Genetic Variation between Biomphalaria alexandrina Snails Susceptible and Resistant to Schistosoma mansoni Infection

Much effort has been made to control schistosomiasis infection in Egypt. However, enduring effects from such strategies have not yet been achieved. In this study, we sought to determine the genetic variability related to the interaction between Biomphalaria alexandrina snails and Schistosoma mansoni. Using RAPD-PCR with eight (10 mers) random primers, we were able to determine the polymorphic markers that differed between snails susceptible and resistant to Schistosoma mansoni infection using five primers out of the eight. Our results suggest that the RAPD-PCR technique is an efficient means by which to compare genomes and to detect genetic variations between schistosomiasis intermediate hosts. The RAPD technique with the above-noted primers can identify genomic markers that are specifically related to the Biomphalaria alexandrina/Schistosoma mansoni relationship in the absence of specific nucleotide sequence information. This approach could be used in epidemiologic surveys to investigate genetic diversity among Biomphalaria alexandrina snails. The ability to determine resistant markers in Biomphalaria alexandrina snails could potentially lead to further studies that use refractory snails as agents to control the spread of schistosomiasis.


Introduction
Schistosomiasis is the second most common parasitic cause of mortality in tropical countries, second only to malaria, and has been a target for increased control by the World Health Organization [1]. Great effort has been made to control the transmission of the disease. However, there is little evidence that the prevalence of the disease has decreased globally. Indeed, it continues to spread to new geographic areas [2]. Of the countries in which neglected tropical diseases are prevalent, Egypt is one of the many counties that have suffered greatly under the burden of tropical diseases, including schistosomiasis [3]. Schistosoma infection is one of the most significant public health problems facing the country.
Human infection with Schistosoma mansoni is closely related to the existence of its intermediate snail host of the genus Biomphalaria. Biomphalaria alexandrina is the only snail host in Egypt [4,5]. This snail has extended its distribution from the Nile Delta and is now present throughout the country along the tributaries of the Nile. By 1979, B. alexandrina had colonized the Nile from the Delta to Lake Nasser [6]. Recently, B. alexandrina snails have invaded the water sources in some reclaimed areas, which has resulted in Schistosoma infection in previously uninfected populations and will eventually lead to an increase in schistosomiasis transmission throughout Egypt [7]. The situation may be further complicated by the increasing number of irrigation projects whose goal is to improve the Egyptian economy along with the increasing mobility of infected agricultural workers who migrate to neighboring areas [8,9]. Schistosomiasis is a specific target for control by the Egyptian Ministry of Health, international agencies, and by the World Bank. This has been done via the mass administration of the medication praziquantel to human populations in conjunction with the use of niclosamide for snail control. However, schistosomiasis control has not yet been achieved [10]. With the appearance of praziquantel resistance among schistosomes [11] and the absence of an available vaccine for use [12], the use of alternative control strategies is necessary. The high degree of specificity of schistosomes for their intermediate snail host led Coelho et al. [13] to suggest a control strategy based on the use of snails resistant to parasitic infection as biological competitors of existing susceptible snails in endemic areas. However, the application of this strategy requires an indepth understanding of the role of genetics in the interaction between parasites and snails.
Newton [14], a pioneer in the study of the genetics of Biomphalaria glabrata, considered the susceptibility or resistance of a snail to infection to be a heritable characteristic. Resistance is found to be a dominant character in many Biomphalaria spp. However, some loci of susceptible snails could carry a certain amount of resistance alleles [15,16]. The increase in the number of inherited resistant genes could lead to an increased immune response to the parasite and decrease the degree of susceptibility to infection [16][17][18].
Resistance in Biomphalaria snails has been found to be age dependent. In B. glabrata, the resistant phenotype in juvenile snails is controlled by four genes, each of which has multiple alleles, while in adulthood, there is only a single dominant gene that determines this trait [19,20]. In B. tenagophila, two dominant genes determine resistance [17]. The susceptibility and resistance of B. alexandrina, as with other Biomphalaria spp., were found to be heritable characteristics, with the resistance trait being dominant [16,21]. However, the number of genes that determine this characteristic has not yet been identified.
One of the factors limiting progress in understanding the molecular genetics of B. alexandrina relative to other Biomphalaria species has been the lack of specific sequence information for the application of polymerase chain reaction (PCR). An alternative powerful and highly applicable technique that does not require prior sequence information is the random-amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Unlike conventional PCRbased analyses, this approach uses oligonucleotide primers of an arbitrary sequence to start DNA strand synthesis under conditions of low stringency. Each primer allows for the amplification of specific fragments that are randomly distributed throughout the genome of the snail, thus allowing for unbiased comparison [22,23]. Many investigators have used this technique to distinguish between susceptible and resistant phenotypes in different Biomphalaria species due to its high applicability [24][25][26][27]. In this study, we sought to determine the genetic variability related to the susceptibility and resistance of B. alexandrina snails to S. mansoni using the RAPD-PCR technique with different primers.

Snails Stocks.
All snails originated from water channels in the Alexandria governorate (Egypt). They were maintained for one year in our laboratory under suitable environmental conditions in glass aquaria containing snail-conditioned water, and they were fed lettuce leaves and calcium carbonate.
The parasite strain was originally obtained from shedding cercariae from naturally infected B. alexandrina snails collected from Alexandria water courses. Maintenance of S. mansoni life cycles was conducted between the snails and Swiss strain albino mice. Separation of Schistosoma resistant and susceptible snail stocks was performed according to the method described by Zanotti-Magalhães et al. [28], which is as follows: 100 juvenile snails (four to six mm in diameter) were used. Each snail was exposed for four hours to eight freshly hatched miracidia of S. mansoni. Susceptibility was monitored weekly from four up to 10 weeks postexposure through cercariae shedding which was favored by lamp light. Nonshedding snails were reexposed individually to eight miracidia/snails. Any snails in which the cercareia were observed were considered infected, and those that did not shed cercariae after two exposures were considered uninfected. The infection rate was calculated.
Snails that remained uninfected after two exposures were isolated and reared singly for self-reproduction under laboratory conditions. Their progeny was the resistant stock. Snails that yielded high infection rates were isolated and reared singly for self-reproduction, and their progeny was the susceptible stock. Thirty days post infection, six snails from each stock were used in the present study.

DNA Extraction.
Snail tissue (both susceptible and resistant) was dissected, and the head-foot of each snail was preserved in absolute ethanol for the extraction of high molecular weight DNA [4,29]. EZ-10 spin column genomic DNA extraction kit for animal tissue (Bio Basic Inc., Canada) was used. Thirty mg of the snail tissue was placed in a 1.5 mL centrifuge tube, and 300 L of Animal Cell Lysis Solution and 20 L proteinase K were added. The mixture was incubated at 55 ∘ C with occasional vortexing until the tissue was completely lysed (at least three hours) and cooled to room temperature. The tube was mixed by vortex for 20 seconds and then centrifuged at 14500 ×g for five minutes. 300 L of the supernatant was then pipetted into an EZ-10 spin column, and 300 L of AB buffer solution was added. The preparation was mixed well and allowed to sit for two minutes. Centrifugation was completed at 1600 ×g for two minutes, and the flow through was discarded. 500 L of the wash solution was added, and spinning was conducted at 6400 ×g for one minute. Washing was repeated, and the flowthrough was discarded. Spinning was completed at 10060 ×g for an additional minute to remove the residual amount of the wash solution. The column was placed into a clean 1.5 mL microfuge tube, and 45 L elution buffer (2.0 mM Tris-HCl pH 8.0-8.5) was added to the central portion of the membrane in the column. The tube was incubated at 50 ∘ C for two minutes. Spinning was conducted at 10060 ×g for one minute to elute DNA from the column.
The quantity of DNA was measured spectrophotometrically by UV absorption at A260 (1.0 OD unit is equivalent of 50 g). The quality of genomic DNA was assessed via an analytical 0.7% agarose gel.   [24,26,31,32], B. tenagophila [25], in both B. glabrata and B. tenagophila [33], and B. alexandrina [21]. To our knowledge, the remaining random primers had not yet been tried in Biomphalaria spp.
The lyophilized primers were purchased from Eurofins (mwg/operon). They were reconstituted by adding sterile water to a final concentration of 100 pmoL/ L distributed in aliquots and stored at −20 ∘ C. The amplification conditions were as follows [21]: the tubes were transferred to the thermal cycler where they were subjected to one cycle of initial denaturation at 95 ∘ C for five minutes, then 40 cycles of 95 ∘ C for one minute, 30 ∘ C for one minute, 72 ∘ C for one minute followed by a final extension cycle at 72 ∘ C for 10 minutes.

Agarose Gel Electrophoresis and Staining.
The PCR products were separated on 2% agarose gel stained with ethidium bromide, visualized on a UV Transilluminator, and photographed by Gel Documentation System (Alpha Imager M1220, Documentation and Analysis System, Canada).

Analysis of Polymorphisms on Agarose Gels.
The genetic variability of the susceptible and resistant strains was determined by analysis of the electrophoretic profiles of the bands visualized on the gels. The identification of polymorphic bands was based on the comparison of the band patterns on the same gel for the susceptible and the resistant strains, and only those detected in all individuals of the same strain were considered polymorphic [24,33]. The similarity coefficient was calculated as described by Dice [34] (Table 1).

Results
The susceptibility or resistance of B. alexandrina snails to S. mansoni infection was studied starting four weeks postinfection and continuing weekly thereafter for up to 10 weeks after miracidia exposure. The obtained results showed that 68% of the examined snails (100 snails) were susceptible, while 32% of the snails were resistant.
In this study, the susceptible and resistant snail lines were selected on the basis of well-characterized resistance/susceptibility phenotypes after uninfected snails were exposed to S. mansoni miracidia twice. The RAPD-PCR method was used to differentiate between susceptible and resistant snail lines of B. alexandrina. A total of eight primers were tested, and polymorphic markers were obtained with five of them (OPA-02, OPB-18, OPC-11, OPD-10, and OPD-18). We found that with these primers, the method was reliable for the identification of stable genomic markers between the two phenotypes, that is, the susceptible and resistant traits within the same snail species. The other three primers, OPA-01, OPA-06 and OPM-04, did not produce any results.
The genomic DNA amplified by using the primer OPA-02 (

Discussion
The infection rate obtained in the present study demonstrates that B. alexandrina is an efficient intermediate host for S. mansoni and consequently plays an important role in schistosomiasis transmission in Egypt. The RAPD-PCR technique was used in the current work to determine the susceptibility/resistant genetic variability of B. alexandrina snails. Eight ten base pair (bp) oligonucleotide primers were each added to an individual sample of DNA, which was then subjected to PCR. The resulting amplified DNA bands were polymorphic segments with different band sizes depending upon the genomic DNA and the primer. The RAPD-PCR products were analyzed using agarose gel, which represents an effective and cheap method [33,35]. However, other authors have used the alternative polyacrylamide gel electrophoresis method [21,33,36].
The results of the current work reveal the low coefficient of similarity between the susceptible and resistant snails studied (Table 1). This may be due to the short period (one year) allotted for snail rearing in the laboratory. In a study by Spada et al. [26] in which snails were reared for 20 years in the laboratory, the authors yielded a high coefficient of similarity. These authors expected low genetic variability of their strains and concluded that laboratory strains undergo much less intense selective pressures than the field isolates have.
Of the eight primers used in the present study, three primers, namely, OPA-01, OPA-06, and OPM-04, whose sequence of oligonucleotides are (5 -CAGGCCCTTC-3 ), snails. However, these primers were efficient in distinguishing genetic variability of the resistant and susceptible lines of B. glabrata and B. tenagophila [24,26,32,33]. These primers might amplify very restricted areas of the genomes of B. glabrata and B. tenagophila specific for resistance or susceptibility, which are not present in B. alexandrina snails. Interestingly, one of these primers, the OPA-01 primer, resulted in high genetic variability between B. alexandrina strains from different provinces in Egypt using the RAPD-PCR technique [37]. The present study showed that with five out of the eight utilized primers, specific bands of different lengths were amplified in the resistant and susceptible phenotype snails. To our knowledge, four of the five utilized primers (OPB-18, OPC-11, OPD-10, and OPD- 18) had not yet been studied with Biomphalaria spp. The fifth primer OPA-02 (5 -TGCCGAGCTG-3 ) had been tried previously with different Biomphalaria species [21,25,26]. In the current study, this primer resulted in three polymorphic bands with nearly 330, 430, and 680 bp in the resistant strains and one band with 730 bp in the susceptible strain. Abdel-Hamid et al. [21] used the same primer on B. alexandrina originating from the Delta region of Egypt to determine markers of resistance. They obtained only one band with 430 bp in the resistant strains, which we also obtained in the current study. The OPA-02 primer was also used by Abdel-Hamid et al. [25] and Spada et al. [26] for the analysis of the genetic variability between resistant and susceptible lineages of B. tenagophila and B. glabrata. Using this primer, the authors were unable to detect any polymorphic markers between resistant and susceptible lines. This marker may be a specific marker distinguishing the resistant and susceptible B. alexandrina from other species.
In the current work, we searched for primers capable of differentiating susceptible and resistant strains. As mentioned above, five of the eight utilized primers were able to amplify specific bands of different lengths between the resistant and susceptible phenotype snails. In addition to the OPA-02 primer, which was used in previous studies, we tried four random primers that, to our knowledge, have not been used before with Biomphalaria species. These primers are OPB-18 (5 -CCACAGCAGT-3 ), OPC-11 (5 -AAAGCTGCGG-3 ), OPD-10 (5 -GGTCTACACC-3 ), and OPD-18 (5 -GAGAGCCAAC-3 ). Each of these primers was able to demonstrate one or more differentiating polymorphic bands in the resistant and susceptible strains in B. alexandrina.
We found that the detection of susceptible and resistant bands using the RAPD-PCR technique is a suitable and efficient methodological approach for the analysis of genetic variability among schistosomiasis intermediate hosts. However, future studies should sequence the PCR products to predict the function of the identified DNA sequences and to assist in explaining the levels of polymorphism reported between the susceptible and resistant snails.
As has been concluded by other researchers, the RAPD-PCR technique was found to be sensitive, fast, and easy to perform [32,38]. Using the RAPD technique with the abovementioned primers can identify genomic markers that are specifically related to the B. alexandrina/S. mansoni relationship in the absence of specific nucleotide sequence information. Consequently, this approach can be used in epidemiologic surveys investigating the genetic diversity of B. alexandrina snails, as it is a time-saving technique when compared to the conventional parasitological methods. The ability to determine resistant markers in B. alexandrina snails could pave the way for further studies based on the use of refractory snails as a means by which to biologically control the transmission of disease, although this issue remains a matter of discussion. However, the use of this approach in concert with other measures could create new strategies for the control of schistosomiasis transmission.

Conflict of Interests
The current authors paid all the expenses of the study, with no financial support from the university or from any other institutions. There are no personal relationships with other people or organizations that could inappropriately influence (bias) the work. No one of the authors of this paper have any direct financial relation with the commercial identities mentioned in the paper that might lead to any conflict of interests for any of the authors.