One important aspect of reproductive development in trematode parasites is the formation of a hardened eggshell which allows the zygote to develop into a miracidium in a hostile environment. The miracidium then can transfer the germline from the vertebrate host to snail intermediate host. Schistosome parasites, unlike other trematodes, have separate sexes and female reproductive development is known to depend on the presence of a male parasite. These facts make the blood flukes ideal material to study the mechanisms that underlie female reproductive development and eggshell formatian. We reasoned that the morphological and biochemical differences between the male and female must be reflected at the molecular level in the differential expression of sexually regulated genes. Radioactive single stranded cDNA was first transcribed from female RNA; and then sequences common to both male and female were removed by hybridization to an excess of male RNA. This probe was used to screen a cDNA library made from mRNA of adult worm paris. One hybridizing clone, pSMf 61-46, was shown to correspond to a 0.9 kilobase mRNA that is present only in mature female worms and is not detectable in female schistosomes from single-sex infections, in male worms or in eggs. Thus expression of the gene was female-specific. During normal bisexual infection this mRNA is first detected 28 days after infection (the time of worm pairing) and increases to a high level at 35 days postinfection, coinciding with egg production. Thus the temporal expression of the gene was dependent on paining with male worm. The nucleotide sequence of the gene shows an open reading frame that encodes a 16 kDA polypeptide that shows strong homology with eggshell proteins on insects. A second female-specific cDNA clone, F-4, represents a 1.6 kilobase mRNA whose expression is also correlated with worm pairing and subsequent egg production, encodes a different putative eggshell component of 44 kDA. The amino acid composition of the 16 kDA and 44 kDA polypeptides show a strong correlation with the actual amino acid composition of the schistosome eggshell. Thus these two polypeptides appear to the major components of the schistosome eggshell. Analysis of the genomic arrangement of the eggshell genes show that pl6 is represented by 5 gene copies and p48 is represented by 2-5 copies. The eggshell genes are expressed in the vitelline cell as recently demonstrated by in hybridization and immunocytochemical localization. The eggshell genes are being expressed in bacteria. The gene products will be used to study the biochemistry of eggshell formation.
