RNA-seq analysis of parasitism by Intoshia linei (Orthonectida) reveals protein effectors of defence, communication, feeding and growth.

Orthonectida is a group of multicellular endoparasites of a wide range of marine invertebrates. Their parasitic stage is a multinuclear shapeless plasmodium infiltrating host tissues. The development of the following worm-like sexual generation takes place within the cytoplasm of the plasmodium. The existence of the plasmodial stage and the development of a sexual stage within the plasmodium are unique features to Bilateria. However, the molecular mechanisms that maintain this peculiar organism, and hence enable parasitism in orthonectids, are unknown. Here, we present the first-ever RNA-seq analysis of the plasmodium, aimed at the identification and characterization of the plasmodium-specific protein-coding genes and corresponding hypothetical proteins that distinguish the parasitic plasmodium stage from the sexual stage of the orthonectid Intoshia linei Giard, 1877, parasite of nemertean Lineus ruber Müller, 1774. We discovered 119 plasmodium-specific proteins, 82 of which have inferred functions based on known domains. Thirty-five of the detected proteins are orphans, at least part of which may reflect the unique evolutionary adaptations of orthonectids to parasitism. Some of the identified proteins are known effector molecules of other endoparasites suggesting convergence. Our data indicate that the plasmodium-specific proteins might be involved in the plasmodium defense against the host, host-parasite communication, feeding and nutrient uptake, growth within the host, and support of the sexual stage development. These molecular processes in orthonectids have not been described before, and the particular protein effectors remained unknown until now.

The Mitochondrial Genomes of Siboglinum plumosum and Oligobrachia dogieli (Annelida: Siboglinidae) and Their Phylogenetic Analysis.

Frenulates are a group of sedentary Annelida within the family Siboglinidae that inhabit the ocean floor and present a unique challenge for comprehensive molecular and phylogenetic investigations. In this study, we focused on the frenulates, specifically assembling the mitochondrial genomes of Siboglinum plumosum and Oligobrachia dogieli. The phylogenetic reconstruction placed S. plumosum as a sister taxon to S. ekmani, and O. dogieli as a sister taxon to S. fiordicum, supporting the non-monophyletic nature of the genus Siboglinum. Overall, this study supports the phylogeny of the family Siboglinidae while highlighting the need for additional molecular data within frenulates.

Evolution of Orthonectida body plan.

Orthonectida is an enigmatic group of animals with still uncertain phylogenetic position. Orthonectids parasitize various marine invertebrates. Their life cycle comprises a parasitic plasmodium and free-living males and females. Sexual individuals develop inside the plasmodium; after egress from the host they copulate in the external environment, and the larva, which has developed inside the female infects a new host. In a series of studied orthonectid species simplification of free-living sexual individuals can be clearly traced. The number of longitudinal and transverse muscle fibers is gradually reduced. In the nervous system, simplification is even more pronounced. The number of neurons constituting the ganglion is dramatically reduced from 200 in Rhopalura ophiocomae to 4-6 in Intoshia variabili. The peripheral nervous system undergoes gradual simplification as well. The morphological simplification is accompanied with genome reduction. However, not only genes are lost from the genome, it also undergoes compactization ensured by extreme reduction of intergenic distances, short intron sizes, and elimination of repetitive elements. The main trend in orthonectid evolution is simplification and miniaturization of free-living sexual individuals coupled with reduction and compactization of the genome.

Plasmodium structure of Intoshia linei (Orthonectida).

Orthonectids are enigmatic parasitic bilaterians whose exact position on the phylogenetic tree is still uncertain. Despite ongoing debate about their phylogenetic position, the parasitic stage of orthonectids known as "plasmodium" remains underexplored. There is still no consensus on the origin of the plasmodium: whether it is an altered host cell or a parasitic organism that develops in the host extracellular environment. To determine the origin of the orthonectid parasitic stage, we studied in detail the fine structure of the Intoshia linei orthonectid plasmodium using a variety of morphological methods. The orthonectid plasmodium is a shapeless multinucleated organism separated from host tissues by a double membrane envelope. Besides numerous nuclei, its cytoplasm contains organelles typical for other bilaterians, reproductive cells, and maturing sexual specimens. Reproductive cells, as well as developing orthonectid males and females, are covered by an additional membrane. The plasmodium forms protrusions directed to the surface of the host body and used by mature individuals for egress from the host. The obtained results indicate that the orthonectid plasmodium is an extracellular parasite. A possible mechanism for its formation might involve spreading parasitic larva cells across the host tissues with subsequent generation of a cell-within-cell complex. The cytoplasm of the plasmodium originates from the outer cell, which undergoes multiple nuclear divisions without cytokinesis, while the inner cell divides, giving rise to reproductive cells and embryos. The term "plasmodium" should be avoided and the term "orthonectid plasmodium" could be temporarily used instead.