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.

No time to relax: Age-dependent infectivity of cercariae in marine coastal ecosystems.

Age dynamics of the ability of cercariae of two digenean species, Himasthla elongata (Himasthlidae) and Renicola parvicaudatus (Renicolidae), to infect the second intermediate host (SIH), mussels (Mytilus edulis), was investigated experimentally. This is the first study of this kind made on cercariae transmitted in the intertidal of the northern seas. The larvae of all tested ages (from 0.5 to 6 hr) were equally successful in infecting mussels. This finding disagrees with the literature data on cercariae of several freshwater digeneans, which are practically incapable of infecting the SIH during the first 1-3 hr of life. The presence of a time delay before the attainment of the maximum infectivity (TDMI) may be associated with the need for physiological maturation of cercariae in the very beginning of their life in the environment, the need for their broad dispersion, and the prevention of superinfection of the downstream host. The absence of TDMI in the cercariae examined in our study could be associated with the instability of environmental factors in the marine intertidal (wave impact, tidal currents). These factors promote a broad dispersion of cercariae in the intertidal biotope and prevent superinfection of potential SIHs. Biological and behavioural features may also play a role. We hypothesize that the presence or absence of TDMI does not depend on the taxonomic affiliation of the cercariae but is determined by the transmission conditions.

Overview of renicolid digeneans (Digenea, Renicolidae) from marine gulls of northern Holarctic with remarks on their species statuses, phylogeny and phylogeography.

Renicolid digeneans parasitize aquatic birds. Their intramolluscan stages develop in marine and brackish-water gastropods, while metacercariae develop in molluscs and fishes. The systematics of renicolids is poorly developed, their life cycles are mostly unknown, and the statuses of many species require revision. Here, we establish based on integrated morphological and molecular data that adult renicolids from gulls Larus argentatus and Larus schistisagus and sporocysts and cercariae of Cercaria parvicaudata from marine snails Littorina spp. are life-cycle stages of the same species. We name it Renicola parvicaudatus and synonymized with it Renicola roscovitus. An analysis of the cox1 gene of R. parvicaudatus from Europe, North America and North Asia demonstrates a low genetic divergence, suggesting that this species has formed quite recently (perhaps during last glacial maximum) and that interregional gene flow is high. In Littorina saxatilis and L. obtusata from the Barents Sea, molecular analysis has revealed intramolluscan stages of Cercaria littorinae saxatilis VIII, a cryptic species relative to R. parvicaudatus. In the molecular trees, Renicola keimahuri from L. schistisagus belongs to another clade than R. parvicaudatus. We show that the species of this clade have cercariae of Rhodometopa group and outline morphological and behavioural transformations leading from xiphidiocercariae to these larvae. Molecular analysis has revealed 3 main phylogenetic branches of renicolids, differing in structure of adults, type of cercariae and host range. Our results elucidate the patterns of host colonization and geographical expansion of renicolids and pave the way to the solution of some long-standing problems of their classification.

The Pacific Notocotylus atlanticus (Digenea: Notocotylidae).

Recently two unidentified Notocotylus species have been recovered from a mallard in Hokkaido, Japan. Preliminary data indicated that one of them may belong to N. atlanticus-a species found in Europe and on the Atlantic coast of North America. Now we have sequenced partial cox1 for several European N. atlanticus isolates and performed a barcode gap analysis. It has shown the conspecificity of European and Japanese worms, bringing up discussion on distribution, transmission and host specificity in this species.

Germinal development in embryonic rediae of the hemiuroid digenean Bunocotyle progenetica: an ultrastructural study.

Embryonic development of reproductive organs in rediae of the digenean Bunocotyle progenetica was studied using transmission electron microscopy. The germinal primordium becomes morphologically distinct in early embryos as a weakly separated cell mass with a forming cavity. It consists of undifferentiated, differentiating, and supporting cells. As embryos develop, the supporting cells form a wall around the enlarging cavity. Other cells of the germinal primordium are incorporated into the wall as solitary cells or as small cell aggregations. Those situated posteriorly give rise to an incipient germinal mass functioning during postembryonic development. Undifferentiated and differentiating cells in the middle and the anterior part of the primordium ensure a considerable growth of the cavity wall, which incorporates solitary germinal cells. In advanced embryonic rediae, these cells mature, cleave, and give rise to germinal balls, which enter the forming brood cavity. In the most mature embryonic rediae, all these early cercarial embryos reside in a brood cavity, which is lined by that time with a syncytium continuous with the supporting tissue of the incipient germinal mass. Based on our results and the literature data, we suggest that the morphogenesis of the reproductive apparatus of the daughter parthenitae in hemiuroid digeneans may be characterized by (1) emergence of an incipient brood cavity within the germinal primordium, (2) formation of the cavity lining from the cells of the germinal primordium, (3) fragmentation and uneven distribution of the germinal material of the germinal primordium around the cavity, and (4) an anticipatory development of some of this germinal material.

A month for the mission: using a sentinel approach to determine the transmission window of digenean cercariae in the subarctic White Sea.

In the digenean life cycle the cercaria ensures an important transmission stage, from the first intermediate host to the second or the definitive host. In regions with pronounced seasonality, this process occurs within a certain interval, the transmission window. In high latitudes, the size of transmission window has previously been determined only by comparing data on seasonal dynamics of infection level in various categories of hosts or extrapolating the results of laboratory experiments on cercarial biology to natural conditions. In this study, we evaluated the dynamics of infection of the second intermediate hosts (mussels Mytilus edulis) with cercariae of two digenean species, Himasthla elongata (Himasthlidae) and Cercaria parvicaudata (Renicolidae), at a littoral site at the White Sea by exposing cages with uninfected mussels during the warm season. This is the first such study in a subarctic sea. Mussel infection was observed from May to mid-September, but its intensity was the greatest only for approximately a month, from some moment after 10 July to mid-August, when water temperature was within the optimal range for cercarial emergence in both studied species (15‒20°C). During this time, the mussels accumulated 66.3 ± 6.2% metacercariae of H. elongata and 79.7 ± 5.3% metacercariae of C. parvicaudata out of the total number accumulated during the experimental period. We suggest that climate warming at high latitudes may prolong the period when the water temperatures are optimal for cercariae emergence, thereby intensifying digenean transmission in coastal ecosystems.

Elucidation of Himasthla leptosoma (Creplin, 1829) Dietz, 1909 (Digenea, Himasthlidae) life cycle with insights into species composition of the north Atlantic Himasthla associated with periwinkles Littorina spp.

Trematodes of the genus Himasthla are usual parasites of coastal birds in nearshore ecosystems of northern European seas and the Atlantic coast of North America. Their first intermediate hosts are marine and brackish-water gastropods, while second intermediate hosts are various invertebrates. We analysed sequences of partial 28S rRNA and nad1 genes and the morphology of intramolluscan stages, particularly cercariae of Himasthla spp. parasitizing intertidal molluscs Littorina spp. in the White Sea, the Barents Sea and coasts of North Norway and Iceland. We showed that only three Himasthla spp. are associated with periwinkles in these regions. Intramolluscan stages of H. elongata were found in Littorina littorea, of H. littorinae, in both L. saxatilis and L. obtusata, and of Cercaria littorinae obtusatae, predominantly, in L. obtusata. Other Himasthla spp. previously reported from Littorina spp. in North Atlantic are either synonymous with one of these species or described erroneously. Based on a comparison of newly generated 28S rDNA sequences with GenBank data, rediae and cercariae of C. littorinae obtusatae were identified as belonging to H. leptosoma. Some previously unknown morphological features of young and mature rediae and cercariae of the three Himasthla spp. are described. We provide a key to the rediae and highlight characters important for identification of cercariae. Genetic diversity within the studied species was only partially determined by their specificity to the molluscan host. The nad1 network constructed for H. leptosoma lacked geographical structure, which is explained by a high gene flow owing to highly vagile definitive hosts, shorebirds.

It is marine: distinguishing a new species of Catatropis (Digenea: Notocotylidae) from its freshwater twin.

The morphology of sexual adults is the cornerstone of digenean systematics. In addition, life cycle data have always been significant. The integration of these approaches, supplemented with molecular data, has allowed us to detect a new species that many researchers may have previously seen, but not recognized. Sexual adults from common eiders that we found in northern European seas were extremely similar to other notocotylids, but the discovery of their intermediate host, a marine snail, revealed the true nature of this material. Here we describe sexual adults, rediae and cercariae of Catatropis onobae sp. nov. We discuss how 'Catatropis verrucosa' should be regarded, justify designation of the new species C. onobae for our material and explain why it can be considered a cryptic species. The phylogenetic position of C. onobae within Notocotylidae, along with other evidence, highlights the challenges for the taxonomy of the family, for which two major genera appear to be polyphyletic and life cycle data likely undervalued.

Short communication: New data support phylogeographic patterns in a marine parasite Tristriata anatis (Digenea: Notocotylidae).

Intraspecific diversity in parasites with heteroxenous life cycles is guided by reproduction mode, host vagility and dispersal, transmission features and many other factors. Studies of these factors in Digenea have highlighted several important patterns. However, little is known about intraspecific variation for digeneans in the marine Arctic ecosystems. Here we analyse an extended dataset of partial cox1 and nadh1 sequences for Tristriata anatis (Notocotylidae) and confirm the preliminary findings on its distribution across Eurasia. Haplotypes are not shared between Europe and the North Pacific, suggesting a lack of current connection between these populations. Periwinkle distribution and anatid migration routes are consistent with such a structure of haplotype network. The North Pacific population appears ancestral, with later expansion of T. anatis to the North Atlantic. Here the parasite circulates widely, but the direction of haplotype transfer from the north-east to the south-west is more likely than the opposite. In the eastern Barents Sea, the local transmission hotspot is favoured.

Transatlantic discovery of Notocotylus atlanticus (Digenea: Notocotylidae) based on life cycle data.

Digenean parasites feature a series of stages with a distinct appearance, reproduction mode, and lifestyle that together constitute their well-known, complex life cycle. Species descriptions of Digenea have always been based on one of these stages-the marita, or sexually reproducing adult in the final host. However, in some cases, data on the life cycle are essential for the differential diagnosis of closely related species. Here, we present the case of Notocotylus atlanticus, where different stages of its life cycle were discovered for the first time since the species description, and across the Atlantic. We used a material from a naturally infected intertidal marine snail, Ecrobia ventrosa, and several waterfowl species and also carried out infection experiments. For morphological studies, we employed light microscopy, SEM, and CLSM; molecular data obtained include sequences of ITS1 and 28S rRNA gene. We demonstrate that N. atlanticus adult worm morphology is barely sufficient to distinguish it from several other species. Cercariae morphology and identity of the first intermediate hosts provide crucial additional information. According to our preliminary phylogenetic reconstructions, two notocotylid lineages are associated with two major gastropod lineages-the Caenogastropoda and the Heterobranchia. The traditional character to identify notocotylid genera (structure of ventral organs) fails to explain the phylogeny and thus requires reassessment. Further reliable morphological, life cycle and molecular data on other species are likely to reveal more patterns in notocotylid systematics, host specificity, and evolution.

Reproduction of trematodes in the molluscan host: an ultrastructural study of the germinal mass and brood cavity in daughter rediae of Tristriata anatis Belopolskaia, 1953 (Digenea: Notocotylidae).

This study describes the fine structure of the germinal mass in daughter rediae of Tristriata anatis. The germinal mass consists of undifferentiated cells, germinal cells and supporting cells and contains numerous cercarial embryos up to tail bud stage. Supporting cells and their outgrowths form a tight meshwork of the germinal mass. In its basal part, this meshwork serves as scaffolding for undifferentiated and germinal cells, naked cell aggregates and early germinal balls. More mature embryos are located apically. The hypertrophied supporting tissue appears to be involved in an intensive transport of substances, as indicated by abundant gap junctions between cell outgrowths and numerous pinocytotic vesicles and microtubules in their cytoplasm. Germinal cells contain annulate lamellae and the nuage, typical organelles of animal oocytes. In young rediae containing embryonic cercariae at the tail bud stage, the supporting tissue starts to degenerate in the apical part of the germinal mass, and a primordial brood cavity emerges though it develops fully only in mature rediae containing late embryonic cercariae. An unusual feature of the germinal mass in T. anatis rediae is an enhancement of the embryo brooding function. At the same time, the performance of this function by the brood cavity is reduced. This is the first time such a redistribution of the embryo brooding function between the germinal mass and the brood cavity has been reported.

Microphallus ochotensis sp. nov. (Digenea, Microphallidae) and relative merits of two-host microphallid life cycles.

A new digenean species, Microphallus ochotensis sp. nov., was described from the intestine of Pacific eiders (Somateria mollissima v-nigrum) from the north of the Sea of Okhotsk. It differs from other microphallids in the structure of the metraterm, which consists of two distinct parts: a sac with spicule-like structures and a short muscular duct opening into the genital atrium. Mi. ochotensis forms a monophyletic clade together with other congeneric species in phylograms derived from the 28S and ITS2 rRNA gene. Its dixenous life cycle was elucidated with the use of the same molecular markers. Encysted metacercariae infective for birds develop inside sporocysts in the first intermediate host, an intertidal mollusc Falsicingula kurilensis. The morphology of metacercariae and adults was described with an emphasis on the structure of terminal genitalia. Considering that Falsicingula occurs at the Pacific coast of North America and that the Pacific eider is capable of trans-continental flights, the distribution of Mi. ochotensis might span the Pacific coast of Alaska and Canada. The range of its final hosts may presumably include other benthos-feeding marine ducks as well as shorebirds. We suggest that a broad occurrence of two-host life cycles in microphallids is associated with parasitism in birds migrating along sea coasts. The chances that migrating birds would stop at a site where both first and second intermediate hosts occur are relatively low. The presence of a single molluscan host in the life cycle increases the probability of transmission.

Life cycle and biology of Tristriata anatis (Digenea: Notocotylidae): morphological and molecular approaches.

Notocotylids are common digeneans parasitising birds and mammals. They have a two-host life cycle with cercariae encysting in the open. Particular life cycles remain unknown for majority of notocotylid species, including a common parasite of sea ducks Tristriata anatis. Here we resolve the life cycle of T. anatis by means of D2 LSU, ITS1 and CO1 DNA sequence analysis, showing that the first intermediate hosts for this species are periwinkles Littorina spp. Morphological descriptions of rediae and cercariae are provided for the first time, and we also supplement the existing morphological data on adults. Apart from differential diagnosis, we discuss some features of cercariae and rediae biology, geographical distribution and host range. Our molecular data confirm that genus Tristriata is monotypic and that T. anatis has circumpolar distribution. CO1 sequence analysis has shown that isolation exists between the Atlantic and Pacific populations of T. anatis, suggesting that there are geographical races. We suppose that their formation may be linked to the Last Ice Age events, when trans-Arctic bird migrations ceased and periwinkle ranges shrunk. These made transfer of parasites across the Arctic impossible, and it still has not resumed. We discuss the possible influence of host vagility and adults' lifespan on digeneans' potential for geographical colonisation.

Trematode reproduction in the molluscan host: an ultrastructural study of the germinal mass in the rediae of Himasthla elongata (Mehlis, 1831) (Digenea: Echinostomatidae).

The germinal mass in Himasthla elongata rediae was studied in detail using transmission electron microscopy. It was shown to be a specialized reproductive organ consisting of germinal cells at various maturation stages, supporting cells and stem cells. The germinal mass also contains early cercarial embryos emerging as a result of cleavage division of mature germinal cells. The stem cells that give rise to germinal cells have heterochromatin-rich nuclei with distinct nucleoli and scarce cytoplasm containing mainly free ribosomes and few mitochondria. The differentiating germinal cells undergo a growth, which is accompanied by an emergence of annulate lamellae and the nuage in their cytoplasm, a noticeable development of RER and Golgi apparatus and an increase in the number of mitochondria. The mitochondria form a large group at one of the cell poles. During differentiation, the nucleus and nucleolus of the germinal cell enlarge while the chromatin becomes gradually less condensed. The supporting tissue of the germinal mass is made up of cells connected by septate junctions. These supporting cells are distinctly different in cellular shape and nuclear ultrastructure. Their outgrowths form a tight meshwork housing stem cells, germinal cells and early cercarial embryos. The cytoplasm of the supporting cells in the mesh area is separated into fine parallel layers by labyrinthine narrow cavities communicating with the intercellular space. The supporting tissue contains differentiating and degenerating cells which indicates its renewal. The results of this ultrastructural study lend support to the hypothesis that the germinal cells of digeneans are germ line cells.

Life cycles, molecular phylogeny and historical biogeography of the 'pygmaeus' microphallids (Digenea: Microphallidae): widespread parasites of marine and coastal birds in the Holarctic.

The 'pygmaeus' microphallids (MPG) are a closely related group of 6 digenean (Platyhelminthes: Trematoda) Microphallus species that share a derived 2-host life cycle in which metacercariae develop inside daughter sporocysts in the intermediate host (intertidal and subtidal gastropods, mostly of the genus Littorina) and are infective to marine birds (ducks, gulls and waders). Here we investigate MPG transmission patterns in coastal ecosystems and their diversification with respect to historical events, host switching and host-parasite co-evolution. Species phylogenies and phylogeographical reconstructions are estimated on the basis of 28S, ITS1 and ITS2 rDNA data and we use a combination of analyses to test the robustness and stability of the results, and the likelihood of alternative biogeographical scenarios. Results demonstrate that speciation within the MPG was not associated with co-speciation with either the first intermediate or final hosts, but rather by host-switching events coincident with glacial cycles in the Northern Hemisphere during the late Pliocene/Pleistocene. These resulted in the expansion of Pacific biota into the Arctic-North Atlantic and periodic isolation of Atlantic and Pacific populations. Thus we hypothesize that contemporary species of MPG and their host associations resulted from fragmentation of populations in regional refugia during stadials, and their subsequent range expansion from refugial centres during interstadials.

New data on Microphallus breviatus Deblock & Maillard, 1975 (Microphallidae: Digenea) with emphasis on the evolution of dixenous life cycles of microphallids.

Infection by intramolluscan stages of Microphallus breviatus Deblock & Maillard, 1975 are common in Hydrobia ventrosa mudsnails in Iceland. Cercariae encyst inside the daughter sporocysts and develop there into metacercariae that become infective for the definitive hosts which are probably charadriiform waders. The adult stage was obtained in 1-day-old chicks that were experimentally infected with metacercariae from naturally infected hydrobians. New data are presented on the morphology and biology of the cercariae, and the adult is described for the first time. Comparisons are made between M. breviatus and closely related species. Differential diagnosis of M. breviatus is given. The morphological specializations in larvae of the hermaphroditic generation of the microphallids accompanying transition from trixenous life cycles to dixenous ones are considered, and the applicability of the term "life-cycle truncation" to microphallids with dixenous life cycles is discussed. Also, reasons for the broad distribution of dixenous life cycles within the family Microphallidae are reviewed.

Factors influencing the distribution of digenetic trematode infections in a mudsnail (Hydrobia ventrosa) population inhabiting salt marsh ponds in Iceland.

This study analyzed the influence of several abiotic and biotic variables on the distribution of digenetic trematode infections in a mudsnail, Hydrobia ventrosa, population inhabiting 12 ponds on the Melabakkar salt marsh in Iceland, the northwestern limits of the geographical distribution. Nine trematode species were found to infect the snail population, which included Microphallus pirum, Microphallus breviatus, Microphallus claviformis, Maritrema subdolum (Microphallidae), Cercaria Notocotylidae sp. 11 Deblock, 1980, C. Notocotylidae sp. 12 Deblock, 1980, C. Notocotylidae sp. 13 Deblock, 1980 (Notocotylidae), Cryptocotyle concavum (Heterophyidae), and Psilostomum brevicolle (Psilostomatidae). Correlations between biotic variables (snail density in the ponds and vegetation cover), abiotic variables (distance of each pond from the sea, pond elevation above chart datum, size, average depth, salinity, and some characters of the littoral zone and sediments), and trematode infections were analyzed. These variables indirectly affected the trematode infections because some determined how attractive the ponds were for the final hosts, which were various species of marine and shore birds. We propose that their habitat use and defecating habits are the main determinants of the trematode distribution in the area.