
 parasitological study - laboratory breeding scheme for 
 snails - the snails used in this study were originally obtained from water 
 channels running in alexandria governorate ( egypt ) and were maintained for two years in 
 the parasitology department , faculty of medicine , alexandria university , egypt .
the 
 snails were housed in glass aquaria containing snail - conditioned water and were fed 
 lettuce leaves , tetramine fish food and calcium carbonate , under suitable environmental 
 laboratory conditions , using the method described by eveland and haseeb ( 2011 ) . 

source and maintenance of the parasite - s. 
 mansonicercariae were originally harvested from shedding b. 
 alexandrinasnails naturally infected with an egyptian strain of s. 
 mansoni and collected from alexandria water channels .
maintenance of the 
 s. mansoni life cycle was conducted between our laboratory snails 
 and swiss strain albino mice ( el naga et al . 

groups of 10 snails were placed in 200 ml beakers containing aged 
 de - chlorinated tap water ( dtw ) under direct sunlight for 2 h. each cercarial suspension 
 was carefully shaken to obtain an even mixture and the average number of cercariae per 
 ml was estimated .
mice were infected with 100 cercariae / mouse using the paddling 
 tail - immersion technique according to the method described by dettman et al .
all work with laboratory animals was 
 conducted in accordance with the egyptian national animal welfare standards and was 
 approved by the ethical committee of the faculty of medicine , alexandria university . at 
 seven weeks post - infection ( p.i . )
, mice were sacrificed and their livers were used as a 
 source of parasite eggs .
the snails were exposed individually to eight-10 vigorously swimming freshly 
 hatched miracidia under direct sunlight for 3 - 4 h and then were kept in the dark and 
 maintained under previously described conditions ( el 
 naga et al .
2010 , eveland & haseeb 
 2011 , mostafa & el - dafrawy 
 2011 ) . 

selection of susceptible and resistant snail stocks - separation of 
 resistant and susceptible isolates was achieved according to zanotti - magalhes ( 1997 ) . for the selection of resistant 
 individuals , snails that remained uninfected after exposure to infection
susceptible individuals were selected by isolation 
 of snail progeny that yielded high infection frequencies ( f1 ) and were then 
 reared singly for self - reproduction ; these progeny were selected as the experimental 
 susceptible group ( zanotti - magalhes et al . 

experimental design - two groups ( f2 ) were used in our 
 experiments : 400 susceptible snails ( group i ) and 400 resistant snails ( group ii ) .
each 
 group was further subdivided equally into two subgroups : 200 young susceptible ( subgroup 
 ia ) and 200 young resistant ( subgroup iia ) snails were infected at the age of two months 
 ( before egg laying was initiated ) , at the size of 3 - 4 mm in diameter .
another 200 adult 
 susceptible ( subgroup ib ) and 200 adult resistant ( subgroup iib ) snails were infected at 
 the age of four months ( after egg laying was initiated ) , at the size of 8 - 10 mm in 
 diameter .
afterwards , the infected snails of each subgroup were returned to separate 
 plastic aquaria and kept in darkness .
two weeks later , 100 snails from each experimental subgroup were subjected to enzyme 
 assay testing .
the other 100 snails in each subgroup were used for the determination of 
 parasitological parameters , starting from the 28th day post - exposure , twice weekly for 
 three weeks .
1:a scheme showing the followed experimental design with regards to the 
 parasitological parameters . 
 

2:a scheme showing the followed experimental design with regards to 
 cytosolic superoxide dismutase enzyme assay . 
 

determination of susceptibility and resistance of individual snails of the four 
 subgroups - at four weeks post - exposure to the parasite , the snails from 
 each experimental subgroup were individually checked for cercarial shedding twice 
 weekly , repeatedly for three weeks .
the susceptible and nonshedding snails of each 
 subgroup were separated and returned to different containers . on the first day of 
 detecting cercariae , the susceptible snails in each subgroup
were isolated individually 
 in small transparent plastic containers , each in 100 ml of aged dtw , and were kept in 
 darkness throughout the shedding period .
each of the containers contained only one 
 susceptible snail and was labelled with the experimental subgroup , the snail number , the 
 pre - patent period ( ppp ) and the number of shed cercariae at each shedding assessment 
 ( zanotti - magalhes et al .
nonshedding individuals from each subgroup were returned together into a separate 
 aquarium and were tested twice weekly for cercarial shedding until the 49th day p.i . at 
 the end of the three weeks dedicated to shedding
, nonshedding snails were examined under 
 the dissecting microscope to make sure there are no arrested developmental stages 
 present . moreover ,
if any snail died during the ppp , it was squeesed between two slides , 
 entirely dissected and inspected under a stereo - dissecting microscope to detect 
 developing sporocysts , which appear as opaque discrete masses within the snail tissue 
 ( corra & paraense 1971 , niemann & lewis 1990 ) .
each shedding snail in all experimental subgroups was investigated for the following 
 parasitological parameters . 

ppp - ppp is defined as the period from the day of snail exposure to 
 miracidia to the day immediately before the first shedding of cercariae ( haroun 1996 , yousif 
 et al .
starting from the 28th day post - exposure , all subgroups were 
 examined twice weekly until the 49th day post - exposure ; each snail was tested 
 individually . 

infection rate ( ir ) - the snail ir was calculated by dividing the 
 number of shedding and positive crushed snails by the number of exposed snails .
the 
 percentage of susceptible and resistant snails in each subgroup was then determined 
 ( frandsen 1979 , yousif et al .
the snail ir was calculated at the end of the 
 experiment using the following equation : 
 mean cercarial output ( mco ) - on the first day of cercariae detection , 
 positive snails were separated individually and placed in plastic cups .
the cercariae 
 shed from each susceptible snail were counted under a stereomicroscope twice weekly for 
 three successive weeks ( chu & dawood 1970 , 
 frandsen 1979 ) . 

mortality rate ( mr ) - the number of dead snails for each subgroup was 
 counted starting from the day of exposure to the parasite until the 49th day p.i .
the 
 number of dead snails in the breeding aquaria was recorded twice weekly . according to 
 yousif
( 1998 ) , the mr was calculated 
 using the following equation : 
 sod1 enzyme assay - one hundred snails from each experimental subgroup 
 were used for the enzyme assay .
each snail was cleaned gently , dissected and examined under a 
 stereomicroscope to detect developing sporocysts .
snails that did not show any 
 sporocysts under the microscope were considered to be resistant .
two control groups were used in the biochemical study : uninfected young snails and 
 uninfected adult snails .
the soft tissue of the dissected snail was perfused with normal saline , blotted on 
 tissue paper and weighed .
the tissue was homogenised with phosphate - buffered saline ( ph 
 7.2 ) by sonication for 2 min in a glass test tube ; to prevent temperature elevation to 
 protect the enzymes present , the tube was placed in a beaker containing ice .
the 
 homogenised tissue was centrifuged at 500 g for 4 min and the 
 supernatant containing the proteins was stored in aliquots at -20c until use .
the collected supernatants were used for an sod1 
 enzyme assay . the protein concentration for each homogenate supernatant
the sod1 activity was 
 determined by adding the substrate of the enzyme to the snail tissue homogenate and 
 measuring the increase in absorbance over time during the reaction with a 
 spectrophotometer according to todd and gomez 
 ( 2001 ) .
a sod1 enzyme assay kit , purchased from biodiagnostic ( egypt ) , was 
 used and relies on the ability of sod1 to inhibit the phenazine methosulphate - mediated 
 reduction of nitroblue tetrazolium .
calculation : where  a control = the change in absorbance at 560 nm over 5 min following the addition 
 of 5-methylphenazinium methyl sulfate ( pms ) ( r4 ) to the reaction mixture in the absence 
 of the sample ,  a sample = the change in absorbance at 560 nm over 5 min following the 
 addition of pms to the reaction mixture in the presence of the sample . 

statistical analysis - data were analysed using the ibm spss software 
 package v.20.0 ( kirkpatrick & feeney 2013 ) . 

qualitative data are described using a number and percent and quantitative data are 
 described using the range ( minimum and maximum ) , mean , standard deviation and median . 

comparisons between the different studied subgroups were tested using the chi - square 
 test .
when more than 20% of the cells showed an expected count of less than 5 , 
 correction for chi - square was performed using monte carlo correction .
the distributions 
 of quantitative variables were tested for normality using the kolmogorov - smirnov test , 
 the shapiro - wilk test and the dagstino test .
when a normal data distribution was revealed , parametric tests were 
 applied ; if the data were abnormally distributed , nonparametric tests were used . for 
 normally distributed data , comparisons between the different studied subgroups were 
 analysed using the f - test ( anova ) and the post - hoc test ( scheffe ) for pairwise 
 comparisons .
the 
 significance of the obtained results was judged at the 5% level ( kotz et al .
ethics - all work with laboratory animals was conducted in accordance 
 with the egyptian national animal welfare standards and was approved by the ethica 
 committee of the faculty of medicine , alexandria university .

 parasitological study - ppp - snails of each subgroup were individually 
 examined for cercarial shedding twice weekly , starting from the 28th day after exposure 
 to infection until the 49th day ( table i ) . 

table inumber of shedding snails in each of the examined pre - patent period ( ppp ) 
 days in the studied subgroupsppp ( n = 
 100)day 
 28th32nd36th40th44th49thia372716500iia2 
 3 
 5 
 79 
 11 
 ib11 
 14 
 16 
 17 
 9 
 7 
 iib0 
 0 
 0 
 0 
 0 
 0 
  
 79.451 
 45.965 
 23.200 

< 0.001 
 < 0.001 

 p < 0.001 

 p < 0.001 

 a : significance with subgroup ia ( young susceptible ) ; 
 b : significance with subgroup iia ( young resistant ) ; 
 c : significance with subgroup ib ( adult susceptible ) ; 
 d : statistically significant at p  0.05 ; iib : adult 
 resistant subgroup ; mc : monte carlo test ; n : number of members in each 
 subgroup ;  : value for chi - square test . 
 
 a : significance with subgroup ia ( young susceptible ) ; 
 b : significance with subgroup iia ( young resistant ) ; 
 c : significance with subgroup ib ( adult susceptible ) ; 
 d : statistically significant at p  0.05 ; iib : adult 
 resistant subgroup ; mc : monte carlo test ; n : number of members in each 
 subgroup ;  : value for chi - square test .
the maximum number of shedding snails on the 28th day among the four subgroups was 
 observed in the young susceptible subgroup ( ia ) .
these differences were demonstrated 
 among the susceptible young ( ia ) and susceptible adult ( ib ) subgroups at all studied 
 time points , except on the 36th day . in contrast , there were no significant differences 
 between the young resistant ( iia ) and adult resistant ( iib ) subgroups at the earlier 
 time points .
significant differences were found at the 40th , 44th and 49th days . additionally , significant differences between the young subgroups ia and iia at all the 
 time points , except on the 40th day , were also observed .
furthermore , significant 
 differences were found between the susceptible and resistant adult subgroups ( ib and 
 iib ) on all days of shedding . 

ir % - the percentage of susceptible and resistant snails in each 
 subgroup was determined .
irs were 92% , 74% , 37% and 0% in the subgroups ia , ib , iia and 
 iib , respectively .
statistically significant differences among the susceptible and 
 resistant groups at different snail ages and among young and adult subgroups with 
 different compatibility patterns were evident ( table 
 ii ) . 

table iiinfection rate ( ir ) among the studied subgroups  iaiiaibiib 
 pir ( % ) 9237740200.315 
 < 0.001 
 sigia - iia , ia - ib , ia - iib , ib - iia , ib - iib , iia - iib 
 -- 
 a : statistically significant at p  0.05 ; ia : young 
 susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant 
 subgroup ; iib : adult resistant subgroup ; sig : significance between subgroups 
 using chi - square test (  ) . 
 

a : statistically significant at p  0.05 ; ia : young 
 susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant 
 subgroup ; iib : adult resistant subgroup ; sig : significance between subgroups 
 using chi - square test (  ) . 

total cercarial production ( tcp ) and mco per susceptible snail - the 
 total number of cercariae produced by the susceptible snails in subgroups ia , ib and iia 
 was counted twice weekly over a period of three weeks .
the mean number of cercariae shed 
 by each susceptible snail in the different studied subgroups was calculated and is 
 presented in tables iii , iv .
the young susceptible subgroup ( ia ) yielded the highest mco , at 
 298.1  132.13 ( tables iii , iv ) . 

table iiicercarial roduction in the studied subgroupssubgroupsshedding snails 
 ( n)tcpmcoia85151,002298.1  132.13iia379,87744.49  32.35ib7441,73294.13  44.17iib000.0  0.0ia : young ceptible bgroup ; ib : adult ceptible bgroup ; iia : young resistant 
 subgroup ; iib : adult resistant subgroup ; mco : mean cercarial output ( mean 
 number of cercariae / snail / shedding time ) ; tcp : total number of 
 cercariae / subgroup .
ia : young ceptible bgroup ; ib : adult ceptible bgroup ; iia : young resistant 
 subgroup ; iib : adult resistant subgroup ; mco : mean cercarial output ( mean 
 number of cercariae / snail / shedding time ) ; tcp : total number of 
 cercariae / subgroup .
table ivmean cercarial shedding over three successive weeks per susceptible snail 
 among the studied subgroups  iaiiaibiibmin - max97 - 59418 - 17320 - 2140 - 0mean  sd298.1  132.144.5  32.3594.13  44.20.0
 0.0median271.033.5090.500.0f test234.333 
 p < 0.001 
 sigia - iia , ia - ib , ia - iib , iia - ib , ib - iib , iia - iib 

 a : statistically significant at p  
 0.05;b : statistically significant at p  0.001 ; ia : young 
 susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant 
 subgroup ; iib : adult resistant subgroup ; sig : significant difference between 
 subgroups using post - hoc test ( scheffe ) .
normally distributed data were 
 expressed in mean  standard deviation ( sd ) and was compared using f test 
 ( anova ) . 
 

a : statistically significant at p  
 0.05;b : statistically significant at p  0.001 ; ia : young 
 susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant 
 subgroup ; iib : adult resistant subgroup ; sig : significant difference between 
 subgroups using post - hoc test ( scheffe ) .
normally distributed data were 
 expressed in mean  standard deviation ( sd ) and was compared using f test 
 ( anova ) . 

mr % - the number of dead snails for each subgroup was counted from the 
 day of exposure to infection until the 49th day p.i .
the mr for each subgroup was 47% , 
 35% , 27% , 14% for subgroups
a positive correlation between both infection and 
 mrs of each subgroup was demonstrated , with mortality increasing with infection ( table vi ) . 

table vmortality rate ( mr ) among the studied subgroups at the different studied 
 pre - patent periods with compatibility status of the dead snailsmr 
 ( % ) day 
 28th32nd36th40th44th49thia ( 47)7 ( 7s)02 ( 2s)5 ( 5s)14 ( 14s)19 ( 19s)iia ( 27)2 ( 2r)3 ( 3r)3 ( 1r , 2s)5 ( 2r , 3s)6 ( 2r , 4s)8 ( 4r , 4s)ib ( 35)0 
 03 ( 3s)6 ( 6s)11 ( 10s , 1r)15 ( 13s , 2r)iib ( 14)1 ( 1r)1 ( 1r)2 ( 2r)3 ( 3r)3(3r)4(4r) 
 27.085 
 11.897 
 0.4101.059.368 
 13.461 
 p <
0.001 

 p = 0.015 

 p = 1.000 
 p = 0.8740.025 
 0.004 

 a : significance with subgroup ia ( young susceptible ) ; 
 b : significance with subgroup ib ( adult susceptible ) ; 
 c : statistically significant at p  0.05 ; iia : young 
 resistant subgroup ; iib : adult resistant subgroup ; mc : monte carlo test ; r : 
 resistant ; s : susceptible ;  : value for chi - square test . 
 
 a : significance with subgroup ia ( young susceptible ) ; 
 b : significance with subgroup ib ( adult susceptible ) ; 
 c : statistically significant at p  0.05 ; iia : young 
 resistant subgroup ; iib : adult resistant subgroup ; mc : monte carlo test ; r : 
 resistant
table vicorrelation between infection rate ( ir ) and mortality rates ( mr ) in the 
 studied subgroupscorrelationir ( % ) mr ( % ) pia9247 < 0.001 
 iia37270.130ib7435 < 0.001 
 iib014 < 0.001 
 r
( p)0.983 ( 0.017)- 
 a : statistically significant at p  0.05 ; ia : young 
 susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant 
 subgroup ; iib : adult resistant subgroup ; p : p - value for chi - square test ; r : 
 pearson coefficient . 
 

a : statistically significant at p  0.05 ; ia : young 
 susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant 
 subgroup ; iib : adult resistant subgroup ; p : p - value for chi - square test ; r : 
 pearson coefficient . 
 enzyme assay - the sod1 enzyme activities in the noninfected ( young and 
 adult ) subgroups were 0.26  0.04 and 0.30  0.03 u / g tissues , respectively . in the 
 infected subgroups ,
the highest level of sod1 enzyme activity was obtained in the adult 
 resistant subgroup ( iib ) , with a mean of 0.90  0.07 u / g tissues , whereas the lowest 
 enzyme activity was in the susceptible individuals of the young susceptible subgroup 
 ( ia ) , at 0.32  0.03 .
the enzyme activity was higher in the resistant snails , regardless 
 of their age , in comparison to susceptible and noninfected snails .
furthermore , the sod1 
 activity was higher in the adult subgroups than in the young subgroups ( table vii ) . 
 table viithe level of cytosolic superoxide dismutase enzyme ( sod1 ) per snail s gram 
 tissue in u / g tissues among the studied subgroupssod1 
 ( u / g)cacbia sia riia siia rib sib riibmin - max0.20 - 0.310.25 - 0.350.25 - 0.350.43 - 0.630.34 - 0.450.66 - 0.830.21 - 0.440.61 - 0.730.79 - 0.99mean  sd0.26  0.040.30  0.030.32  0.030.55  0.070.40  0.030.76  0.050.34  0.070.68  0.040.90  0.07median0.260.300.320.580.400.770.360.670.91f 
 - 

 - 

 - 

 - 
 209.018 


- 

 - 

 - 

 - 
 p 
 - 

 - 

 - 

 - 
 < 0.001 

 - 

 - 

 - 

 - 
 sig--- 
 b - d 

 b , c , e 

 b - f 

 e , g 

 b - f , h 

 b - i 

 a : statistically significant at p  
 0.05;b : significance with group uninfected 
 young;c : significance with group uninfected adult ; d : 
 significance with subgroup ia ( young susceptible 
 members);e : significance with subgroup ia ( young resistant 
 members ) ; f : significance with subgroup iia ( young 
 susceptible members ) ; g : significance with subgroup iia 
 ( young resistant members ) ; h : significance with subgroup ib 
 ( adult susceptible members);i : significance with subgroup 
 ib ( adult resistant members ) ; ca : young noninfected ; cb : adult noninfected ; 
 ia r : young susceptible subgroup ( resistant members ) ; ia s : young 
 susceptible subgroup ( susceptible members ) ; ib r : adult susceptible subgroup 
 ( resistant members ) ; ib s : adult susceptible subgroup ( susceptible members ) ; 
 iia r : young resistant subgroup ( resistant members ) ; iia s : young resistant 
 subgroup ( susceptible members ) ; sig : significance between subgroups was done 
 using post - hoc test ( scheffe ) .
normally distributed data were expressed in 
 mean  standard deviation ( sd ) and was compared using f test ( anova ) . 
 

a : statistically significant at p  
 0.05;b : significance with group uninfected 
 young;c : significance with group uninfected adult ; d : 
 significance with subgroup ia ( young susceptible 
 members);e : significance with subgroup ia ( young resistant 
 members ) ; f : significance with subgroup iia ( young 
 susceptible members ) ; g : significance with subgroup iia 
 ( young resistant members ) ; h : significance with subgroup ib 
 ( adult susceptible members);i : significance with subgroup 
 ib ( adult resistant members ) ; ca : young noninfected ; cb : adult noninfected ; 
 ia r : young susceptible subgroup ( resistant members ) ; ia s : young 
 susceptible subgroup ( susceptible members ) ; ib r : adult susceptible subgroup 
 ( resistant members ) ; ib s : adult susceptible subgroup ( susceptible members ) ; 
 iia r : young resistant subgroup ( resistant members ) ; iia s : young resistant 
 subgroup ( susceptible members ) ; sig : significance between subgroups was done 
 using post - hoc test ( scheffe ) .
normally distributed data were expressed in 
 mean  standard deviation ( sd ) and was compared using f test ( anova ) .
infection of biomphalaria species by the larval stage of s. 
 mansoni leads to an intimate , long - term association that largely challenges 
 the snail s physical integrity and severely compromises its biological fitness ( sandland et al .
although the infective stage 
 ( miracidia ) is small relative to the host , once inside the host s body , miracidia 
 initiate major developmental changes to support their extensive growth and 
 transformation to more specialised larval stages ( de jong - brink 1995 ) .
based on a postulation of the short life span of miracidium 
 in water , which at the same time gives rise to hundreds of cercariae , schistosome 
 development inside its snail intermediate host can be considered the weakest link in the 
 parasite s life cycle . additionally ,
although a given parasite strain can only develop 
 within a compatible snail , no such discrimination occurs in the human host .
therefore , 
 snail control is highly effective in eliminating schistosomiasis and is also easier than 
 changing human habits . for these reasons , searching for snail factors that prevent 
 parasite development will help in designing tools that can be utilised for controlling 
 schistosomiasis ( knight et al .
2014 ) . genetic background and the ids are snail - related factors that are known to affect the 
 compatibility of biomphalaria to s. mansoni ( richards 1975 , negro - corra et al . 2007 ) .
age - dependent variability in susceptibility to 
 s. mansoni has been studied in b. glabrata(richards 1984 , spada 
 et al .
2010 ) 
 and it was found that the number of genes controlling resistance differs with the age of 
 the snail , which in turn affects the compatibility pattern ( richards 1977 , richards et al . 

moreover , the number of genes controlling resistance varies 
 amongbiomphalaria species ( richards 
 1977 , richards et al .
2005 ) , which highlights the importance 
 of studying the effect of age on the genetic outcome of b. 
 alexandrina . 

these cells produce the enzyme cytosolic sod1 , an important enzyme that 
 catalyses the conversion of o2- to h2o2 .
the 
 h2o2 produced by biomphalaria snails kills 
 s. mansoni through its potential oxidative damage , thereby affecting 
 snail susceptibility ( bender et al .
goodall et al . ( 2006 ) found that sod1 enzyme 
 expression in b. glabrata differs among susceptible and resistant 
 snails .
however , the effect of the snail s age on the activity of this enzyme has not 
 been evaluated . within this context ,
the current work examined the effect of the age of b. 
 alexandrina snails on their genetic outcome and on the activity of sod1 ( as 
 a representative of ids ) .
the present work was conducted using approaches involving both 
 parasitological and biochemical techniques and was achieved by including 200 snails in 
 each of four experimental subgroups : young susceptible ( ia ) , adult susceptible ( ib ) , 
 young resistant ( iia ) and adult resistant ( iib ) . in each subgroup ,
100 snails were 
 dedicated for parasitological examination that began four weeks post - exposure to 
 miracidia and continued for the following three weeks .
the other 100 snails were used 
 for the biochemical study ( sod1 enzyme assay ) , which was performed at two weeks 
 post - exposure to infection .
moreover , the effect of the interaction of snail age , genetics and internal defence on 
 the outcome of infection was also examined .
our results revealed that younger snails , belonging to either a susceptible or resistant 
 group , showed higher susceptibility when compared to adult snails .
young susceptible 
 snails exhibited the shortest range of ppp , with the majority of shedding snails being 
 recorded by the 28th day , at 37 of 85 shedding snails .
moreover , the remainder of 
 shedding snails in the same subgroup showed their first shedding before the 40th day 
 p.i . :
27 snails shed cercariae for the first time on the 32nd day , 16 snails shed on the 
 36th day and five snails shed on the 40th day . regarding the adult susceptible subgroup ( ib ) , of 74 shedding snails , only 11 shed for 
 the first time on the 28th day p.i .
the maximum number of shedding snails was 16 and 17 
 recorded by the 36th and the 40th days , respectively .
( 2011 ) stated that high snail compatibility to 
 trematode infection is characterised by a short ppp .
the significant differences 
 observed between subgroups ia and ib with regard to shedding durations indicate that 
 subgroup ia ( young susceptible ) carries higher susceptibility to s. 
 mansoni infection , accompanied by rapid development of the parasite inside 
 the snail . as for subgroup iia ( young resistant ) shedding members , the maximum number of shedding 
 snails for the first time was 11 that were recorded by the 49th day with only two snails 
 shed for the first time by the 28th day .
conversely , the snails belonging to the adult 
 resistant subgroup iib displayed only the resistant phenotype , with no recoded ppp . 
 subgroup iia ( young resistant ) showed a significant difference compared to subgroup iib 
 ( adult resistant ) , which contained only resistant individuals , indicating higher 
 susceptibility of the former .
these significant differences between subgroups ia and ib 
 and between iia and iib can be attributed to the effect of age .
it was not surprising that the compatibility pattern also affected the outcome of 
 infection , as evidenced by the significant differences between subgroups ia and iia and 
 between subgroups ib and iib .
moreover , the significant differences recorded between 
 young resistant , susceptible snails and adult susceptible , susceptible snails ( iia and 
 ib ) may be attributed to the effect of the interaction between age and genetics on 
 compatibility patterns .
this was represented by a longer ppp in the susceptible 
 individuals in subgroup iia than in snails of the aforementioned adult susceptible 
 subgroup . in the current study
, some individuals of subgroups iia and ib displayed a ppp of 49 
 days .
the delay in parasite development in these snails indicates their lower 
 susceptibility to infection . a comparison between the young resistant ( iia ) subgroup and 
 adult susceptible ( ib ) subgroup revealed that 11 snails shed for the first time on the 
 49th day in the former group , but only seven in the latter .
however , this difference was 
 not statistically significant , indicating that both age and compatibility pattern are 
 important in determining the outcome of infection . 

richards ( 1984 ) stated that genetic factors 
 determining b. glabrata juvenile nonsusceptibility operate throughout 
 the snail s life , masking the presence of the factors for adult susceptibility and , 
 thus , unfortunately complicating studies on the influence of age on snail 
 susceptibility .
the highest ir in the present study was exhibited by the snails belonging to the young 
 susceptible subgroup ia . in total , 92% of subgroup
ia ( young susceptible ) , 74% of 
 subgroup ib ( adult susceptible ) and 37% of subgroup iia ( young resistant ) were 
 susceptible .
it should be 
 noted that even the snails that died during the course of the experiment were included 
 in the statistical analysis ; these snails were examined for developing sporocysts .
although our experiment was carried out on snails resulting from self - reproduction , some 
 resistant snails were obtained in the progeny of susceptible individuals , indicating the 
 dominance of the resistance character in b. alexandrina . in their 
 studies , both shoukry et al .
( 2010 ) observed the appearance of 
 resistant snails originating from cross of susceptible parents .
a probable explanation 
 for the resistant members obtained in the susceptible subgroups is that , although 
 resistance alleles are sometimes not expressed in the snail phenotype , they can 
 accumulate through generations to produce a resistant phenotype that appears in later 
 progeny .
that is , the parents of these resistant snails harboured unexpressed resistance 
 genes , which subsequently accumulated among successive generations ( el naga et al .
similarly , the resistance 
 character in b. glabrata snails is inherited in a dominant manner 
 ( richards & merritt 1972 , lewis et al .
abdel - hamid et al . ( 2006 ) and el - nassery et 
 al . (
2013 ) found that both susceptibility and resistance in b. 
 alexandrina are hereditary characters and are genetically controlled . in contrary to lewis et al .
( 2002 ) , who found 
 that susceptible b. glabrata parents did not give rise to any resistant 
 progeny , susceptible subgroups in the current study resulted in 8 - 26% resistant 
 individuals in the two age groups .
( 2010 ) that a susceptible population 
 of b. alexandrina may contain more resistance alleles than b. 
 glabrata , thus accounting for the appearance of resistant progeny 
 originating from completely susceptible parents .
although the two susceptible subgroups 
 contained resistant snails , the significant difference noted between them suggests that 
 the resistant alleles obtained from f1 parents were potentiated by the impact 
 of age , resulting in the appearance of more resistant individuals in the adult 
 susceptible subgroup ( ib ) . in the current work ,
this result was also observed in other studies using different species 
 ( lewis et al .
however , 37 susceptible members were recorded in our 
 young resistant subgroup ( iia ) , which reinforces our assumption on the effect of the age 
 of b. alexandrina on snail compatibility pattern .
the appearance of 
 susceptible individuals in the young resistant subgroup could also be explained by its 
 weaker internal defence when compared to the adult resistant subgroup .
this was proven 
 by the significant difference noted in sod1 enzyme activity between resistant snails in 
 the young and adult resistant subgroups , at 0.68  0.04 and 0.90  0.07 u / g tissues , 
 respectively . 

anderson et al . ( 1982 ) attributed the lower irs of 
 adult snails compared to younger snails to a number of different factors . first
, older 
 snails possess physical barriers to penetration imposed by their tough thickened body 
 covering .
additionally , they produce greater quantities of mucus , which replete with 
 chemicals that confuse searching miracidia .
older snails also move faster and are thus more difficult to locate by 
 miracidia .
furthermore , egg - laying capacity could be among the factors causing more 
 resistance in adult snails , wherein the snail expends all its energy on reproduction , as 
 stated by richards ( 1977 ) .
our main goal was to ascertain whether resistant alleles transmitted from susceptible 
 parent snails to their young progeny are more functional at the adult stage and our 
 results suggest that they are .
we propose that resistance alleles in the susceptible 
 population may become more functional through the snail aging process , which is 
 responsible for the lower susceptibility in adult b. 
 alexandrinasnails .
the highest susceptibility in the young susceptible subgroup was also evidenced by the 
 highest tcp among the four studied subgroups , at 151,002 .
this was followed by the adult 
 susceptible subgroup , which produced 41,732 cercariae over the three weeks of shedding . 

according 
 to the frandsen classification , the three subgroups can be categorised in classes 4 , 2 
 and 1 ( well compatible , poorly compatible and not very compatible , respectively ) . 
 regarding the adult resistant subgroup ,
no cercariae were produced at all , placing this 
 subgroup in frandsen class 0 or resistant snails ( frandsen 1979 ) .
the recorded mrs in the present study were 47% , 35% , 27% and 14% in subgroups ia , ib , 
 iia and iib , respectively and were directly proportional to the irs in the different 
 subgroups .
the higher mrs noted in the susceptible compared to the resistant groups 
 could be explained by the devastating effects of s. mansoni on snail 
 health .
s. mansoni is considered a parasite that exerts a selective 
 pressure on the susceptible population . during its development ,
s. mansoni 
 sporocysts invade biomphalaria s digestive gland and the 
 adjacent gonads , leading to destruction of these organs ( lemos & andrade 2001 ) .
thus , infection becomes an element of natural 
 selection , which is highly negative for infected snails ( rosa et al .
moreover , the disturbance in metallic ion concentrations 
 due to trematode infection is considered another cause of mortality in parasitised 
 snails ( mostafa & dajem 2010 ) .
furthermore , 
 the exposure of susceptible snails to a high dose of infection ( 8 - 10 miracidia ) per 
 snail at the same time may explain the higher mortality recorded in the laboratory , as 
 the majority of these miracidia are thought to develop without much restraint . in 
 contrast , under natural conditions , the snail is not likely to be exposed to such a high 
 number of miracidia at a given time ( corra & 
 paraense 1971 ) .
the mrs recorded in the two resistant subgroups were 35% and 14% , subgroups iia ( young 
 resistant ) and iib ( adult resistant ) , respectively .
the mortality of the resistant 
 snails can be attributed to a high concentration of the sod1 enzyme , which produces 
 h2o2 , a highly ros .
although these reactive species are 
 beneficial for combating the invading parasite , they also seriously damage biological 
 membranes in the host tissue , the so - called " cost of resistance " ( bender et al . 2007 , bonner et al . 

2012 ) ; hence , ros are considered a double - edged sword ( beckman & ames 1998 ) .
( 2010 ) stated that exposure alone of b. alexandrina 
 snails to s. mansoni miracidia has a negative impact on snail 
 survival rates . in the present work ,
we functionally investigated the activity of the sod1 enzyme in a 
 whole - snail soft tissue homogenate at two weeks after exposure to infection .
for the 
 control noninfected snails , the results were 0.26  0.04 and 0.30  0.03 u / g of sod1 
 activity in the noninfected young and adult groups , respectively .
cytosolic sod1 is present 
 during the course of the snail lifetime to protect cells against damage by the free 
 radicals that are generated due to normal metabolism ( bender et al . 2005 ) .
regarding the results of the experimental subgroups in the current study , the combined 
 effects of both age and compatibility pattern resulted in the highest sod1 activity in 
 the adult resistant subgroup , with a mean of 0.90  0.07 u / g tissue .
in contrast , the 
 lowest enzyme activity was found in the susceptible snails of the young susceptible 
 subgroup , at 0.32  0.03 u / g tissue .
furthermore , as shown intable vii , the sod1 activities were higher in the resistant 
 individuals of the studied subgroups than in the corresponding susceptible individuals . 

this indicates the importance of this enzyme in determining a snail s compatibility 
 status .
our results of higher sod1 activity levels in resistant vs. susceptible snails 
 are in agreement with hahn et al .
( 2004 ) , mahmoud and rizk ( 2004 ) , bender et 
 al .
( 2005 ) and bayne ( 2009 ) , who 
 reported an increased number of haemocytes generating superoxide in s. 
 mansoni resistant snails .
the resulting free radicals were efficient for 
 encapsulating or eliminating the parasite in resistant snails , but failed in the case of 
 susceptible snails ( mahmoud & rizk 2004 ) .
it is worth mentioning that studies on the genes of a resistant b. 
 glabrata strain have revealed that polymorphisms in the gene encoding 
 cytosolic sod1 are associated with susceptibility / resistance ; such results reinforce the 
 role of sod1 in schistosome resistance ( goodall et al . 

the results of the present work showed that the age of b. alexandrina 
 snails affects sod1 activity levels .
significant differences in sod1 activity levels 
 between young and adult snails obtained from the same ( f1 ) parent were 
 observed , with a higher activity at the adult stage .
goodall et al . ( 2006 ) reported thatbiomphalaria sod1 is 
 encoded by three alleles and one was found to be significantly associated with 
 resistance .
furthermore , some alleles controlling compatibility were found to be 
 affected by snail age ( richards 1977 , richards & shade , richards et al .
, it would be interesting 
 to assess whether the genes encoding sod1 are among the other genes that are modified by 
 snail age . in summary ,
the significant differences in all parasitological and biochemical 
 parameters between young and adult snails in the same group were attributed to the age 
 effect , which made resistance alleles more functional in adults , with higher sod1 enzyme 
 activity . moreover , the complex interaction between age , genetic background and ids 
 between the susceptible and resistant subgroups results in great variability in 
 compatibility patterns .
this interaction led to the appearance of the highest 
 significant difference between the young susceptible ( ia ) and adult resistant ( iib ) 
 subgroups .
the results presented herein can have potential epidaemiological implications 
 inbiomphalaria control . by determining the age
at which a snail is 
 most susceptible to s. mansoni infection , the optimal timing for the 
 application of molluscicides could be identified .
this in turn would increase the 
 efficacy of the applied method , potentiating schistosomiasis control .
moreover , 
 identification of the most resistant snails is important for use in later biological 
 snail control , after studying the compatibility of successive generations .
investigations of genes encoding enzymes involved in the respiratory burst are 
 recommended . in particular , the genes responsible for the production of 
 h2o2 should be targeted as potential candidates for genetic 
 loci causally related to the susceptibility / resistance ofb . 

 of the approximately 34 identified biomphalaria 
 species , biomphalaria alexandrina represents the 
 intermediate host of schistosoma mansoni in egypt . using 
 parasitological and sod1 enzyme assay , this study aimed to elucidate the impact of 
 the age of b. alexandrina snails on their genetic variability and 
 internal defence against s. mansoni infection . 
 susceptible and 
 resistant snails were reared individually for self - reproduction ; four subgroups of 
 their progeny were used in experiment . 
 the young susceptible subgroup showed the 
 highest infection rate , the shortest pre - patent period , the highest total cercarial 
 production , the highest mortality rate and the lowest sod1 activity . among the young 
 and adult susceptible subgroups , 8% and 26% 
 were found to be resistant , indicating 
 the inheritance of resistance alleles from parents . 
 the adult resistant subgroup , 
 however , contained only resistant snails and showed the highest enzyme activity . 
 the 
 complex interaction between snail age , genetic background and internal defence 
 resulted in great variability in compatibility patterns , with the highest significant 
 difference between young susceptible and adult resistant snails . 
 the results 
 demonstrate that resistance alleles function to a greater degree in adults , with 
 higher sod1 activity and provide potential implications for biomphalaria 
 control . 
 the identification of the most susceptible snail age enables 
 determination of the best timing for applying molluscicides . 
 moreover , adult 
 resistant snails could be beneficial in biological snail control . 
MATERIALS AND METHODS
RESULTS
DISCUSSION