ULTRASTRUCTURE OF EGG ENVELOPES AND EARLY EMBRYOS OF ROHDELLA AMAZONICA (TREMATODA: ASPIDOGASTREA) PARASITIC IN BANDED PUFFER FISH, COLOMESUS PSITTACUS.

Egg structure and early embryonic development of the aspidogastrean, Rohdella amazonica, a basal trematode, were studied by transmission electron microscopy (TEM) to gain insight into functional, developmental, and phylogenetic characteristics. Gravid worms were removed from the intestine of naturally infected banded puffer fish Colomesus psittacus, collected from the Bay of Marajó, Paracauari River (Pará, Brazil) and processed by standard TEM methods. By the time of pronuclear fusion, the fertilized zygote was already enclosed in a thick, electron-dense pre-operculate eggshell and an underlying layer of vitellocytes that fused into a vitelline syncytium as they were still secreting their shell granules. When cleavage commenced, a small number of macromeres moved to the area just underneath the eggshell, where they fused to form a single syncytial embryonic envelope. Simultaneously, the smaller blastomeres continued to divide as they maintained contact with each other, but remained separate from the vitelline syncytium. Concurrent with these cellular changes, a thickened knob expanded at one pole of the eggshell and began to form an opercular suture. By the time the operculum was fully formed, the vitelline syncytium had mostly degenerated, while the smaller blastomeres had become cohesive as a single mass that preceded the differentiation and morphogenesis of the cotylocidium larva. The general pattern of cleavage and eggshell formation resembles that of other trematodes and polylecithal cestodes, but the single embryonic envelope has been reported only in a few basal taxa. The only other aspidogastrean studied in detail to date is very similar, indicating close phylogenetic affinity and conservatism within this basal neodermatan and neoophoran group.

Ultrastructure of digenean trematode eggs (Platyhelminthes: Neoophora): A review emphasizing new comparative data on four European Microphalloidea.

Despite their tremendous diversity and their medical and veterinary importance, details of egg ultrastructure among the digenean trematodes has been studied rather little. The available literature is spread over several decades and several species, but has not been adequately reviewed to reveal patterns of similarity and divergence. We present this review to synthesize and analyse what is known from the available literature reporting studies using both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To support our general review of existing literature, we also have synthesized our own previously published descriptions, and present herein our new previously unpublished data. From these new electron micrographs, we provide a comparative analysis of the intrauterine eggs of four digenean species, representing four genera and three families of the superfamily Microphalloidea, collected from four different host wildlife species in four European countries: 1) Mediogonimus jourdanei (Prosthogonimidae) from Myodes glareolus (Mammalia: Rodentia), collected in France; 2) Maritrema feliui (Microphallidae) from Crocidura russula (Mammalia: Soricimorpha), collected in Spain; 3) Brandesia turgida (Pleurogenidae) from Pelophylax ridibundus (Amphibia: Anura: Ranidae), collected in Russia; and 4) Prosotocus confusus (Pleurogenidae) from Rana lessonae (Amphibia: Anura: Ranidae), collected in Belarus. All were studied by preparing whole worms by various techniques for TEM, so that eggs could be studied in situ within the uterus of the parent worm. Based on the literature review and the new data presented here, we describe basic similarities in patterns of embryogenesis and egg formation among all trematode species, but substantial variations in timing of larvigenesis, sculpturing of egg shell surfaces, and some other features, especially including accessory cocoon coverings outside the egg shells of B. turgida and P. confusus. In the future, many more studies are needed to explore egg ultrastructure in other digenean taxa, to explore potential phylogenetic patterns in egg development and structure, and to correlate structure with function in the life cycle.

Molecular and biochemical characterization of Paragonimus westermani tyrosinase.

Trematode tyrosinases (TYRs) play a major role in the tanning process during eggshell formation. We investigated the molecular and biochemical features of Paragonimus westermani TYR (PwTYR). The PwTYR cDNA was composed of 1568-bp encompassing a 1422-bp-long open reading frame (474-amino acid polypeptide). A strong phylogenetic relationship with Platyhelminthes and Deuterostomian orthologues was evident. The recombinant PwTYR expressed in prokaryotic cells promptly oxidized diphenol substrates, with a preferential affinity toward ortho-positioned hydroxyl groups. It demonstrated fairly weak activity for monophenol compounds. Diphenol oxidase activity was augmented with an increase of pH from 5.0 to 8.0, while monophenol oxidase activity was highest at an acidic pH and gradually decreased as pH increased. Transcription profile of PwTYR was temporally upregulated along with worm development. PwTYR was specifically localized in vitellocytes and eggs. The results suggested that conversion of tyrosine to L-dihydroxyphenylalanine by PwTYR monophenol oxidase activity might be rate-limiting step during the sclerotization process of P. westermani eggs. The pH-dependent pattern of monophenol and diphenol oxidase activity further proposes that the initial hydroxylation might slowly but steadily progress in acidic secreted vesicles of vitellocytes and the second oxidation process might be rapidly accelerated by neural or weak alkaline pH environments within the ootype.

Ultrastructural and cytochemical aspects of the germarium and the vitellarium in Syndesmis patagonica (Platyhelminthes, Rhabdocoela, Umagillidae).

The cytoarchitecture of the female gonad of the endosymbiont umagillid Syndesmis patagonica has been investigated using electron microscopy and cytochemical techniques. The female gonad consists of paired germaria and vitellaria located behind the pharynx in the mid-posterior region of the body. Both the germaria and the vitellaria are enveloped by an outer extracellular lamina and an inner sheath of accessory cells which contribute to the extracellular lamina. Oocyte maturation occurs completely during the prophase of the first meiotic division. Oocyte differentiation is characterized by the appearance of chromatoid bodies and the development of endoplasmic reticulum and Golgi complexes. These organelles appear to be involved in the production of round granules, about 2-2.5 µm in diameter, with a homogeneous electron-dense core surrounded by a granular component and a translucent halo delimited by a membrane. These egg granules migrate to the periphery of mature oocytes, are positive to the cytochemical test for polyphenol detection, are unaffected by protease and have been interpreted as eggshell granules. The mature oocytes also contain a small number of yolk granules, lipid droplets, and glycogen particles scattered throughout the ooplasm. The vitellaria are branched organs composed of vitelline follicles with vitellocytes at different stages of maturation. Developing vitellocytes contain well-developed rough endoplasmic reticulum and small Golgi complexes involved in the production of eggshell and yolk globules. Eggshell globules are round, measure 4-5 µm in diameter, and have a mosaic-like patterned content which contains polyphenols. The yolk globules, 2-3 µm in diameter, show a homogeneous protein content of medium electron density, devoid of polyphenols, and completely digested by protease. The mature vitellocytes also contain glycogen as further reserve material. The presence of polyphenolic eggshell granules in the oocytes and of polyphenolic eggshell globules with a mosaic-like pattern in the vitellocytes have been considered apomorphic features of the Rhabdocoela + Prolecithophora.