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.     

Life cycle and identification of an eyefluke from Israel transmitted by Melanoides tuberculata (Müller, 1774)

A philophthalmid species from Israel using the freshwater snail Melanoides tuberculata as intermediate host was studied. The biological and morphological characteristics of all developmental stages of the life cycle of this philophthalmid were described, and compared to those of Philophthalmus lucipetus Rudolphi, 1819 from Israel, Philophthalmus gralli Mathis and Leger, 1910 from Jordan, Philophthalmus palpebrarum Looss, 1899, Philophthalmus nocturnus Looss, 1907, Cercaria distomatosa Looss 1896 from Egypt, and Philophthalmus lucknowensis Baugh, 1962 from India. The possible identity with 1 of these species is discussed. On the basis of comparative analysis of the data for all parasite stages in the life cycle, geographical distribution, snail hosts, and snail host specificity, we propose to designate the Israeli Melanoides tuberculata-transmitted eye fluke to Philophthalmus distomatosa n. comb. (Looss, 1896), (Digenea: Philophthalmidae).     

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 Melano?des tuberculata snails from Israel.     

Chaetotaxy and surface structures of the miracidia of Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825) and Apatemon gracilis (Szidat, 1928) (Digenea,Strigeidae)

Silver nitrate staining was used to investigate the number and arrangement of argentophillic structures (epidermal plates, sensilla, excretory pores) of three species of strigeid miracidia, Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825) and Apatemon gracilis (Szidat, 1928). The epidermal plates of all three species of miracidia were arranged in four tiers according to the formula (6 + 9 + 4 + 3) = 22, while the number and distribution of sensilla were also found to be identical. Modifications to the nomenclature of Dimitrov et al. (1989) for miracidial chaetotaxy are proposed.Scanning electron microscopy (SEM) was also employed to examine the surface structures of deciliated miracidia of Ichthyocotylurus spp., confirming the position and revealing the form of body sensilla.     

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.     

Fasciola hepatica: surfaces involved in movement of miracidia and cercariae

Rapid freezing and substitution with fixative prior to scanning electron microscopy was used to demonstrate the pattern of beat and recovery of the cilia of free swimming miracidia of Fasciola hepatica. There were stages of dexioplectic metachronal co-ordination and the power stroke was approximately 15 degrees anticlockwise from the anterior-posterior axis. Around the circumference of the body of the miracidia there were approximately 12 metachronal waves of power and recovery. Free-swimming cercariae were recorded by time-lapse photography and, after conventional fixation, by scanning electron microscopy. Cercarial tail-beats were to the posterior of the body in the lateral plane at a rate of 8 Hz. The tail has paired lateral ridges positioned to act as leading edges. There is an array of 32 sensory papillae on the mid-ventral surface of the tail. The tegument of the most distal part of the tail is described: it is free of sensory endings and the surface shows a spiral pattern.     

The life cycle of Cloacitrema michiganensis McIntosh, 1938 (Trematoda: Philophthalmidae)

Experimental infections of Cloacitrema michiganensis were established in chicks by inoculation per cloaca with excysted metacercariae. Ovigerous adults were present 20 days later with eggs containing ocellate miracidia after 21 days. The eggs hatch in utero; the miracidium contains a fully developed redia. Rediae of 2 size groups were present in the digestive gland of the brackish-water snail, Cerithidea californica. Megalurous cercariae are released and encyst on submerged objects. The stages of C. michiganensis resembled those of Parorchis acanthus, a philophthalmid also infecting C. californica, except for differences in the epidermal plate count of their miracidia and in their cercarial stage which was larger in P. acanthus and had a spinuous tegument with a collar of spines.     

Associations between two trematode parasites, an ectosymbiotic annelid, and Thiara (Tarebia) granifera (Gastropoda) in Jamaica

This work describes associations of Thiara (Tarebia) granifera, its larval trematode community, and Chaetogaster limnaei limnaei at a freshwater reservoir in Jamaica. Larvae of 2 trematodes were present, i.e., a notocotylid (15.3%) and Philophthalmus sp. (1.3%), in 3,575 T. granifera examined. The prevalence of both infections increased with snail shell length (H = 56, P < 0.01, H = 23.1, P < 0.01, respectively). Only 3.0% (n = 595) of infected snails possessed reproductive stages, compared with 90.3% (n = 2,980) of uninfected snails (χ(2) = 2,059.8, df = 1, P < 0.001); both trematodes negatively impacted snail reproduction. Chaetogaster l. limnaei occurred within the mantle cavity of T. granifera with a prevalence of 2.3% (n = 3,575); intensity ranged from 1 to 6 annelids. Notocotylid larvae occurred in 32.5% (n = 83) of snails also harboring C. l. limnaei, compared with 14.9% (n = 3,492) of snails lacking the annelid (χ(2) = 18.127; P < 0.001). Chaetogaster l. limnaei appears not to influence the recruitment of egg-transmitted, notocotylid infections to snails. Ingestion of emergent cercariae by the annelid was observed; this may impact transmission of the parasite. The article presents the first report of a notocotylid and C. l. limnaei in T. granifera, and of Philophthalmus sp. in Jamaica.     

The longevity and hatchability of Philophthalmus megalurus and P. gralli miracidia in different environmental conditions.

The effects of salinity, pH, and temperature on the longevity and hatchability of miracidia of Philophthalmus megalurus and P. gralli were determined. Miracidia of both species are able to hatch and survive at saline concentrations much above physiological levels, although these processes are reduced in 2.0--2.4% saline and completely inhibited at 2.6%. The greatest hatching rates for both species were found near neutrality (pH 6--8) but some miracidia hatched at the extreme pH levels of 3 and 12. Philophthalmus megalurus miracidia exhibited longer half-lives under acid conditions (pH 2--6) than P. gralli miracidia; conversely, P. gralli miracidia showed longer half-lives in alkaline conditions (pH 8--11). Hatching and longevity were much greater below room temperature (5--20 C) than above (30--50 C) for miracidia of both species. Temperatures above 50 C proved lethal for eyefluke eggs. Except in acid pH, P. gralli miracidia showed longer half-lives than miracidia of P. megalurus. Comparison to studies on schistosomes revealed that the inhibitory effects of physiological saline and host body temperature on the hatching process of schistosome eggs does not occur in these 2 species of eyeflukes.     

Experimental study of the biology of the miracidium of Philophthalmus rhionica Tichomirov, 1976 (Trematoda, Philophthalmidae)]

Some peculiarities of the biology of miracidia of Philophthalmus rhionica have been studied: penetration of the parasite's eggs into the environment, hatching of larvae, the miracidia motion, their life span and changes in the invasion ability character in time.     

Schistosoma mansoni: ultrastructural demonstration of a miracidial glycocalyx that cross-reacts with antibodies raised against the cercarial glycocalyx

Cercariae are covered by a glycocalyx that is highly antigenic. Here, we have examined the surface of miracidia for a similar structure. The miracidia are covered by epithelial plates and syncytial ridges. By transmission electron microscopy, the plates and ridges were covered by a 0.5-micron-thick glycocalyx composed of a mesh of 9- to 10-nm fibrils that were stained by ruthenium red delivered in the aldehydes or ferrocyanide-reduced osmium tetroxide. Rabbit antibodies prepared against phenol extracted and chromatographed cercarial glycocalyx were detected by immunoelectron microscopy with secondary antibodies conjugated to horseradish peroxidase. Reaction product bound to both the miracidial and cercarial glycocalyx. In addition, the outer leaflets of the cercarial tegumental membrane and membranes of the miracidial surface structures, including plates, ridges, terebratorium, and sensory papillae, had reaction product. Controls incubated with nonspecific rabbit serum had no reaction product. By indirect immunofluorescence, antibodies against the cercarial glycocalyx stained both plates and ridges. As the miracidia transformed to sporocysts, the glycocalyx remained associated with the plates as they were sloughed. These studies demonstrate that miracidia possess a glycocalyx similar in structure and antigenicity to the cercarial glycocalyx.     

Various stages of schistosoma express Lewis(x), LacdiNAc, GalNAcbeta1-4 (Fucalpha1-3)GlcNAc and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins

We report here that fucosylated epitopes such as Lewis(x), LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon resonance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis(x)-, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis(x)epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope antibodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection.     

Scanning and transmission electron microscopy of oral sucker papillae of Philophthalmus megalurus

Edwards H. H., Nollen P. M. &; Nadakavukaren M. J. 1977. Scanning and transmission electron microscopy of the oral sucker papillae of Philophthalmus megalurus. International Journal for Parasitology7: 429–437. Scanning electron microscopy reveals papillae on both the inside and outside of the oral sucker of the eyefluke, Philophthalmus megalurus. In the transmission electron microscope the papillae can be divided into 3 different types with 2 of these types occurring on the outside of the oral sucker. One type of outer papilla contains a bulb cell which is terminated by a cilium having an apparent typical arrangement of microtubules. This type is a presumed sensory receptor having tango- and/or rheoreceptor function. The second type of outer papilla contains a gland cell which secretes electron-dense granules to the outside of the fluke. The third type of papilla is found only on the inside of the oral sucker and contains a bulb cell terminated by a cilium which does not have the typical 9 + 2 arrangement of microtubules. Instead, their numbers increase to over 60 randomly arranged microtubules. This inner papilla is felt to have chemoreceptor function because of its location and the presence of the modified cilium. The bulb cell of the inner papilla contains 2 newly described features. The first is granular, and associated with microtubules; it may be a possible nucleating site for microtubule synthesis. The second structure is a crystalline inclusion of unknown function and origin.     

Cercariae of a species of Philophthalmus detected in a freshwater snail, Semisulcospira libertina, in Japan

Cercariae belonging to the genus Philophthalmus (Digenea: Philophthalmidae) were detected in a freshwater snail Semisulcospira libertina collected from Innai Town, Oita Prefecture, Kyusyu, Japan. This is the first record of the cercaria of this genus from East Asia and from Semisulcospira. Daughter rediae and cercariae are briefly described and morphologically compared with similar but unidentified ones reported from Japan. Cercariae were found encysting on the surface of the bottom of the container and metamorphosed into a flask-like metacercaria characteristic of Philophthalmus.     

The distribution and incidence of larval trematodes in the freshwater fauna of the Wentloog level, South Wales

The Wentloog level, in South Wales, is a low-lying coastal plain dissected by a network of drainage channels. The latter contain a rich aquatic invertebrate fauna which harbour a wide range of larval digenetic trematodes. Few of the host species examined were not infected with larval trematodes. Miracidia together with their associated sporocysts, rediae and cercariae were more host specific than metacercariae but many of the miracidia which developed in pulmonate molluscs could do so in more than one genus. Metacercarial host specificity is less rigid, and usually species are only restricted to members of one phylum although several were found which could develop in two or more phyla. Most larval stages showed some host preference when they occurred in several host species and this was attributed to ecological or physiological factors or to mechanical barriers. Seasonal variation of infection rate occurs in all larval stages: infection with developing cercariae showed peaks during June and August, but metacercarial infection rates varied according to the host. In molluscs there may be two peaks of infection with metacercariae (as in the cercarial stages), but for many arthropods infections show a gradual increase through the year. The factors mainly responsible for these seasonal changes are considered to be the life-cycle of the intermediate hosts and the environmental temperature, the latter acting on the rate of development of the trematodes. Rainfall, and patterns of egg production of adult trematodes in the definitive hosts are also important.     

Influence of saccharose on the development of cercariae from Schistosoma mansoni strains BH and SJ.

The development of cercariae from Schistosoma mansoni strains BH and SJ in Biomphalaria glabrata and Biomphalaria tenagophila treated with saccharose was studied. The molluscs were maintained in dechlorinated tap water containing 0.01% saccharose. After one week of treatment with saccharose, B. glabrata and B. tenagophila were exposed to ten S. mansoni miracidia, from BH and SJ strains respectively. Control snails of both species were maintained in dechlorinated tap water without saccharose and exposed to the same number of miracidia. There was no significant difference between the infection rates of snails treated or not with saccharose. However, the two groups of B. glabrata had significantly greater infection rates than the corresponding B. tenagophila groups. Molluscs treated with saccharose had a lower survival rate, with the greatest mortality occurring immediately before and at the beginning of cercariae release. Treatment with saccharose did not result in the release of more cercariae, but larvae from molluscs so treated showed a greater capacity to penetrate mouse skin, which was attributed to the greater energy supply during larval development in the mollusc.     

Comparative scanning electron microscope (SEM) study of miracidia of four human schistosome species

The miracidia of four human blood flukes, Schistosoma haematobium, S. intercalatum, S. mansoni and S. japonicum, have been studied by means of the scanning electron microscope (SEM). Differences have been observed in their respective dimensions, in the configuration of their terebratoria (apical papillae), in the shape of the epidermal plates, and in the distribution of the sensory receptors. The most evident differences have been noticed on the terebratoria where two main patterns of organization of the anastomosing membrane foldings have been described: (1) a ‘rosette’ pattern observed in S. haematobium and S. intercalatum and (2) a ‘honeycomb’ pattern in S. mansoni and S. japonicum. The structure and the taxonomic importance of these morphological features are analysed.     

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.     

Schistosoma mansoni: male-biased sex ratios in snails and mice

Adult sex ratios of Schistosoma mansoni, in mice, were shown to be biased toward males (3:1) despite the finding that sex ratios of miracidia were 50:50. The adult male bias was caused by greater male infectivity of miracidia for snails and cercariae for mice. A significantly higher percentage of male miracidia developed to cercarial production in unimiracidial infections (57 male, 34 female), and a significantly higher percentage of male cercariae developed to adulthood in mice (143 male, 79 female worms resulted from 900 male and 900 female cercariae). No significant differences were found between male and female parasites for longevity of miracidia (both sexes, 10 hr) and cercariae (males 21.3 +/- 5.75 hr, females 25.0 +/- 7.02 hr), prepatent periods in snail hosts (male 34 +/- 2.92 days, females 33 +/- 2.36 days), longevity of snail infections (males 96.6 +/- 25.15 days, females 115.2 +/- 82.43 days), and the numbers of cercariae produced per snail lifetime (males 30,751.44 +/- 18,064.33, females 34,083.00 +/- 33,732.82). Present results provide a better understanding of the life cycle of S. mansoni, are of theoretical significance for theories of biased sex ratios (which at present cannot account for the male-biased ratio of S. mansoni), and also suggest that schistosomiasis transmission models assuming a 50:50 sex ratio at all stages of the life cycle should be reassessed.