abd El-Baky et al. 2009). Essack et al. (2014) pointed out that the molluscicidal activity of cyanobacteria was due to four compounds: barbamide, tanikolide, cyanolide A, and comnostin B. Barbamide has a molluscicidal activity toward the snail, Biomphalaria glabrata (LC₁₀₀ = 10 μg/mL) (Orjala and Gerwick 1996). Also, comnostin B isolated from Nostoc commune showed molluscicidal activity against B. glabrata, with a minimum inhibitory concentration of 20 μg/mL (Jaki et al. 2000). Therefore, the high molluscicidal activity of C-PC extracts from tested strains in this study could be due to one or multiple chemicals. Further studies will be required to elucidate the selective active components against snail hosts.

Conclusions

In an attempt to find a new, highly effective biopesticide against the snail intermediate host of S. mansoni, we have tested phycocyanin extracts from three cyanobacteria species: A. oryzae SOS13, N. muscorum SOS14, and S. platensis SOS13 for their molluscicidal potency. The LC₅₀ values suggest that most of the tested cyanobacteria have good potential for future use in schistosomiasis control. C-PC extracts are the first promising microbial biopesticides for the control of freshwater B. alexandrina snails. Further studies are required to elucidate the active component or compounds specific to snail hosts, with special attention on Anabaena C-PC extract (the most active in this study), and to use them effectively.

Footnotes

Author Disclosure Statement

No conflicting financial interests exist.

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