The responses of Philophthalmus gralli and P. megalurus miracidia to light, gravity and magnetic fields

Miracida of an eyefluke of birds, Philophthalmus gralli, which are positively geotactic, exhibited a positive north-seeking magnetotaxis when subjected to magnetic field strengths from 3 × 10^?4 to 2 × 10^?2 T. A closely-related species, P. megalurus, which is positively geotactic only in complete darkness, exhibited no magnetotaxis under similar conditions. A positive phototactic response overrode the north-seeking magnetotaxis when P. gralli miracidia were subjected to both stimuli in a competing system. No detector mechanisms for magnetic fields are known in miracidia of digenetic trematodes. The order of responsiveness for P. gralli miracidia then is: geotaxis > phototaxis > magnetotaxis.     

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.     

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.     

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.     

Inseminatory behavior of Philophthalmus megalurus and Philophthalmus hegeneri in concurrent infections of chicks

Sperm labeled with ³H-thymidine and³H-tyrosine detected by autoradiography were used to determine inseminatory patterns in concurrent infections of Philophthalmus megalurus and Philophthalmus hegeneri. In infections of one labeled P. megalurus and 1 to 3 unlabeled P. hegeneri self-insemination but not cross-insemination was detected. When P. hegeneri was labeled and transplanted with 1 to 3 unlabeled P. megalurus no insemination was found. Transplantation of one labeled P. megalurus with various numbers of unlabeled adults of both species resulted in exclusive cross-insemination ofP. megalurus. When one labeled P. hegeneri was in the presence of unlabeled adults of both species, only cross-insemination of P. hegeneri was detected. Thus no insemination between species was found precluding hybrid production. This may be due to a separation of feeding sites within the microenvironment of the conjunctival sac of the chick's eye. Both species carried on normal inseminatory behavior in the presence of the other species. Recovery rates of both species from concurrent infections were not greatly different than those from single species infections. However, the recovery rate forP. megalurus (61·2 %) was much greater than for P. hegeneri (44·7 %).     

The effects of crowding on adults of Philophthalmus gralli (Trematoda) grown in chickens

Chickens were infected with six, 10, 30, 50, or 100 metacercariae of Philophthalmus gralli per eye and the adults were harvested 9, 14, 21, 35, and 50 days later. The worms recovered were measured and assessed for maturity. Significantly more adults from infections of six and 10 metacercariae per eye were recovered than from infections of 100 per eye. When metacercariae were placed in one eye, over one-fourth of the resulting adults were found in the opposite eye. Worms recovered from groups of zero to 10 were significantly longer than those in groups of 41 to 50 and 51 to 60 at all ages sampled. The normal movement of worms from the conjunctival sac to the outside of the nictitating membrane was hindered by crowding; when more than 10 were present, some worms developed in the sac to mature adults. Worms in all groups produced eggs in which miracidia developed at the same rate.     

Argentophilic structures of the miracidia and cercariae of Philophthalmus distomatosa n. comb. from Israel

The morphology and patterns of distribution of the argentophilic structures of miracidia and cercariae of Philophthalmus distomatosa n. comb. are described. The epidermal plate arrangement of the vast majority (94.2%) of miracidia studied conformed to the formula 6:8:4:2 = 20. The rest (5.8%) displayed the following patterns of arrangement of epidermal plates: 6:7:4:2 = 19; 6:6:4:2 = 18; and 6:4:4:2 = 16. Twenty papillalike structures were observed on the terebratorium. They were arranged along 1 axis, in 3 groups. Most commonly, 16 papillae were present on the body, located between epidermal plates of the first and second rows. The eye spots were located dorsally, near interepidermal space S1. The numbers, arrangements, and locations of the excretory pores varied. Patterns of distribution of the tegumentary papillae of P. distomatosa n. comb. cercariae are given, including those of the cephalic regions, periacetabular region, and tail. Argentophilic structures of the mentioned larval stages and of other parameters clearly distinguishing between the presently described species and those of P. lucipetus from Israel are summarized. On the basis of comparison of the presently described larval stages and those of other Melanoides tuberculata-associated larval stages of Philophthalmus in Israel and Jordan, the possibility exists that a third species occurs in Israel.     

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.     

Molecular characterization of Oxyspirura mansoni and Philophthalmus gralli collected from the eyes of domestic chickens in Bangladesh

A variety of helminths have been found in domestic chickens in Bangladesh, but little is known about their gene sequences. Here, parasitic nematodes and trematodes were collected from the eyes of domestic chickens and analyzed for their morphological and morphometric characteristics, and characterized molecularly. The helminths were identified as Oxyspirura mansoni and Philophthalmus gralli. The ITS1 and ITS2 sequences of O. mansoni were 532 bp and 306 bp in length, respectively, and showed low identity (50.7–62.7%) with those of O. petrowi and O. conjunctivalis. Furthermore, the O. mansoni CO1 sequences (393 bp) showed five haplotypes (97.5–99.5% similarity) that formed a monophyletic clade. With respect to P. gralli, the ITS1 (452 bp) and ITS2 (736 bp) sequences showed 100% similarity with the reference sequences in GenBank. Both the ND1 and CO1 phylograms showed that P. gralli from Bangladesh, Costa Rica and Peru form a monophyletic clade, distinct from the clades of P. lucipetus and P. lacrymosus. Our data show that, Philophthalmus gralli isolates from Bangladesh, Costa Rica and Peru are genetically close to each other.     

Development, growth, and migration of Philophthalmus gralli (Trematoda) from Jordan in the eyes of chicks

The growth of Philophthalmus gralli from Jordan was studied experimentally by inoculating 10 excysted metacercariae around the right eye of 47 day-old chicks. The growth rate was slow in the first three days during which flukes did not exceed 0.30 mm long. Then growth became rapid reaching 2.70 mm long on day 24. This was followed by a low growth rate during which the flukes grew from 2.70 mm to 2.90 mm on day 40. The flukes started to appear in the left eye on day 12 post-inoculation of metacercariae in the right eye. The recovery of flukes in the left eye increased from 5% on day 12 to 30% on day 32. The development of P. gralli from Jordan was the same as of other species of Philophthalmus. However, a sixth stage, post-maturation stage, is suggested in addition to the 5 developmental stages of Philophthalmus. The ratio of the transverse diameter of the ovary to that of testes in the post-maturation stage was 1:1.1-1:1.2.     

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.     

Argentophilic structures of miracidia and cercariae of Philophthalmus lucipetus (Philophthalmidae: Trematoda) from Israel

Argentophilic structures of Philophthalmus lucipetus miracidia and cercariae from Israel are described. Eighty-four of 87 miracidia examined displayed an epidermal plate arrangement of 6:8:4:2 = 20, similar to other Philophthalmus species. Twenty papilla-like structures are arranged on the terebratorium in 3 groups, along 1 axis. Sixteen body papillae are located at the bases of epidermal plates of row 1. Eyespots are mediodorsal, between rows 1 and 2. Excretory pores are lateral, between rows 2 and 3. Features common to Israeli and Bulgarian isolates, differentiating them from other species, include the presence of 16 body papillae as opposed to 10 in other species, and a maximum of 20 papillae on the terebratorium as opposed to 19 in the others. About 3% of the miracidia displayed different plate arrangements. Among the argentophilic structures of P. lucipetus cercariae, the Israeli and Bulgarian P. lucipetus show a common pattern of 2-4 excretory pores in the tail, but arrangement of cephalic CI3 and CI5 papillae in the 2 isolates is insufficiently unequivocal for species determination. The data presented show that miracidial characteristics, rather than those of cercariae, aid in determining the species of philophthalmids. They also support former evidence attesting to the identity of the Bulgarian and Israeli species.