Cytological and histochemical criteria for evaluating development of trematodes and pseudophyllidean cestodesin vivoandin vitro

Abstract

1. Maturation of the plerocercoid of the pseudophyllidean cestodeDiphyllobothriumsp. and the metacercaria of the strigeid trematodeDiplostomum phoxinifollow a similar pattern, which may be divided into the following phases: (1) cell multiplication; (2) segmentation or body shaping; (3) organogeny; (4) early gametogeny; (5) late gametogeny; (6) egg-shell formation and vitellogenesis; (7) oviposition.2. Cytological or histochemical criteria have been developed by means of which each developmental phase may be recognized. Developmentin vitromay be considered comparable with that occurringin vivoif the same phases are completed with the same incubation time and without the appearance of cytological abnormalities.3. The cell multiplication phase is characterized by high mitotic activity. This activityin vitrocan be evaluated by incubating larvae after 24 hr. culture for a standard time (5 hr.Diphyllobothrium; 4 hr.Diplostomum) in 10⁻⁴colchicine and comparing the number of mitoses seen in a defined area of an aceto-orcein squash with those found in tissue maturedin vivofor the same period. Use of this criterion enables the growth-producing properties of a medium to be assessed after 24 hr. culture.4. Phases 2–4 are characterized by the appearance of the tubular genitalia, the rudiments of which are readily recognized in aceto-orcein squashes or whole mount preparations.5. The presence of spermatids and mature spermatozoa (phases 4–5) may be detected in aceto-orcein squashes or in fresh tissue teases. The quantity and activity of spermatozoa also serve as important criteria.6. Recognition of phase 6, egg-shell formation, is carried out by means of diazo reagents which give orange or orange-red reactions with the phenolic egg-shell precursors in the vitellaria, as seen in whole mount preparations. Abnormal egg-shell development is reflected in the failure of vitellaria to give a normal diazo reaction.7. The appearance of eggs which are macroscopically normal constitutes the final maturation phase. Early detection of eggs (especially abnormal ones) is facilitated by the use of the diazo reaction or the catechol test for polyphenol oxidase.8. Embryonation and hatching of eggs to produce morphologically normal infective coracidia or miracidia serve as a final criteria.9. Criteria for the maintenance of the adult condition are also considered.