On the development of body-wall musculature Diplostomum chromatophorum metacercariae (Trematoda: Diplostomidae)

The body-wall musculature of invasive cercariae and metacercariae of Diplostomum chromatophorum at different intervals after the penetration into the experimental intermediate host Cyprinus carpio (1, 3, 5, 6, 7, 10, 12, 20, 34, 40 days) has been investigated with the help of TEM technique. During the first 10 days after the invasion (in conditions of our experiment), the cercarial subtegumental muscle fibres degenerate. These muscles are replaced by newly formed ones. Mass differentiation of myoblasts beneath the tegument was observed in 7-10-day-old metacercariae. Obtained data indicate the metamorphosis of body-wall musculature during the morphogenesis in Diplostomum chromatophorum metacercariae.     

Cultivation of excysted metacercariae of Echinostoma caproni (Trematoda) to ovigerous adults on the chick chorioallantois

Excysted metacercariae of Echinostoma caproni were cultivated on the chick chorioallantoic membrane (CAM) maintained at 38.5 +/- 1 C and a relative humidity of 60-65%. Of 59 6-day-old embryos, each inoculated with 25 metacercariae, 29 (49.2%) were infected 2-12 days postinoculation. The total number of worms recovered from the infected eggs was 163 or 22.5% of the 725 inoculated metacercariae. Eggs contained from 1 to 12 (average 5.6) worms per CAM. Worm length increased rapidly from an average of 0.5 mm at 2 days to about 3.0 mm at 6 days postinoculation. Ovigerous worms first were seen on day 8 PI, but fluke eggs did not develop embryos. Worm development in ovo lagged about 1 day behind that of in vivo worms. One worm maintained for 17 days on 2 successive CAMs reached 6 mm in length, contained about 100 eggs in its uterus, and laid an additional 100 eggs on the CAM surface.     

Growth, development, pathogenicity, and transplantation of Echinostoma caproni (Trematoda) on the chick chorioallantois.

Excysted metacercariae of Echinostoma caproni were cultivated on the chorioallantoic membrane (CAM) of 6-day-old domestic chick embryos for 2-13 days postinoculation. There was no significant difference in the body area of fixed and stained preovigerous worms from the CAM versus those grown in domestic chicks. However, ovigerous worms from the CAM were significantly smaller than those from chicks. Worm development, i.e., gonadal differentiation, uterine curling, vitellinogenesis, ovigerousness, and oviposition, took 1 day longer on the CAM than in the chick. Histopathologic studies of worms attached to the CAM were done on cryostat and paraffin sections stained with hematoxylin and eosin. Some worms attached to the CAM by their collar spines and acetabulum, whereas others penetrated the chorionic epithelium and encapsulated in the mesenchyme. Pathogenicity to the CAM included hyperplasia of the chorionic epithelium, hemorrhagia, reduced fibrocytes and blood vessels, but increased lymphocytes and eosinophils in the mesenchyme. Attempts to transplant 11-day-old CAM worms to new CAMs were unsuccessful.     

Site-finding and pairing of Echinostoma revolutum (Trematoda) on the chick chorioallantois

Site-finding of 14-day-old Echinostoma revolutum from the domestic chick was studied by inoculating single worms into various sites on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. Regardless of the site of inoculation, single worms were attracted significantly to the area of the CAM above the embryo. More worms were found in this site at 24 than at 1 hr postinoculation. Worm-pairing was studied in chick embryos by inoculating 2 worms in separate windows, 2 cm apart. Worm-pairing, i.e., worms in contact or within 5 mm of each other, was very evident at 24 hr. The percentage of paired worms on the CAM above the embryo was considerably less than single worms.     

Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda)

Fried B. and Huffman J. E. 1982. Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda). International Journal for Parasitology12: 427–431. Encysted metacercariae of Sphaeridiotrema globulus were obtained from naturally infected Goniobasis virginica snails in Morris County, New Jersey, U.S.A. These metacercariae were successfully excysted within l h in an alkaline bile salts trypsin medium maintained at 41°C in the absence of acid pepsin pretreatment. Encysted metacercariae treated in 3% sodium bicarbonate produced successful infections in day-old domestic chicks and worms became ovigerous in the lower ileum by day 4. Excysted metacercariae transferred to the chorioallantois of 6–10-day-old chick embryos maintained at 41°C developed into ovigerous adults by day 4.     

Cultivation of the cercaria of Himasthla quissetensis (Trematoda) on the chick chorioallantois

Cercariae of Himasthla quissetensis were cultivated on the chick chorioallantois maintained at 38 ± 1°C and a relative humidity of 70–75%. Of 68, 6-day-old eggs, each inoculated with 100 cercariae, 28 (41%) were examined 1–9 days post-inoculation. Of the 28 eggs, 23 (82%) were infected with a total of 224 live worms plus 10 encysted metacercariae. Worms contained blood or hematin-like material in their intestinal caeca and showed considerable development of reproductive structures. A total of 58, 6- or 7-day-old chorioallantoic-worms were serially transferred to new 6-day-old embryos, but only a single live worm was recovered after 13 days on two membranes. Based on fixed and stained specimens, 7-day-old chorioallantoic-worms increased their body area about 4× compared to cercariae and the 13-day-old worm increased its body area about 10×.     

The culture of Diplostomum spathaceum metacercariae on the chick chorioallantois

Metacercariae (2,828) obtained from the lens of naturally infected Aplodinotus grunniens were transplanted onto chorioallantoic membranes (CAM's) of 94 eggs (means 30/egg) 5 to 12 days old. Membranes were examined 2 to 8 days later and 381 flukes were recovered. Two hundred fifty-nine chorioallantoic-grown worms were transferred to 28 additional embryos 3 to 8 days after the initial inoculation. These eggs were examined 3 to 11 days later. Fifty-three serially transferred worms were recovered after 7 to 14 days on CAM's. Six stages of development were recognized: stage 1, immature; stage 2, genital rudiment; stage 3, testes; stage 4, follicular ovary; stage 5, vitellaria; stage 6, ovigerous. The intestinal ceca of many worms (except stage 1) contained ingested blood. Trematode eggs oviposited on CAM's were embryonated in tap water, and viable miracidia were observed in 15. No previous study has achieved gonadal development in Diplostomum spathaceum on the CAM. Furthermore, inasmuch as metacercaria matured and produced fertile eggs, this form is shown to be a useful model for the study of trematode differentiation. The development of metacercariae to ovigerous adults capable of producing viable miracidia suggests that the CAM shares characteristics with the intestine of the definitive host, a piscivorous bird.     

Cultivation of excysted metacercariae of Echinostoma revolutum (Trematoda) in chick embryos

Three techniques were used to study post-metacercarial growth and development of chemically excysted metacercariae of Echinostoma revolutum on the chorioallantoic membrane of domestic chick embryos. The in ovo technique of Zwilling (1959) and the in vitro technique of Auerbach et al. (1974) provided for better worm recovery in chick embryos than the in ovo technique of Woodruff &; Goodpasture (1931). Regardless of the technique used, postmetacercarial development was obtained for this species, and 6- to 8-day-old chorioallantoic-wornis achieved sexual development to the coiled uterus stage comparable to worms grown in domestic chicks for 7 days. However, somatic growth of worms from the chorioallantois was stunted when compared to worms grown in chicks. Worms grown on the chorioallantois voided their excretory concretions and showed histochemical lipid staining identical to that seen in worms grown in chicks.     

In vivo and in vitro excystation of Zygocotyle lunata (Trematoda) metacercariae and histochemical observations on the cyst.

Encysted metacercariae of Zygocotyle lunata (Trematoda) excyst within 2 hr postexposure in the lower ileum of the domestic chick. Optimal in vitro excystation of this species occurs following pretreatment of the cyst for 15 min in 1% acidified pepsin, treatment in 0.02 M sodium dithionite (a reductant) for 1 to 2 min and then 2 hr treatment in an excystation medium containing 1% sodium glycocholate plus 1% trypsin in Earle's BSS adjusted to pH 8.8 with tris and maintained at 41 C. The cyst of this species is a dome-shaped hemisphere containing an inner and outer wall. The outer wall contains mainly acid mucopolysaccharides, whereas the inner wall is mainly proteinaceous. The cyst contains a ventral lid which only was visualized during excystation.     

Excystation in vitro of Echinostoma liei and E. revolutum (Trematoda) metacercariae

Metacercariae of Echinostoma liei and E. revolutum were excysted in an alkaline bile-trypsin medium at 41 C in the absence of acid-pepsin pretreatment. After 60 min at a pH of 7.8 or 8.0, excystation of E. liei reached 98%; optimal excystation of E. revolutum occurred at pH 8.2 and was 70% after 60 min. The rate of excystation was very rapid in E. liei, reaching 91% at 30 min, and less rapid in E. revolutum reaching 40% at 30 min. Almost 100% of the E. liei cysts stored for 5.5 mo at 4 C in Locke's 1:1 excysted in the medium, compared to 40% for E. revolutum treated identically.