Interrelationships of vitelline and muscle cells within the vitelline follicles of the blood fluke Aporocotyle simplex (Digenea, Aporocotylidae) and morphological evidence for the modification of vitelline material for eggshell formation

Digenea Carus, 1863 represent a highly diverse group of parasitic platyhelminths that infect all major vertebrate groups as definitive hosts. Morphology is the cornerstone of digenean systematics, but molecular markers have been instrumental in searching for a stable classification system of the subclass and in establishing more accurate species limits. The first comprehensive molecular phylogenetic tree of Digenea published in 2003 used two nuclear rRNA genes (ssrDNA = 18S rDNA and lsrDNA = 28S rDNA) and was based on 163 taxa representing 77 nominal families, resulting in a widely accepted phylogenetic classification. The genetic library for the 28S rRNA gene has increased steadily over the last 15 years because this marker possesses a strong phylogenetic signal to resolve sister-group relationships among species and to infer phylogenetic relationships at higher levels of the taxonomic hierarchy. Here, we have updated the database of 18S and 28S rRNA genes until December 2017, we have added newly generated 28S rDNA sequences and we have reassessed phylogenetic relationships to test the current higher-level classification of digeneans (at the subordinal and subfamilial levels). The new dataset consisted of 1077 digenean taxa allocated to 106 nominal families for 28S and 419 taxa in 98 families for 18S. Overall, the results were consistent with previous higher-level classification schemes, and most superfamilies and suborders were recovered as monophyletic assemblages. With the advancement of next-generation sequencing (NGS) technologies, new phylogenetic hypotheses from complete mitochondrial genomes have been proposed, although their power to resolve deep levels of trees remains controversial. Since data from NGS methods are replacing other widely used markers for phylogenetic analyses, it is timely to reassess the phylogenetic relationships of digeneans with conventional nuclear rRNA genes, and to use the new analysis to test the performance of genomic information gathered from NGS, e.g. mitogenomes, to infer higher-level relationships of this group of parasitic platyhelminths.

Helminth eggs have resisted analysis by electron microscopy because fixatives, dehydrating agents, and embedding media penetrate these eggs poorly. Slam-freezing at liquid nitrogen temperature followed by freeze-substitution and Spurr's medium embedment provides preservation of the internal structure of the Schistosoma mansoni egg shell, developing miracidium, and perimiracidial structures. The egg shell consists of the three previously described layers (outer microspinous, middle intermediately dense, and inner dense layers) with cribriform pores. A newly described layer (Reynolds' layer) develops subjacent the egg shell and is comprised of densely-packed branching filaments. A single layer of squamous cells (von Lichtenberg's envelope) closely adheres to Reynolds' layer. Between von Lichtenberg's envelope and the embryo is a space (Lehman's lacuna); this space is initially filled with electron-lucent fluid, but subsequently masses of granulofloccular material (Cheever bodies) develop; Cheever bodies are partially membrane bound. Epidermal plates differentiate from superficial cells of the embryonal cell mass, while epidermal ridges differentiate from cells just below the surface of the embryonal cell mass. The cytoplasmic layer (von Lichtenberg's envelope) interposed between the host extracellular fluid and the developing miracidia effect a barrier against a simple passive diffusion; this infers that complex macromolecules, such as schistosomal egg antigen, undergo active, and perhaps selective, transport in or out of the egg.