Parvatrema spp. (Digenea, Gymnophallidae) with parthenogenetic metacercariae: diversity, distribution and host specificity in the palaearctic.

There are several species of gymnophallid digeneans in the genus Parvatrema that are unique in developing metacercariae that reproduce by parthenogenesis in the second intermediate host. Transmission of these digeneans takes place in coastal ecosystems of the North Pacific and North Atlantic seas. The first intermediate hosts are bivalves, the second ones are gastropods, and the definitive hosts are migratory birds. We integrated data accumulated over 25 years of research and differentiated a complex of five closely related species. They differ in the molluscan second intermediate hosts, distribution ranges, and life cycles patterns. The type I life cycle includes two generations of parthenogenetic metacercariae, followed by development of metacercariae which are invasive for the definitive host. In the type II life cycle, the number of generations of parthenogenetic metacercariae is unlimited, and they can also produce cercariae. These cercariae emerge into the environment and can infect new individuals of the second intermediate host. We conclude that the type I life cycle is a derived option that has evolved as a better fit to transmission in the unstable conditions in the intertidal zone. Another evolutionary trend in Parvatrema is transition from inhabiting the extrapallial space of the gastropod second intermediate host to endoparasitism in its mantle and internal organs. rDNA sequence analysis highlighted that Parvatrema spp. with parthenogenetic metacercariae form a monophyletic clade and suggested the Pacific origin of the group, with two transfers to the North Atlantic and colonisation of new second intermediate host species. Apparently the group formed in the late Pliocene-Pleistocene and diversified as a result of recurrent isolation in inshore refugia during glacial periods. We argue that parthenogenetic metacercariae in Parvatrema may serve as a model for early digenean evolution, demonstrating the first steps of adopting the molluscan first intermediate host and becoming tissue parasites.

Tricks of the puppet masters: morphological adaptations to the interaction with nervous system underlying host manipulation by rhizocephalan barnacle Polyascus polygeneus.

Background: Rhizocephalan interaction with their decapod hosts is a superb example of host manipulation. These parasites are able to alter the host's physiology and behavior. Host-parasite interaction is performed, presumably, via special modified rootlets invading the ventral ganglions. Methods: In this study, we focus on the morphology and ultrastructure of these special rootlets in Polyascus polygeneus (Lützen & Takahashi, 1997), family Polyascidae, invading the neuropil of the host's nervous tissue. The ventral ganglionic mass of the infected crabs were fixed, and the observed sites of the host-parasite interplay were studied using transmission electron microscopy, immunolabeling and confocal microscopy. Results: The goblet-shaped organs present in the basal families of parasitic barnacles were presumably lost in a common ancestor of Polyascidae and crown "Akentrogonida", but the observed invasive rootlets appear to perform similar functions, including the synthesis of various substances which are transferred to the host's nervous tissue. Invasive rootlets significantly differ from trophic ones in cell layer composition and cuticle thickness. Numerous multilamellar bodies are present in the rootlets indicating the intrinsic cell rearrangement. The invasive rootlets of P. polygeneus are enlaced by the thin projections of glial cells. Thus, glial cells can be both the first hosts' respondents to the nervous tissue damage and the mediator of the rhizocephalan interaction with the nervous cells. One of the potential molecules engaged in the relationships of P. polygeneus and its host is serotonin, a neurotransmitter which is found exclusively in the invasive rootlets but not in trophic ones. Serotonin participates in different biological pathways in metazoans including the regulation of aggression in crustaceans, which is reduced in infected crabs. We conclude that rootlets associated with the host's nervous tissue are crucial for the regulation of host-parasite interplay and for evolution of the Rhizocephala.

Polymorphic parasitic larvae cooperate to build swimming colonies luring hosts.

Parasites have evolved a variety of astonishing strategies to survive within their hosts, yet the most challenging event in their personal chronicles is the passage from one host to another. It becomes even more complex when a parasite needs to pass through the external environment. Therefore, the free-living stages of parasites present a wide range of adaptations for transmission. Parasitic flatworms from the group Digenea (flukes) have free-living larvae, cercariae, which are remarkably diverse in structure and behavior.1,2 One of the cercariae transmission strategies is to attain a prey-like appearance for the host.3 This can be done through the formation of a swimming aggregate of several cercariae adjoined together by their tails.4 Through the use of live observations and light, electron, and confocal microscopy, we described such a supposedly prey-mimetic colony comprising cercariae of two distinct morphotypes. They are functionally specialized: larger morphotype (sailors) enable motility, and smaller morphotype (passengers) presumably facilitate infection. The analysis of local read alignments between the two samples reveals that both cercaria types have identical 18S, 28S, and 5.8S rRNA genes. Further phylogenetic analysis of these ribosomal sequences indicates that our specimen belongs to the digenean family Acanthocolpidae, likely genus Pleorchis. This discovery provides a unique example and a novel insight into how morphologically and functionally heterogeneous individuals of the same species cooperate to build colonial organisms for the purpose of infection. This strategy bears resemblance to the cooperating castes of the same species found among insects.5.

Functional role of lacunar and muscular systems in the externa of Peltogasterella gracilis (Cirripedia: Rhizocephala).

One of the most conspicuous traits of parasitic organisms is a well-developed reproductive system. In Rhizocephala ("Crustacea": Cirripedia) it is believed to be nested in the externa-a "reproductive part" located outside of the host. However, it is not clear how nutrients are transported to the externa. Several authors described a system of lacunae in the externa, and muscular contractions probably enable transport through these cavities. The aim of our study was to visualize (using microcomputed tomography and confocal laser scanning microscopy) and describe lacunar and muscular systems in the externa of Peltogasterella gracilis (fam. Peltogasterellidae). The lacunar system consists of "ventral" lacuna and several protrusions. The "ventral" lacuna is probably responsible for visceral mass nutrition, and mantle protrusions are associated with the mantle nutrition. The gross organization of the muscular system mostly corresponds to previous descriptions in other rhizocephalan species. Nonetheless, we observed several features of the externa morphology that had not been described before such as a muscular thickening in the proximal externa's part and a stalk plug disk. The muscular thickening might play a role of a propulsatory organ, helping to transport liquid through the lacunar system. The plug disk might fill the hole in the host's cuticle after the old externa drop off. The results allow us to make first assumptions on transport mechanisms in Rhizocephala.

Cancer spares no one: First record of neoplasm in parasitic barnacles (Arthropoda: Rhizocephala).

Cancer-like neoplasms are extremely rarely present in arthropods, particularly in crustaceans. Thus, it is assumed that these animals have some efficient cancer-preventing mechanisms. However, several cases of cancer-like neoplasms are described in crustaceans, though only for the Decapoda. We identified a tumor in the parasitic barnacle Peltogaster paguri (Cirripedia: Rhizocephala), and described its histological structure. A spherical cell mass consisting mostly of roundish cells with big translucent nuclei, prominent nucleoli, and sparse chromatin, and of cells with condensed chromosomes, was found in the main trunk of the P. paguri rootlet system. Numerous mitoses were observed in this area. Such tissue organization is utterly uncharacteristic of the Rhizocephala. Based on acquired histological data, we assume that this tumor is a cancer-like neoplasm. This is the first report of a tumor identified in the rhizocephalans, as well as in non-decapod crustaceans as a whole.

Species complexes and life cycles of digenetic trematodes from the family Derogenidae.

The best way to study digenean diversity combines molecular genetic methods, life-cycle studies and elaborate morphological descriptions. This approach has been barely used for one of the most widespread digenean taxa parasitizing fish ­ the superfamily Hemiuroidea. Here, we applied the integrative approach to the hemiuroideans from the family Derogenidae parasitizing fish at the White and Barents Seas. Analysis of 28S, 18S, 5.8S rDNA, ITS2 and cox1 gene sequences from sexually adult worms (maritae) showed genetic heterogeneity for 2 derogenid species known from this area: Derogenes varicus and Progonus muelleri. Thus, 2 pairs of genetic lineages were found: DV1 and DV2, PM1 and PM2, respectively. Data from other regions indicate that 2 more lineages of D. varicus probably exist. Based on previous records from the White and Barents Seas, we hypothesized that the cercariae found in the moonsnails (family Naticidae) belong to the Derogenidae and may help to differentiate these lineages as species. According to our results, Cercaria appendiculata from Cryptonatica affinis matched DV1, similar nameless cercariae from Euspira pallida and Amauropsis islandica matched DV2, and Cercaria octocauda from C. affinis matched PM1. We provide new data on the structure of these cercariae and discuss the life-cycle pattern of the studied digeneans.

The interna of the rhizocephalan Peltogaster reticulata: Comparative morphology and ultrastructure.

Specialized morphology of diverse parasitic crustaceans reflects their adaptations to an endoparasitic lifestyle. Rhizocephalan barnacles are one of the most highly modified obligatory parasites of other crustaceans. Comprehension of the functional morphology of rhizocephalans could elucidate the main evolutionary trends not only inside parasitic barnacles, but in parasitism as a whole. Despite that, the available morphological information on the rhizocephalans is very fragmented. In this study, we examined the organization and ultrastructural features in different parts of the interna of Peltogaster reticulata (fam. Peltogastridae). The main trunk cuticle is much thicker than that of the side branches due to the different functions of these body parts. The central lumen in the main trunk is lined by an extracellular matrix, while the side branches are not. Muscular fibers are only present in the body wall of the main trunk, where they are organized as a "wicker basket". Furthermore, functional differentiation can be found at the ultrastructural level in the cells of the rootlets: there are distinct cell types both in hypodermal and axial cell layers. The rootlets of P. reticulata are covered by a network of the host's neurons and capillaries.

From head to rootlet: comparative transcriptomic analysis of a rhizocephalan barnacle Peltogaster reticulata (Crustacea: Rhizocephala).

Background: Rhizocephalan barnacles stand out in the diverse world of metazoan parasites. The body of a rhizocephalan female is modified beyond revealing any recognizable morphological features, consisting of the interna, a system of rootlets, and the externa, a sac-like reproductive body. Moreover, rhizocephalans have an outstanding ability to control their hosts, literally turning them into "zombies". Despite all these amazing traits, there are no genomic or transcriptomic data about any Rhizocephala. Methods: We collected transcriptomes from four body parts of an adult female rhizocephalan Peltogaster reticulata: the externa, and the main, growing, and thoracic parts of the interna. We used all prepared data for the de novo assembly of the reference transcriptome. Next, a set of encoded proteins was determined, the expression levels of protein-coding genes in different parts of the parasite's body were calculated and lists of enriched bioprocesses were identified. We also in silico identified and analyzed sets of potential excretory / secretory proteins. Finally, we applied phylostratigraphy and evolutionary transcriptomics approaches to our data.  Results: The assembled reference transcriptome included transcripts of 12,620 protein-coding genes and was the first for any rhizocephalan. Based on the results obtained, the spatial heterogeneity of protein-coding gene expression in different regions of the adult female body of P. reticulata was established. The results of both transcriptomic analysis and histological studies indicated the presence of germ-like cells in the lumen of the interna. The potential molecular basis of the interaction between the nervous system of the host and the parasite's interna was also determined. Given the prolonged expression of development-associated genes, we suggest that rhizocephalans "got stuck in their metamorphosis", even at the reproductive stage. Conclusions: The results of the first comparative transcriptomic analysis for Rhizocephala not only clarified but also expanded the existing ideas about the biology of these extraordinary parasites.

Life cycle truncation in Digenea, a case study of Neophasis spp. (Acanthocolpidae).

Truncated life cycles may emerge in digeneans if the second intermediate host is eliminated, and the first intermediate host, the mollusc, takes up its role. To understand the causes of this type of life cycle truncation, we analyzed closely related species of the genus Neophasis (Acanthocolpidae) with three-host and two-host life cycles. The life cycle of Neophasis anarrhichae involves two hosts: wolffishes of the genus Anarhichas as the definitive host and the common whelk Buccinum undatum as the intermediate host. Neophasis oculata, a closely related species with a three-host life cycle, would be a suitable candidate for the comparison, but some previous data on its life cycle seem to be erroneous. In this study, we aimed to redescribe the life cycle of N. oculata and to verify the life cycle of N. anarrhichae using molecular and morphological methods. Putative life cycle stages of these two species from intermediate hosts were linked with adult worms from definitive hosts using ribosomal molecular data: 18S, ITS1, 5.8S-ITS2, 28S. These markers did not differ within the species and were only slightly different between them. Intra- and interspecific variability was also estimated using mitochondrial COI gene. In the constructed phylogeny Neophasis spp. formed a common clade with two other genera of the Acanthocolpidae, Tormopsolus and Pleorchis. We demonstrated that the first intermediate hosts of N. oculata were gastropods Neptunea despecta and B. undatum (Buccinoidea). Shorthorn sculpins Myoxocephalus scorpius were shown to act as the second intermediate and definitive hosts of N. oculata. The previous reconstruction of the two-host life cycle of N. anarrhichae was reaffirmed. We suggest that life cycle truncation in N. anarrhichae was initiated by an acquisition of continuous morphogenesis in the hermaphroditic generation and supported by a strong prey-predator relationship between A. lupus and B. undatum.

Specialised rootlets of Sacculina pilosella (Rhizocephala: Sacculinidae) used for interactions with its host's nervous system.

Parasitic rhizocephalan barnacles induce morphological, physiological, and behavioural changes in their hosts. The mechanisms of these intimate host-parasite interactions remain unknown. We have shown previously that rootlets of the internae of Peltogasterella gracilis and Peltogaster paguri penetrate the ganglion's envelope of their hermit crab hosts and form specialised structures in the ganglion periphery, the so-called goblet-shaped organs. Here, we examine the gross morphology and ultrastructure of these goblet-shaped organs in the interna of Sacculina pilosella. They consist of three layers of cells; in the intermediate layer of the organs, unusual lamellar bodies and muscle cells were found. Extensive degeneration of the host nervous tissue was observed in the funnel of the goblet-shaped organs. We conclude that the ability to penetrate into the host's nervous tissue could be a common trait in rhizocephalans. The goblet-shaped organs may play a key role in the host-parasite relationships by enabling the parasite to influence the host via hormones and neurotransmitters.

Muscular system in interna of Peltogaster paguri (Rhizocephala: Peltogastridae).

Rhizocephalan parasites have a peculiar life cycle, and their adults lost almost all traits found usually in Crustacea. Despite some data on anatomy and ultrastructure of interna of Peltogastridae, some crucial aspects of morphology are still unknown. For example, there is only one mentioning of myocytes found in interna of Rhizocephalans (Sacculina carcini). So we aimed at studying the muscular system of the interna of Peltogaster paguri using serial histological sectioning and fluorescent staining (TRITC-labeled phalloidin) with confocal microscopy. Within the wall of the main trunk we found striated muscular fibers. The majority of these fibers form a unidirectional single spiral. There are additional small fibers that connect the coils of the large spiral. The density of muscular fibers is highest near the externa stalk, and the number of muscle fibers decreases towards the distal part of the main trunk. We suggest that such a muscular system could provide peristaltic movements of the main trunk and the transport of nutrients through the interna.