Life history and identification of Philophthalmus lucipetus from Israel.

A Philophthalmus species whose larval stages have been isolated from Melanopsis praemorsa snails collected in Israel and formerly identified as probably belonging to Philophthalmus palpebrarum has now been reevaluated as belonging to Philophthalmus lucipetus. The present determination is based on a detailed study of all the stages of the parasite's life cycle--eggs, intramolluscan stages, cercariae, and adults. They were compared to the original species P. lucipetus, recently reexamined and redescribed from source material in Vienna, Austria. The identity of the eyefluke from Israel and P. lucipetus was further confirmed by successful cross-infections employing miracidia of the Israeli isolate in European (Bulgarian) Fagotia acicularis acicularis snails on the one hand and miracidia of P. lucipetus from Bulgaria in Israeli Melanopsis praemorsa on the other hand, but none infected Melanoïdes tuberculata snails from Israel.

Mating behaviour of Echinostoma trivolvis and E. paraensei in concurrent infections in hamsters.

Young adults of Echinostoma trivolvis and E. paraensei were recovered from hamsters previously infected with metacercarial cysts. Some worms of each species were exposed for 1 h to 3-H-tyrosine to label sperm and transplanted singly to uninfected hamsters with several unlabelled worms of the same or opposite species or both species. After 5 days, recovered worms were processed for paraffin sectioning and autoradiography. The resulting slides were observed for the location of radioactive sperm in the seminal receptacles of donor (labelled) and recipient (unlabelled) worms. When E. trivolvis was the donor with the recipient E. paraensei, self-insemination took place, but only one interspecies mating occurred out of 72 possible recipient worms. When E. paraensei served as the donor, self-insemination again occurred, but no cross-insemination was observed among the 59 E. trivolvis recipient worms. When single donor worms had a choice of either species of recipient worms, no interspecies mating took place, but both self- and cross-insemination occurred in the normal, unrestricted behaviour found in single species mating studies. Rates of both self- and cross-insemination were higher in concurrent infections of both recipient species than in single species mating studies. After transplant, both species localized in their natural habitat within the small intestine, with 1/3 overlapping in the duodenum, making interspecies mating a possibility. The correlation between mating and electrophoretic studies on the genetic relationship between 37-collar-spined echinostomes is discussed.

The effects of salinity, pH and temperature on the half-life and longevity of Echinostoma caproni miracidia.

Miracidia of Echinostoma caproni were exposed to solutions varying in salinity, pH, and temperature in 1 ml concavity slides. Half-lives of the miracidial populations were determined and longevity curves constructed to find maximum life spans of the miracidia in the different conditions. Control miracidia in aquarium water at pH 7.2 and 22 degrees C. had a half-life of 3.6 h and a maximum life span of 9 h. Miracidia of E. caproni were not very tolerant of saline solutions from 0.1% to 0.4%, the latter being lethal within an hour. A bimodal effect was found with exposure to aquarium water of varying pH, with a peak at pH 5 in acid solutions and pH 9 in alkaline solutions. Miracidia tolerated pH ranges from 3 to 11 exhibiting half-lives of 2.4 h or greater in these solutions. At lower than ambient temperatures, E. caproni miracidia lived longer, the greatest being a half-life of 5.0 h and a maximum life span of 15 h at 5 degrees C. At warmer temperatures, the half-life was reduced until lethality was reached at 40 degrees C. ANCOVA analysis of log transformed longevity curves supported the observation that in pH trials many miracidia survived initially with a major die-off after 3.3 h. The opposite trend of early die-off and gradual mortality of the survivors was supported in the temperature trials.

Mating behaviour of Echinostoma caproni and E. trivolvis in concurrent infections in hamsters.

Young adults of Echinostoma caproni and E. trivolvis were recovered from hamsters. Some worms of each species were exposed to [3H]tyrosine for 1 h to label sperm, and these were transplanted singly to uninfected hamsters with various combinations of unexposed worms of either the opposite species or both species. Worms recovered 5 days later were serially sectioned, processed for autoradiography and observed for the location of radioactive sperm. Interspecies mating was detected when E. caproni was the sperm donor and E. trivolvis the recipient, but not the converse. The cross-insemination rate during interspecies mating was very low (13%) when compared to the normal rate of E. caproni intraspecies mating (52%). When single donor adults of either E. caproni or E. trivolvis had a choice of both recipient species, no interspecies mating took place, but both self- and cross-inseminated in a non-restrictive mating pattern typical of echinostome species. After transplantation, both species localized in their normal habitat within the hamster intestine. However, 25% of opposite species recoveries were found within 1 cm of each other, making interspecies mating a possibility.

The effects of light intensity on hatching of Echinostoma caproni Miracidia.

Egg cultures of Echinostoma caproni were exposed to 25-W incandescent light at control intensities (9.7 mumol s-1 m-2) and reduced intensities (1.0, 0.5, 0.25, 0.125, 0.06, 0.03, and 0.01 mumol s-1 m-2). Low intensities of light did not affect the diurnal hatching rhythm of the miracidia, with maximum hatching occurring from 1200 to 1300 hr. However, at 0.5, 0.25, and 0.125 mumol s-1 m-2, hatching was delayed 2 days at each interval. From 0.06 to 0.01 mumol s-1 m-2, hatching still occurred, but at 17 days of development rather than at the 11.3-day average found under illumination with a 25-W incandescent light. No appreciable hatching was observed in cultures kept in the dark.

Mating behaviour of Echinostoma caproni and E. paraensei in concurrent infections in mice.

Ten-day-old adults of Echinostoma caproni and E. paraensei, some of which had been exposed to 3-H-tyrosine to label sperm, were transplanted to mice in various combinations. Mating behaviour was followed on autoradiograms of worms recovered after 5 days by detecting the transfer of labelled sperm from labelled worms to themselves or to unlabelled worms. When single, labelled E. caproni adults were transplanted with unlabelled E. paraensei, they self-inseminated but did not show evidence of interspecies mating. No interspecies mating but self-insemination was observed when single, labelled E. paraensei and unlabelled E. caproni were transplanted together. When the labelled species had a choice of unlabelled adults of its own species or the opposite species, it mated in similar fashion to that seen in single species infections. The labelled species acted as if the opposite species was not present and showed an unrestricted mating pattern where it would both self- and cross-inseminate. Even though after transplant the E. paraensei adults were found in the duodenum and the E. caproni adults were found in the ileum, approximately 25% of the transplanted worms of both species were found within 1 cm of each other. Thus habitat separation was not a major cause of the lack of interspecies mating. Lack of reproductive recognition is another reason to separate E. caproni and E. paraensei into distinct species.

The mating behaviour of Echinostoma paraensei grown in mice.

Adults of Echinostoma paraensei grown in mice were exposed to 3-H-tyrosine and transplanted to uninfected mice singly or with unexposed worms. The presence of sperm labelled with the radioactive tyrosine in the seminal receptacles of labelled and unlabelled worms recovered after 5 days indicated the mating pattern of this species. Of nine isolated, labelled worms recovered, four had self-inseminated (44%). Of the 39 unlabelled found with eight labelled worms, only eight exhibited labelled sperm for a cross-insemination rate of 21%. Six of these eight labelled worms had self-inseminated (75%) in addition to the cross-insemination. This pattern of mating is termed unrestricted, unlike the restricted mating seen in three species of eyeflukes, which will not self-inseminate in groups. In contrast to the other species of echinostomes investigated, very little sperm was found in the seminal receptacles of the serially sectioned E. paraensei. Adults were always found in the duodenum, even when transplanted in the ileum.

A comparison of the adult and miracidial stages of Echinostoma paraensei and E. caproni.

Adult Echinostoma paraensei and Echinostoma caproni were grown in outbred mice and golden hamsters to compare size, growth rates, infectivity, and habitat selection. Antagonistic responses between the 2 species were investigated by concurrent infections in mice. Miracidial stages were compared for developmental stages, hatching responses, and behaviour to light and gravity. Size differences and growth rates were significantly different in both mice and hamsters. Mice proved to be better hosts for E. caproni and hamsters for E. paraensei. In mature infections, E. paraensei adults localized in the duodenum and E. caproni in the ileum of both mice and hamsters. In concurrent infections of mice, E. paraensei adults were significantly smaller than in single species infections beyond 14 days post-infection, while E. caproni adults were either equal to or larger than those in single species infections. On the other hand, E. paraensei were recovered in larger numbers in concurrent infections than in single species infections, while the reverse was found for infectivity of E. caproni adults. Miracidia of E. paraensei developed at the same rate as those of E. caproni in both light and dark cultures, but E. paraensei hatched much sooner when exposed to light. No miracidia hatched from cultures kept in the dark, indicating light is needed to stimulate the hatching process. All light-stimulated cultures exhibited a circadian hatching pattern from 1100 to 1600 hours. Cultures maintained in the dark past 11 days did not hatch when exposed to light. Miracidia of E. paraensei showed a positive phototaxis but no response to gravity. This comparison of life cycle stages leads us to conclude that E. paraensei and E. caproni are distinct species.

The mating behaviour of Zygocotyle lunata adults grown in mice.

Zygocotyle lunata adults grown in mice were treated in vitro with 3-H-adenosine to label sperm and then transplanted to the caecum of mice singly or with 6 to 8 unlabelled adults. After 6 to 10 days, worms were recovered, processed for autoradiography, and observed for radioactive sperm in their seminal receptacles. Adults would not incorporate radioactive thymidine into dividing cells. Tyrosine was utilized only by juvenile worms and then not enough to label germinal cells for mating studies. Four of 13 (31%) worms transplanted singly self-inseminated, whereas 9 of 10 (90%) labelled worms in multiple infections self-inseminated. When in groups, the labelled worms cross-inseminated with 14 of 43 (33%) unlabelled worms. This mating pattern was unrestricted in that both self- and cross-insemination took place in multiple infections, much like the mating pattern in another paramphistomatid, Megalodiscus temperatus, but unlike the restrictive pattern shown by three species of eyeflukes.

The hatching behavior of Echinostoma trivolvis miracidia and their responses to gravity, light and chemicals.

Echinostoma trivolvis eggs developed to mature miracidia in both dark and light culture conditions as early as 7 days. Hatching initially occurred in eggs exposed to incandescent light on day 11. Those exposed to fluorescent light first hatched on day 13. Maximum hatching (50+ miracidia/day) lasted only over a 3-day span with the entire hatching period being at the most 5 days. Eggs stored in complete darkness for 21 days developed to mature miracidia in 9 days, and, when exposed to light, gave a 50+ hatch the next day. Those eggs stored for 28 and 35 days developed normally but never hatched upon exposure to light. Miracidia hatched at all times of the day without a circadian pattern. Miracidia that hatched from these trials exhibited a definite negative geotaxis in a four-tube vertical system. This response was either negated in open tubes or dominated by a positive phototaxis in the open-bottomed tubes. In horizontal chambers without the depth component, a strong positive phototaxis was also demonstrated. Miracidia showed a positive klinokinetic response, as determined in phi-chambers, to acidic compounds such as aspartic, glutamic, hydrochloric, sulfuric, and acetic acids. Snail-conditioned water prepared from Helisoma trivolvis snails also elicited a positive response. The neutral amino acid, leucine, and possible snail excretion products, Mg2+ and ammonia, did not stimulate these echinostroma miracidia.

Echinostoma trivolvis: mating behavior of adults raised in hamsters.

Echinostoma trivolvis adults grown in golden hamsters were treated in vitro with [3H]-tyrosine to label sperm and transplanted to uninfected hamsters alone or with four to six unlabeled adults. After 4-5 days, worms were recovered, processed for autoradiography, and observed for silver grains over their seminal receptacles. Of 11 worms transplanted singly, 6 (55%) self-inseminated. In multiple-worm situations, 7 of 12 (58%) labeled worms self-inseminated, and in the process the 12 labeled worms inseminated only 5 of 35 (14%) possible unlabeled worms. E. trivolvis adults self-inseminated when isolated and both self- and cross-inseminated in groups, suggesting an unrestricted mating pattern. These results were compared with the mating patterns of other digenetic trematodes.

Hatching of Echinostoma caproni miracidia from eggs derived from adults grown in hamsters and mice.

Echinostoma caproni eggs developed fully formed miracidia from hamster-source eggs in 9 days and from mouse-source eggs in 10 days under either light or dark conditions at 27 degrees C. Incubation of egg cultures under constant light resulted in miracidial hatching from hamster-source eggs in 11 days and from mouse-source eggs in 13 days. Exposure to light was essential to trigger hatching, with incandescent light providing more consistent stimulation than fluorescent light. A majority of the miracidia hatched at between 1100 and 1600 hours, indicating a diurnal circadian pattern. Eggs stored in a dark environment for 11 days required 6 days to reach maximal hatching after exposure to light. Eggs stored for 46 and 56 days hatched on the same day of their exposure to light. Miracidia that hatched after 56 days of dark storage exhibited aberrant swimming behavior; those stored in a dark environment for 70 days or longer did not hatch when exposed to light.

Responses of Echinostoma caproni miracidia to gravity, light, and chemicals.

In a four-tube vertical system, Echinostoma caproni miracidia exhibited a strong negative geotaxis which was dominated by a positive phototaxis. In horizontal chambers a positive phototactic response was also demonstrated. These miracidia showed a positive chemoresponse, as determined in phi-chambers, to glutamic and aspartic acids but not leucine. Positive responses were also elicited to snail-conditioned water and sulfuric and acetic acids. Ammonia, Mg2+, and HCl produced no significant reactions. Responses of E. caproni and Schistosoma mansoni miracidia, both of which develop in Biomphalaria glabrata snails, were similar providing further evidence that miracidia mimic the behavioral patterns of compatible snail species.

A comparison of the surface features of Philophthalmus megalurus and Philophthalmus gralli rediae by scanning electron microscopy.

General features observed on the surface of Philophthalmus megalurus and Philophthalmus gralli rediae include 2 rounded ambulatory buds, a tapered tail, a slitlike birth pore, and an oral opening surrounded by uniciliate sensory receptors. The tegument is folded in a ribbed pattern and is covered with small microvilli. The sensory receptors are concentrated on the lip of the oral opening but are less dense inside the buccal cavity. Both the birth pore and the buccal cavity are lined with a honeycomblike surface that may support the structural integrity of these highly extensible orifices. Major differences between the 2 species were noted in the structure of the sensory receptors. In P. megalurus they have flexible cilia that originate from a bulbous tegumental base. Receptors of P. gralli rediae are less dense, appear to have rigid cilia, and lack a bulbous base. In general P. megalurus rediae resemble more closely rediae of the philophthalmid Parorchis acanthus than they do those of P. gralli.

A comparative study of the regenerative processes in a trematode, Philophthalmus gralli, and a planarian, Dugesia dorotocephala.

Adults of Philophthalmus gralli, an eyefluke of birds, were laterally amputated mid-way between the anterior testis and the ventral sucker. Worms were sampled at various short time intervals (30 min-6 h) after in vitro culture and long time intervals (1-8 days) after transplantation back to the host. Specimens were fixed for scanning electron microscopy and light microscopy and compared to the planarian, Dugesia dorotocephala, which was laterally transected in the pharyngeal region and maintained in springwater. It was found that wound closure took place by 2 days in the planaria; however, at the end of 8 days wound closure had been initiated but not yet completed in P. gralli. Replacement of major tissues was observed only in D. dorotocephala. Because calcium had been reported to be critical for planarian regeneration, a histochemical stain for calcium distribution was carried out. At the end of the 2-day study, no differences could be found in calcium distribution between the two organisms. No areas of calcium concentration were noted in any tissue important for the regenerative process.

Echinostoma caproni: mating behavior and the timing of development and movement of reproductive cells.

The development and movement of reproductive cells were determined in Echinsostoma caproni on autoradiograms by labeling nuclei of stem cells during exposure to [3H]thymidine and then transplanting the worms to mice for various times. The development and movement of sperm, primary oocytes, and vitelline cells were much more rapid in E. caproni than other digenetic trematodes investigated previously. Mating behavior was determined by labeling the sperm of 1 adult by in vitro exposure to [3H]tyrosine and transplanting alone or with unlabeled worms to mice for 4 and 6 days. Echinostoma caproni adults self-inseminated when isolated and self- and cross-inseminated when in groups. This behavior is similar to that found for the frog rectal fluke Megalodiscus temperatus but unlike that determined for eyeflukes in the genus Philophthalmus. Worm size was not a barrier to insemination in E. caproni. Cross-insemination could not be detected in 6-day transplants probably because of dilution or elimination of radioactive sperm due to rapid turnover or frequent sperm transfer. An increased number of structural anomalies was noted in the transplanted worms. The most common anomaly was an accumulation of vitelline cells in the vitelline reservoir and ducts.

Escape of rediae from miracidia of Philophthalmus megalurus and Philophthalmus gralli during in vitro culture.

Miracidia of the eyeflukes Philophthalmus megalurus and Philophthalmus gralli contain a preformed larval stage that was identified as a redia by electron microscopy and, on living forms, by the presence of ambulatory buds and an oral opening. This redial stage escaped and actively moved about when the miracidium stopped swimming in pond water. No redial escape was observed in NaCl solutions even though miracidia became immobile, but escape was noted in Hanks' balanced salt solution (HBSS) after 7 hr of exposure. When miracidia were placed in RPMI-1640 and Eagle's minimum essential medium, rediae escaped much earlier than in pond water and HBSS. Redial escape in vitro will provide a good source of material to initiate cultures of this larval stage.

Chemosensitivity of Philophthalmus megalurus (Trematoda) miracidia.

Miracidia of the eyefluke Philophthalmus megalurus were tested in phi-chambers to determine if they reacted similarly to chemicals found stimulative for miracidia of Philophthalmus gralli, a closely related species. Philophthalmus megalurus miracidia were less responsive than P. gralli to the dicarboxylic amino acids and showed a significantly positive response only to 10 mM glutamic acid. These P. megalurus miracidia were chemonegative to 10 mM HCl and H2SO4, chemicals to which P. gralli miracidia gave a significantly positive response. Ammonia and Mg2+ did not elicit any response from P. megalurus miracidia.

Effects of concurrent infections on growth, development, distribution, and infectivity of adult Philophthalmus megalurus and Philophthalmus gralli.

Chickens were infected concurrently with 10 and 20 metacercariae/eye of Philophthalmus megalurus and Philophthalmus gralli. After 14 and 34 days of growth, the adults were removed and worm lengths, return rates, stage of development, and distribution within the host recorded. In a second experiment, chickens infected on day 1 with 10 metacercariae/eye of P. megalurus were concurrently infected with a similar dose of P. gralli on day 14. At day 34, the worms were removed and evaluated for the same parameters as in the first study. The effect of concurrent infections on worm length of P. gralli but not P. megalurus was significant when compared to single species control groups. In all cases recovery rates of P. megalurus in concurrent infections were significantly lower than controls, whereas P. gralli adults were recovered at lower rates only in 20-metacercariae--14-day infections. Maturation of eggs was delayed in both species in concurrent infections at the higher infection levels. Normal distribution was disrupted more for P. gralli at the higher infection levels and longer growth periods. Philophthalmus megalurus adults rarely left their normal habitat in the conjunctival sac. A delayed infective dose of P. gralli affected both species by disrupting normal distribution patterns, delaying egg development, and, in the case of P. megalurus, reducing the recovery rate. The possible role of crowding and antagonistic effects is discussed.

The effects of gossypol on spermatogenesis and development of the eyefluke, Philophthalmus gralli, and its chicken host.

To determine the effects of gossypol, a male antifertility drug, on the eyefluke, P. gralli, this chemical was administered orally to chickens in long-term and short-term regimens. Gossypol acetic acid (GAA), fed to juvenile chickens from 1 to 35 days, caused a decreased weight gain when compared to controls on untreated feed. An FeSO4 supplement to the GAA-fed chickens provided partial protection from the toxic effects of GAA. Worms from GAA-fed chickens were significantly larger than controls, while those from chickens fed GAA + FeSO4 were intermediate in size. Sperm development in these worms was unaffected by GAA. In a second experiment, GAA was administered either in the feed of the hosts from days 35 to 70 or by capsule from days 63 to 77. Worms were exposed to [3H] thymidine, transplanted to the host's eyes, removed on a timed schedule, and processed for autoradiography to determine the rate of spermatogenesis in both GAA-feed and GAA-capsule groups. Early stages of spermatogenesis in both groups were unaffected by GAA and later stages developed at a slightly faster rate than reported for worms from chickens on untreated feed. Higher frequencies of testicular anomalies were observed in both groups including 3 testes, 1 testis, no testes, fused testes, degenerating testes, ovarian tissue in the testes, deformed sperm, and encapsulated sperm. Testes from chickens in both groups showed a significantly lower weight and no signs of spermatogenesis when compared to control chickens.