echinococcus granulosus causative agent of cystic echinococcosis is an important zoonotic worm with global distribution , hygiene and economic importance . the relative disease is prevalent in middle east and many part of our country , iran ( 1 - 4 ) . cultivation of this parasite is very valuable for improvement of different branches of medico - veterinary researches . although many advances have been done with cultivation methods , fertilization is a missing link for in vitro life cycle of e. granulosus ( 5 - 7 ) . regarding previous observations , self - fertilization is considered to be a normal way for sperm transfer in natural intestinal worms but despite several experimental techniques , it has never been seen in cultivated worms ( 6 , 8) . in addition , cross - fertilization has never seen even in intestinal parasite , although theoretically it is possible ( 8 , 9 ) and rarely has been suggested by molecular studies ( 10 , 11 ) . during daily follow up of cultivated worms ( 12 ) , we succeeded to detect some evidences of behavior similar to self - mating ( type 2 ) and cross - mating which can be very important to further understanding of the biology and speciation of the parasite and subsequent researches . here hydatid cysts with more than 80% fertile protoscoleces ( psc ) were collected from infected sheep at shiraz abattoirs , cut opened under aseptic conditions , passed through two layers sterile gauze , washed three times with sterile pbs and then cultured in s10e.h culture media using the same procedures described before with some modifications ( 6 , 12 , 13 ) . all stages of parasite growth were observed during a long term assay ( about two months ) from the first day of growth to adult forms , using an inverted microscope . after around 55 days post cultivation , 50 - 60% of initially pscs were developed to adult worms with at least 3 proglottids in culture flasks . genital pore also was opened and closed rhythmically special in latest segment of some of them . during daily control of the culture flasks we succeeded to detect some behavior similar to mating in reared e. granulosus . in a survey second segment of an individual worm connected to third one of another worm ( fig 1 : a & b ; movie : 1 ) . it was considered that the connection has occurred between cirrus of a worm and genital pore of another one . furthermore , in another observation an individual worm has attempted to close its third segment to the first segment of the same worm ( fig 1 : c & d ; movie.2 ) . although these observations did not cause that we find mature eggs in culture flasks but these evidences may confirm positive potential of the e.granulosus to cross - mating / fertilization . initial efforts were carried out since 1926 and 1928 by dv to grow pscs in cyst fluid ( 14 , 15 ) . smyth succeeded to introduce in vitro cultivation of different larval stages of this parasite ( 16 ) . he also cultivated sexually mature strobilate of this parasite from psc ( 5 ) . despite within several years many advances have been done with cultivation method but fertilization is missing link for in vitro life cycle of e. granulosus ( 5 - 7 ; 12 - 19 ; 21 ) . presence of male and female reproductive systems and common genital pore shows the parasite has potential for both cross and self - fertilization ( 9 ) . probably self - fertilization is an advantage for this small worm that it might be difficult to find another worm particularly in light infections ( 22 ) . this phenomenon has been observed in e. granulosus collected from naturally and experimentally infected definitive host ( 8 , 9 ) . in sections derived of dog worms , the cirrus was inserted into the vagina and morphologically has been proved seminal receptacle of intestinal worms filled with spermatozoa ( 8) . ( self - fertilization has also confirmed by some molecular studies in dog worms(10 , 11 , 23 ) . although cross - fertilization has not been observed in sections obtained of dog intestine ( 8) some researchers have suggested that aggregative behavior observed in intestinal worms are probably as a result of attraction between individual worms ( 24 ) . limited molecular reports have suggested probability of this phenomenon ( 10 , 11 ) while others have rejected it ( 23 ) . the major difference between sexually mature cultured and dog worms , was the failure of insemination to occur in the reared worms in culture media ( 25 ) . in the well - developed genitalia of the mature reared adult worm many spermatozoa were seen in testes , but uterus filled with immature ova and empty seminal receptacle has always been observed ( 6 ) . in present study , we have frequently observed active proglottids with protruded cirrus . genital pore was also opened and closed rhythmically special in latest segment , the situation that has also observed in previous investigations ( 6 , 13).we also succeeded to detect some behavior similar to mating between the same and different worms although mature eggs did not find . our finding may be confirming positive potential for cross - fertilization as it was confirm with detection early stages of the development of a shelled egg in the uterus cavity of a monozoic/ vesicular worm cultured in vitro ( 20 ) . according to the movie number 1(movie 1 ) , we speculate some cultivated parasites show tendency to  cross - mating  in culture which could be due to lack of suitable conditions for self - fertilization . especially the large number of the parasite in culture media may be an advantage in which the small worms can find others . another our finding is confirmed this theory that insemination between different proglottids of the same strobila is highly unlikely to occur ( 9 ) . according to movie number 2(movie 2 ) , we believe the worms have tendency to unsuccessful mating between their individual proglottids . kumaratilake et al , have believed the type ( 2 ) of self - fertilization is unsuccessful because no two proglottids of the same strobila are ever at the same stage of maturation ( 9 ) . in addition to kumaratilake s opinion , we are believed that short stature and low number of the segment are important reasons of failure in connection and then mating / fertilization between two strobila segments of an individual worm . . extremely complexity of requirements for fertilization may cause this physiologic function can not dissolve production of fertile egg . regarding the above observations , these parasites show tendency to unsuccessful self - mating / fertilization ( type 2 ) which failure could be due to anatomical position and physiological maturation . also lack of suitable conditions for self - fertilization causes the worms try to do unsuccessful cross - mating / fertilization in culture media .
backgroundechinococcus granulosus cultivation is very important for improvement of different aspect of medical and veterinary researches . despite many advances in this case , 
 there is a missing link for in vitro life cycle of adult worms and it is fertilization . regarding the researchers observations , self - fertilization can be done in worms living in dog intestine , but despite all sorts of experimental techniques 
 , this phenomenon has never been observed in reared worms in culture media . 
 furthermore , cross fertilization has not been observed in vitro and even in parasites with dog intestinal origin ; although it theoretically is possible . during a follow - up of cultivated adult worms , evidences of behaviors similar to self - mating ( type 2 ) and cross - mating 
 were observed in our lab which will be presented here.methodsprotoscoleces were aseptically removed from sheep hydatid cysts , washed twice with pbs and then cultivated in s.10e.h culture medium . 
 the stages of parasite growth were observed using an inverted microscope for two months and all stages and behaviors were microscopically photographed . 
 different movies have also been made from these behavioral features.resultsafter around 55 days post cultivation , some evidences of behaviors similar to self - mating ( type 2 ) and cross - mating were observed in some of the mature adult worms . 
 however , fertile eggs in these parasites have never been observed.conclusionregarding the above observations , these parasites show tendency to unsuccessful self - mating / fertilization ( type 2 ) which failure could be due to anatomical position and physiological maturation . also lack of suitable conditions for self - fertilization causes the worms try to do unsuccessful cross- mating / fertilization in culture media