Daughter Sporocysts Research Articles

Development and reproduction of sporocysts of Leucochloridium paradoxum (Trematoda)

AbstractWe studied the development of sporocysts of the trematode Leucochloridium paradoxum. For this purpose, their life cycle was traced experimentally. Eggs obtained from adult trematodes raised in chicks (Gallus gallus domesticus) were used to infect snails (Succinea putris, laboratory strain). Histological studies of sporocysts were conducted for 95 days after exposure. During this time, the embryos of sporocysts developed into a small stolon filled with numerous metacercarial embryos. We found that miracidia hatched in the stomach and in the proximal part of the snail's midgut. After the larvae migrated into the area of the hepatopancreas, their body was destroyed, and germinal cells were released. They cleaved to form the embryos of sporocysts, in which only metacercarial embryos developed. Our study showed that these trematodes lack the parasitic phase of the mother sporocyst and have only one generation of parthenitae, the daughter sporocysts.

A single-cell atlas of the miracidium larva of Schistosoma mansoni reveals cell types, developmental pathways, and tissue architecture.

Schistosoma mansoni is a parasitic flatworm that causes the major neglected tropical disease schistosomiasis. The miracidium is the first larval stage of the life cycle. It swims and infects a freshwater snail, transforms into a mother sporocyst, where its stem cells generate daughter sporocysts that give rise to human-infective cercariae larvae. To understand the miracidium at cellular and molecular levels, we created a whole-body atlas of its ~365 cells. Single-cell RNA sequencing identified 19 transcriptionally distinct cell clusters. In situ hybridisation of tissue-specific genes revealed that 93% of the cells in the larva are somatic (57% neural, 19% muscle, 13% epidermal or tegument, 2% parenchyma, and 2% protonephridia) and 7% are stem. Whereas neurons represent the most diverse somatic cell types, trajectory analysis of the two main stem cell populations indicates that one of them is the origin of the tegument lineage and the other likely contains pluripotent cells. Furthermore, unlike the somatic cells, each of these stem populations shows sex-biased transcriptional signatures suggesting a cell-type-specific gene dosage compensation for sex chromosome-linked loci. The miracidium represents a simple developmental stage with which to gain a fundamental understanding of the molecular biology and spatial architecture of schistosome cells.

A single-cell atlas of the miracidium larva of Schistosoma mansoni reveals cell types, developmental pathways, and tissue architecture

Schistosoma mansoni is a parasitic flatworm that causes the major neglected tropical disease schistosomiasis. The miracidium is the first larval stage of the life cycle. It swims and infects a freshwater snail, transforms into a mother sporocyst, where its stem cells generate daughter sporocysts that give rise to human-infective cercariae larvae. To understand the miracidium at cellular and molecular levels, we created a whole-body atlas of its ~365 cells. Single-cell RNA sequencing identified 19 transcriptionally distinct cell clusters. In situ hybridisation of tissue-specific genes revealed that 93% of the cells in the larva are somatic (57% neural, 19% muscle, 13% epidermal or tegument, 2% parenchyma, and 2% protonephridia) and 7% are stem. Whereas neurons represent the most diverse somatic cell types, trajectory analysis of the two main stem cell populations indicates that one of them is the origin of the tegument lineage and the other likely contains pluripotent cells. Furthermore, unlike the somatic cells, each of these stem populations shows sex-biased transcriptional signatures suggesting a cell-type-specific gene dosage compensation for sex chromosome-linked loci. The miracidium represents a simple developmental stage with which to gain a fundamental understanding of the molecular biology and spatial architecture of schistosome cells.

Ultrastructure of an opecoelid daughter sporocyst, Podocotyle sp. (Digenea: Opecoelidae): comparative analysis of the somatic tissues and new insights into the organization of the nervous system

Digenea is a group of widespread parasitic flatworms with a complex life cycle including a successive change of parthenogenetic and hermaphroditic generations. Daughter sporocysts are among the least studied parthenitae in terms of the ultrastructure of their body wall and nervous system. Here we present an ultrastructural and immunocytochemical study of Podocotyle sp. daughter sporocyst (Opecoelidae), parasitizing in Littorina obtusata Linnaeus, 1758 from the White Sea. Our results focus on the structure of the body wall, birth pore, excretory and nervous systems, and include new data on the taxonomic affiliation based on the partial cox1 mitochondrial DNA sequence. The daughter sporocyst of Podocotyle sp. has ultrastructural similarities with different plagiorchiid and diplostomid digeneans and also possesses some specific features. In the studied species, the birth canal is epithelialized, the body wall is composed of nine types of somatic cells and includes a well-developed central nervous system. Neurons of the latter form specific hemidesmosome-like attachment sites, which we found in digeneans for the first time. The obtained results are necessary to complement the comparative morphological analysis of daughter parthenogenetic generations in different digenean lineages.

No Tail No Fail: Life Cycles of the Zoogonidae (Digenea)

The Zoogonidae is the only digenean family where known cercariae lack the tail but actively search for the second intermediate host. However, the data on the zoogonid life cycles are scarce. In the present study, we elucidated and verified life cycles of the Zoogonidae from the White Sea. Using rDNA data, we showed that Pseudozoogonoides subaequiporus utilizes gastropods from the family Buccinidae as the first intermediate host and protobranch bivalves as the second one. This life cycle can be facultatively truncated: some cercariae of P. subaequiporus encyst within the daughter sporocysts. Molecular data also confirmed previous hypotheses on Zoogonoides viviapus life cycle with buccinid gastropods acting as the first intermediate hosts, and annelids and bivalves as the second intermediate hosts. We demonstrated the presence of short tail primordium in the developing cercariae of both species. Based on the reviewed and our own data, we hypothesize that the emergence of tailless cercariae in the evolution of the Zoogonidae is linked to the switch to non-arthropod second intermediate hosts, and that it possibly happened only in the subfamily Zoogoninae. Basally branching zoogonids have retained the ancestral second intermediate host and might have also retained the tail.

Schistosoma mansoni larvae in vitro cultures using Biomphalaria glabrata extracts

Schistosomiasis is one of the most prevalent waterborne parasitic diseases affecting humans. In natural conditions, snails are necessary for maintenance of its lifecycle and also required as intermediate hosts to maintain the lifecycle in laboratory settings. In the present study, the location of S. mansoni larvae in Biomphalaria glabrata snails after infection (inoculation of miracidia) was investigated. Larvae were found located in the head-foot (HF) area of B. glabrata snails at 10 days post-infection (DPI), then their location was predominantly changed to the hepatopancreas and ovotestis (HPOT) area by 56 DPI. Next, the effects of extracts from various organs of B. glabrata snails including HF and HPOT for in vitro culturing of S. mansoni larvae were investigated. The HF extract enabled prolonged culturing of S. mansoni larvae. Furthermore, sequential use of that followed by the HPOT extract supported larval development or reproduction of daughter sporocysts. These results may provide important information for identifying essential factors and molecules for culturing Schistosoma larvae in vitro.

An atlas of the germ ball-cercaria-schistosomulum transition in Schistosoma mansoni, using confocal microscopy and in situ hybridisation

Schistosomes are complex platyhelminth parasites with a genome comprising ∼12,000 protein-coding genes, three distinct generations, and at least seven distinct phenotypes. We chart here cellular and gene expression changes associated with development of the cercaria, in the intramolluscan daughter sporocyst, and its transformation into the skin stage schistosomulum upon infection of the mammalian host. We describe the morphology of the early daughter sporocyst and the increasing complexity of cellular organisation in germ balls as they rapidly develop into cercariae. We show how individual myocytes differentiate and combine to create the complex musculature of the head capsule and body wall. In situ hybridisation reveals that some transcripts encoding the secretory proteins, released during skin penetration, are expressed in gland-cell precursors very early in germ ball development. However, those for the projected anti-inflammatory protein Sm16-stathmin are widely expressed in germ ball tissues, suggesting the protein has intracellular functions. Transcripts for smkk7 are expressed in six cells of the larval body, while the KK7 protein is present throughout the peripheral nerve net, including sensory nerve bulbs, providing a marker for the nerve net in adult worms. We also note that the cercaria-schistosomulum transformation is accompanied by tissue remodelling without growth.

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams, Ruditapes philippinarum, from Aphae-do (Island), Shinan-gun, Korea.

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphae-do (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307–570 (av. 395) μm long and 101–213 (av. 157) μm wide. Most of the daughter sporocysts contained 15–20 furcocercous cercariae each. The cercariae measured 112–146 (av. 134) μm in total length and 35–46 (av. 40) μm in width, with 69–92 (av. 85) μm long body and 39–54 (av. 49) μm long tail. The metacercariae were 210–250 (av. 231) μm in length and 170–195 (av. 185) μm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1–5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8–99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.

Seasonal dynamics of trematode infection in the first and the second intermediate hosts: A long-term study at the subarctic marine intertidal

The purpose of this study was to reveal seasonal reorganizations in groups of parthenitae (rediae or daughter sporocysts) of trematodes in the first intermediate hosts (1IH) and of their larvae (metacercariae) in the second intermediate hosts (2IH) in the nearshore ecosystems of the White Sea. The study involved two trematode species parasitizing seabirds, Himasthla littorinae (Himasthlidae) and Cercaria parvicaudata (Renicolidae), and was based on long-term seasonal monitoring of populations of 1IH (snails Littorina spp.) and 2IH (mussels Mytilus edulis) at two intertidal sites (66°N). We discovered that groups of H. littorinae rediae in the molluscan host were self-sustaining and could function for more than one warm season. Groups of daughter sporocysts of C. parvicaudata caused the death of the molluscan host in the course of a year. Parthenitae groups of both trematode species spend the cold season (7–8 months a year) in the state of developmental arrest. After the water warms up in spring, the surviving mature groups of sporocysts of C. parvicaudata start to emit cercariae, but their molluscan hosts die soon. Most of the mature rediae in groups of H. elongata die in autumn-winter; in spring young overwintered rediae mature and produce cercariae over the next warm period. The maximum prevalence of mature parthenitae groups of H. littorinae and C. parvicaudata in periwinkles was observed in the warmest period, July-August, when they produced and emitted numerous cercariae ensuring mass infection of mussels. Though metacercariae occurred in mussels all year round, the mean abundance usually reached the highest values in summer–autumn. In winter-spring the level of infection decreased because heavily infected molluscs died out and new molluscs were not infected as cercarial emergence from 1IH had stopped. We discuss the components of the transmission success of H. littorinae and C. parvicaudata in the Subarctic and suggest that the parasites’ transmission may intensify owing to the prolongation of the period of functional activity of parthenitae groups under conditions of a warming climate.

In vivo cultivation of tuna blood fluke Cardicola orientalis in terebellid intermediate hosts

Some fish blood flukes of the genus Cardicola (Aporocotylidae) are considered important pathogens of farmed/ranched tuna, Thunnus spp. Infections with Cardicola spp. might obstruct the blood flow in the gills via massive accumulations of eggs and often lead to mass mortalities in captive tuna. At present, oral administration of an anthelminthic drug, praziquantel is the most effective treatment, but the tuna farming industries are seeking non-drug control measures. Development of prophylactic and holistic measures have been difficult, owing to a lack of basic knowledge about these parasites. Unlike other trematodes which use molluscs, blood flukes of marine actinopterygian fish use terebellid polychaetes as intermediate hosts. However, information about the development of Cardicola spp. within intermediate hosts is very limited. Recent success in Cardicola opisthorchis sporocyst transplantation into the host polychaete has opened possibilities for the cultivation of Cardicola in the laboratory. Here, we conducted several transplantation trials with another tuna blood fluke, Cardicol orientalis, into its natural and surrogate polychaete hosts. Cardicola orientalis sporocysts were injected into a total of 195 Nicolea gracilibranchis, the natural host, and clear sporocyst development and reproduction was observed in 32 recipients (overall success rate 16.4%). The production of daughter sporocysts in the transplanted polychaete occurred within 14 days post injection, and one sporocystogenous cycle took approximately 4 weeks. Serial passage culture via transplantation of in vivo-cultured sporocysts was also achieved, but with limited sporocyst reproduction. In addition, sporocysts were successfully retrieved from six and one individuals of the surrogate hosts, Thelepus setosus (n = 10) and Thelepus japonicus (n = 5), respectively. These results indicate that the in vivo cultivation of C. orientalis sporocysts is possible, not only in its natural host but also in other terebellids, although the problems of high mortality and inconsistency in successful transplantation need to be resolved.

DICROCOELIUM LANCEATUM (TREMATODA, DICROCOELIIDAE): THE STUDY OF THE NEURO-MUSCULAR SYSTEM

The work provides data on the presence and localization in the nervous system of the causative agent of dicroceliosis, Dicrocoelium lanceatum serotonergic and FMRFaminergic structures. Using the immunocytochemical method and confocal scanning laser microscopy, the test substances were found in the central and peripheral parts of the parasite's nervous system — in the head ganglia, the central nervous commissure, the longitudinal nerve trunks and the transverse commissures connecting them. The innervation of the nerve fibers of the attachment organs of trematodes by immunoreactive to serotonin and neuropeptide is shown.Trematode Dicrocoelium lanceatum (Stiles and Hassal, 1896) (Dicrocoeliidae) is the causative agent of Dicroceliosis, a widespread helminthiasis of domestic and wild animals. Dwelling in the bile ducts of the liver and in the gallbladder, dicroceliums cause irreversible pathological changes in the host organism, chronic inflammation of the bile ducts, cirrhosis of the liver, and metabolic processes in animals occur. As a result of the disease, animals are depleted, their productivity decreases, growth and development lag, and the nutritional and caloric value of meat decrease.In the cycle of development of the causative agent of dicroceliasis, the intermediate hosts are land mollusks, while the additional hosts are ants. The eggs of the parasite along with the bile enter the host intestine, and then with the feces into the external environment. Mollusks swallow eggs, miracidia come out from the eggs in the intestines of the mollusk, and penetrate the liver, where daughter sporocysts develop, in which cercariae form. Of the daughter sporocysts, cercariae are introduced into the respiratory cavity of the mollusk, where they are covered with sticky mucous substance. The formed mucous lumps are pushed by the respiratory movements of the mollusk into the external environment. Infection of ants occurs when eating these mucous balls. Once in the intestinal tract, cercariae enter the ant's abdominal cavity, where they turn into metacercariae. The definitive owners infect on pastures, swallowing invasive ants along with grass.

Histological description of Cercaria etgesii Shchenkov, 2017 daughter sporocysts (Trematoda: Pleurogenidae): compartmentalization of the brood cavity and germinal mass morphology.

Morphology of the daughter sporocysts of Cercaria etgesii Shchenkov, 2017 (Pleurogenidae) has been described according to conventional histological techniques and subsequent 3D reconstruction with Bitplanelmaris® software. Novel data were obtained concerning the spatial organization of the brood cavity of Pleurogenidae daughter sporocysts. It was shown that the laminated structures of the endocyst are lining the brood cavity and carry out its compartmentalisation by formation of the chambers around developing and disintegrating embryos of cercariae. First description of the germinal mass of the Pleurogenidae daughter sporocysts is given. No mitoses in germinal mass were evident although apparent production of the cercariae embryos by the sporocysts take place. The analogous principle of daughter sporocysts morphofunctional organisation was noted for the studied representatives of closely related taxa of trematodes: Pleurogenidae (C. etgesii) and Microphallidae (Microphallus 'pygmaeus' group).

Transplantation of Cardicola opisthorchis (Trematoda: Aporocotylidae) sporocysts into the intermediate host, Terebella sp. (Polychaeta: Terebellidae)

Cardicola opisthorchis is a blood fluke pathogen significantly affecting cultured Pacific bluefin tuna Thunnus orientalis in Japan. It is known that the intermediate host of C. opisthorchis is a terebellid polychaete Terebella sp. In order to study the intrapolychaete larval development of C. opisthorchis, we transplanted sporocysts, which contained a large number of cercariae, of C. opisthorchis obtained from Terebella sp. into sporocyst-free Terebella sp., which had been maintained at 20°C. The transplanted sporocysts switched from cercarial to sporocystal production by 17days after transplantation (d.a.t.) and daughter sporocysts were released into the polychaete body cavity at 25d.a.t. Subsequently, the released daughter sporocysts produced daughter sporocysts again. Thereafter, daughter sporocysts that contained cercariae appeared at 38d.a.t. and gradually increased. At 51d.a.t., 136 sporocysts that had multiplied from the original two transplanted sporocysts were observed in the body of one polychaete, and cercariae were released from daughter sporocysts inside the polychaete body cavity. Subsequently the cercariae were found to be released outside the polychaete at 57d.a.t. This is the first successful case of in situ observation of the development of a blood fluke within the intermediate host.

Muscle system in rediae and daughter sporocysts of several digeneans

Morphological studies on digenetic trematodes are quite numerous, but the large majority of researchers deal with the hermaphroditic generation (cercariae, metacercariae, adults). Parthenitae (miracidia, sporocysts, rediae), although they constitute a significant part of a digeneans’ life cycle, attract much less attention. The sparse literature concerning the musculature of parthenitae indicates that it differs in many respects from that of digenean hermaphroditic generation and many other flatworms. We have tried to fill the knowledge gap on digenean muscle systems by focusing on daughter parthenitae (three species with rediae and two with daughter sporocysts). The study was performed using TRITC-phalloidin labeling and confocal microscopy and was aimed at both describing general trends and comparing different morphotypes. The basic body-wall muscle layers were confirmed to be composed of circular and longitudinal muscle fibers. Circular fibers form quite a uniform layer, and longitudinal fibers are typically joined into bundles. The density of muscle layers varies widely among studied species, and the possible causes of this variation are discussed. The internal muscle fibers and bundles are present in all studied species, being most prominent in the anterior region. The brood chamber and birth pore channel often have a muscular lining. The rediae pharynx has a typical set of muscle elements, mostly coinciding with one in the pharynx of the hermaphroditic generation. Taken together, our findings suggest that data on the musculature of daughter parthenitae are important from an evolutionary perspective.

Transmission rates of the bacterial endosymbiont, Neorickettsia risticii, during the asexual reproduction phase of its digenean host, Plagiorchis elegans, within naturally infected lymnaeid snails

BackgroundNeorickettsia are obligate intracellular bacterial endosymbionts of digenean parasites present in all lifestages of digeneans. Quantitative information on the transmission of neorickettsial endosymbionts throughout the complex life cycles of digeneans is lacking. This study quantified the transmission of Neorickettsia during the asexual reproductive phase of a digenean parasite, Plagiorchis elegans, developing within naturally parasitized lymnaeid pond snails.MethodsLymnaea stagnalis snails were collected from 3 ponds in Nelson County, North Dakota and screened for the presence of digenean cercariae. Cercariae were identified to species by PCR and sequencing of the 28S rRNA gene. Neorickettsia infections were initially detected using nested PCR and sequencing of a partial 16S rRNA gene of pooled cercariae shed from each parasitized snail. Fifty to 100 single cercariae or sporocysts were isolated from each of six parasitized snails and tested for the presence of Neorickettsia using nested PCR to estimate the efficiency at which Neorickettsia were transmitted to cercariae during asexual development of the digenean.ResultsA total of 616 L. stagnalis were collected and 240 (39%) shed digenean cercariae. Of these, 18 (8%) were Neorickettsia-positive. Six Neorickettsia infections were selected to determine the transmission efficiency of Neorickettsia from mother to daughter sporocyst and from daughter sporocyst to cercaria. The prevalence of neorickettsiae in cercariae varied from 11 to 91%. The prevalence of neorickettsiae in sporocysts from one snail was 100%.ConclusionPrevalence of Neorickettsia infection in cercariae of Plagiorchis elegans was variable and never reached 100%. Reasons for this are speculative, however, the low prevalence of Neorickettsia observed in some of our samples (11 to 52%) differs from the high prevalence of other, related bacterial endosymbionts, e.g. Wolbachia in Wolbachia-dependent filariid nematodes, where the prevalence among progeny is universally 100%. This suggests that, unlike the Wolbachia-filaria relationship, the Neorickettsia-digenean relationship is not obligatory mutualism. Our study represents the first quantitative estimate of the Neorickettsia transmission through the asexual phase of the digenean life cycle.

Associated and digenean fauna of the mussel Mytilus galloprovincialis cultured on shellfish tables in the lagoon of Bizerta (Tunisia)

1 - Shellfish tables used for the breeding in bags of the oyster Crassostrea gigas and the mussel Mytilus galloprovincialis are suitable support for rapid colonization by marine living organisms.2 - During the period, June 2008 to May 2009, monthly samples of M. galloprovincialis were collected from the mytiliculture farm situated in Menzel Jemil (Bizerta Lagoon). Specimens were analyzed to identify their associated and digenean fauna.3 - Overall samples contain 68 taxa belonging to 12 zoological classes. Crustaceans, Polychaetes and Bivalves dominated largely the associated fauna, with frequencies of 34.7%, 22.9% and 10.6% respectively. The Polychaete Myrianida pinnigera was identified for the first time in the Tunisian coast. The remaining groups of associated fauna (Ascidia, Ophiuroids, Gastropods, Actinaria, Gymnolaemata, Demosponges, Calcispongia, Turbellaria, and Echinids,) accounted for only 31.8%. Diversity indices showed a temporal variation of the taxonomic structure of listed groups related to the environmental conditions and the biological cycles.4 - The examination of ~1400 individual mussels showed the occurrence of a low number of Proctoeces maculatus sporocysts (Digenea, Fellodistomodae). Bag-shaped sporocysts containing, according to the maturation stage, germinal balls, daughter sporocysts and or cercariae colonize at the beginning of the infection, the digestive gland then gonads and totality of the mantle.

Characterization of randomly amplified polymorphic DNA (RAPD) fragments revealing clonal variability in cercariae of avian schistosome <i>Trichobilharzia szidati</i> (Trematoda: Schistosomatidae)

Recently we applied randomly amplified polymorphic DNA (RAPD) fingerprinting to detect clonal variability among individual cercariae within daughter sporocysts and rediae of 10 digenean trematodes (Platyhelminthes: Trematoda). The most variable RAPD patterns were obtained for Schistosomatidae representative-avian schistosome Trichobilharzia szidati. In this work, 50 polymorphic DNA fragments of approximately 300-1500 bp from RAPD patterns of individual T. szidati cercariae were cloned and sequenced. As a result genomic DNA sequences (total length of approximately 41,000 bp) revealing clonal variability in T. szidati cercariae were obtained and analyzed. The analysis indicated that these sequences contained tandem, inverted and dispersed repeats as well as regions homological to retroelements of two human parasites, Schistosoma mansoni and S. japonicum. Tandem and inverted repeats constituted 8.9% and 22.1% respectively, while the percentage of dispersed repeats was 21.0%. The average content of these components was 41.7% with the average AT content being 59.0%. About 40% of sequences included regions ranging in length from 96 to 1005 bp which displayed amino acid homology with open reading frame pol products of S. mansoni and S. japonicum retroelements: non-long terminal repeat retrotransposons (nLTRs, 76%), long terminal repeat retrotransposons (LTRs, 14%), and Penelope-like elements (PLEs, 10%). Most of these regions (86.4%) contained frameshifts, gaps, and stop-codons. The largest portion of them was homological to nLTRs of the RTE clade (67%). The number of sequences homologous to the members of CR1 lineage was 7 times smaller (9%). Homology with LTRs of Gypsy/Ty3 and BEL clades was revealed in 5% and 9% of cases respectively. We assume that the repetitive elements including retroelement-like sequences described in the current study may serve as the source of clonal variability detected previously in T. szidati and other digenean trematodes. Such genome regions rapidly accumulate mutations and thus may play an important functional role in the life history of the species.

Maritrema orensenseandMaritrema bonaerense(Digenea: Microphallidae): Descriptions, Life Cycles, and Comparative Morphometric Analyses

We elucidate the life cycle of Maritrema orensense for the first time and experimentally confirm that of the sympatric Maritrema bonaerense. In Argentinean estuaries, both species parasitize the cochliopid snail Heleobia australis as first intermediate host, the grapsid crabs Neohelice granulata and Cyrtograpsus angulatus as second intermediate hosts, and gulls as definitive hosts. Here, we describe the daughter sporocysts and cercariae of M. orensense and redescribe these stages for M. bonaerense. Sporocysts of M. orensense are shorter, with fewer developed cercariae than M. bonaerense. The cercariae of M. orensense have longer, larger, and more undulating cephalic glands than M. bonaerense. We redescribe metacercariae and adults of both species and compare them with the previous descriptions. Intestinal ceca length, vitellaria shape and extension, and egg size are the most relevant characteristics in metacercariae and adults for differentiating the species. Hence, the detailed morphological description and comparative analyses of morphometrics obtained from natural and experimental infections permit clear differentiation of M. orensense and M. bonaerense at each life stage.

Cercaria caribbea LVIII Cable, 1963 (Digenea: Cyathocotylidae) in the Republic of Korea and Its Surface Ultrastructure

Cercaria caribbea LVIII Cable, 1963 (Digenea: Cyathocotylidae) was detected from a brackish water gastropod species (Cerithideopsilla cingulata) in a coatal area of Shinan-gun, Jeollanam-do (Province), the Republic of Korea, and its surface ultrastructure was studied using a scanning electron microscope. The cercariae were found freely swimming or enveloped within daughter sporocysts when the snail host was mechanically broken. They were morphologically characterized by a linguiform and ventrally concave body, a long and bifurcated tail, and the presence of a holdfast (=tribocytic) organ posterior to the ventral sucker. On the whole ventral and dorsal surfaces, peg-like tegumental spines were densely distributed. Around the oral sucker, several sensory papillae, each with a short cilium, were distributed, and on the tail, sensory papillae, each with an extensively long cilium, were observed. This is the first record describing a cyathocotylid cercaria from a brackish water gastropod in the Republic of Korea.

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.