Trematode maturation patterns in a migratory snail host: What happens during upshore residency in a Mediterranean lagoon?

Maturation of trematode larval stages is expected to be temporally and spatially adapted to maximise the encounter with the adequate downstream host, i.e. the host, which will be infected by this parasite stage. Since studies on intramolluscan parasite maturation are scarce but important in the context of parasite transmission, the larval development inside sporocysts was monitored during upshore residency of the snail host Gibbula adansonii (Trochidae), i.e., from March to May (2011 and 2013), when these snails temporarily reside in the intertidal habitat of a Western Mediterranean lagoon (40° 37' 35″ N, 0° 44' 31″ E, Spain). Data on the relative quantity of different maturation stages of Cainocreadium labracis and Macvicaria obovata (Opecoelidae) parasitising the G. adansonii as well as on snail and sporocyst size were explored using linear models and linear mixed models. The effect of the trematodes on snail growth was shown to be species-specific, with snail and sporocyst size acting as proxies of the reproductive capacity of M. obovata but not that of C. labracis. The number of cercarial embryos and germinal balls did not show monthly variation in either parasite species, but a higher number of mature stages and the highest maturity index was found in April. Hence, during the snail's limited spawning-related presence in the upshore waters of the lagoon, continuous production and output of infectious cercariae was observed, which indicates a link between larval maturation and snail migration. The synchronization of snails, mature parasite transmission stages and downstream hosts in time and space guarantees a successful completion of the life cycle.

Same host, same lagoon, different transmission pathways: effects of exogenous factors on larval emergence in two marine digenean parasites.

Due to their shallow and confined nature, lagoons provide excellent conditions for the transmission of digenean trematode parasites that require two or more intermediate hosts for the completion of their complex life cycles. However, these unstable environments are characterised by an internal heterogeneity and a large variation of a range of abiotic variables. We conducted a series of experiments in a comparative framework to assess the effect of a number of exogenous factors known to exhibit marked fluctuations in the lagoonal environment, i.e. temperature, salinity, water level and photoperiod, on larval emergence of two sympatric parasites, Cainocreadium labracis and Macvicaria obovata, which share the snail intermediate host, Gibbula adansonii, and a sit-and-wait downstream host-finding strategy. Our results demonstrated contrasting patterns and rates of larval emergence indicating an overall differential response of the two species to the variation of the environmental factors. Cercariae of M. obovata exhibited increased emergence rates at elevated temperature (with a sharp increase at 15 °C), decreased salinity (35-25 practical salinity units (psu)) and low water levels, whereas larval emergence of C. labracis was unaffected by the experimental variation in temperature and water level and showed decreased rates at decreased salinity levels (35-25 psu). The differential impact of the variable environmental conditions indicates the complexity of the patterns of exogenous control modifying parasite transmission and abundance in the lagoonal environment. Most importantly, the contrasting rhythms of larval emergence indicate endogenous control associated with the different transmission pathways of the two opecoelid digeneans.

Sphaerospora sensu stricto: taxonomy, diversity and evolution of a unique lineage of myxosporeans (Myxozoa).

Myxosporeans (Myxozoa) are eukaryotic parasites, primarily of fish, whose classification is in a state of flux as taxonomists attempt to synthesize the traditional morphology-based system with emerging DNA sequence-based phylogenies. The genus Sphaerospora Thélohan, 1892, which includes pathogenic species that cause significant impacts on fisheries and aquaculture, is one of the most polyphyletic taxa and exemplifies the current challenges facing myxozoan taxonomists. The type species, S. elegans, clusters within the Sphaerospora sensu stricto clade, members of which share similar tissue tropism and long insertions in their variable rRNA gene regions. However, other morphologically similar sphaerosporids lie in different branches of myxozoan phylogenetic trees. Herein, we significantly extend taxonomic sampling of sphaerosporids with SSU+LSU rDNA and EF-2 sequence data for 12 taxa including three representatives of the morphologically similar genus Polysporoplasma Sitjà-Bobadilla et Álvarez-Pellitero, 1995. These taxa were sampled from different vertebrate host groups, biogeographic realms and environments. Our phylogenetic analyses and statistical tests of single and concatenated datasets revealed Sphaerospora s. s. as a strongly supported monophyletic lineage, that clustered sister to the whole myxosporean clade (freshwater+marine lineages). Generally, Sphaerospora s. s. rDNA sequences (up to 3.7 kb) are the longest of all myxozoans and indeed metazoans. The sphaerosporid clade has two lineages, which have specific morphological, biological and sequence traits. Lineage A taxa (marine Sphaerospora spp.) have a single binucleate sporoplasm and shorter AT-rich rDNA inserts. Lineage B taxa (freshwater/brackish Sphaerospora spp.+marine/brackish Polysporoplasma spp.) have 2-12 uninucleate sporoplasms and longer GC-rich rDNA inserts. Lineage B has four subclades that correlate with host group and habitat; all Polysporoplasma species, including the type species, cluster together in one of these subclades. We thus suppress the genus Polysporoplasma and the family Polysporoplasmidae and emend the generic diagnosis of the genus Sphaerospora. The combination of morphological, biological and DNA sequence data applied in this study helped to elucidate an important part of the taxonomic puzzle within the phylum Myxozoa.

'Who's who' in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish--molecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto.

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.

The Acute Immune Responses of the Common Carp Cyprinus carpio to PLGA Microparticles-The Interactions of a Teleost Fish with a Foreign Material.

Poly lactic-co-glycolic acid (PLGA) particles safely and effectively deliver pharmaceutical ingredients, with many applications approved for clinical use in humans. In fishes, PLGA particles are being considered as carriers of therapeutic drugs and vaccine antigens. However, existing studies focus mainly on vaccine antigens, the endpoint immune responses to these (e.g., improved antibody titres), without deeper understanding of whether fishes react to the carrier. To test whether or not PLGA are recognized by or interact at all with the immune system of a teleost fish, we prepared, characterized and injected PLGA microparticles intraperitoneally into common carp. The influx, phenotype of inflammatory leukocytes, and their capacity to produce reactive oxygen species and phagocytose PLGA microparticles were tested by flow cytometry, qPCR, and microscopy. PLGA microparticles were indeed recognized. However, they induced only transient recruitment of inflammatory leukocytes that was resolved 4 days later whereas only the smallest µm-sized particles were phagocytosed. The overall response resembled that described in mammals against foreign materials. Given the similarities between our findings and those described in mammals, PLGA particles can be adapted to play a dual role as both antigen and drug carriers in fishes, depending on the administered dose and their design.

The description of two new species of Chloromyxum from skates in the Argentine Sea reveals that a limited geographic host distribution causes phylogenetic lineage separation of myxozoans in Chondrichthyes.

During a survey on the myxosporean fauna of Rajiformes from the Atlantic coast of Argentina, in waters off Buenos Aires Province (34°-42°S; 53°-62°W), the gall bladders of 217 specimens belonging to seven species of skates, representatives of two families, were examined. As a result, three species of Chloromyxum Mingazzini, 1890, namely C. atlantoraji n. sp., C. zearaji n. sp. and C. riorajum Azevedo, Casal, Garcia, Matos, Teles-Grilo and Matos, 2009 were found infecting three endemic host species, the spotback skate Atlantoraja castelnaui (Arhynchobatidae), the yellownose skate Zearaja chilensis (Rajidae) and the Rio skate Rioraja agassizii (Arhynchobatidae), respectively. These species were described based on myxospore morphology and morphometry characterization, as well as by providing their small subunit ribosomal DNA (SSU rDNA) sequences. The SSU rDNA-based phylogenetic analyses showed that these three species constituted a well-established monophyletic subclade within the marine Chloromyxum clade, while branches subtending the other Chloromyxum species were poorly resolved or unresolved, independently of the host taxonomic identities (Carchariniformes, Myliobatiformes, Orectolobiformes, Pristiophoriformes, Rajiformes, Squaliformes and Torpediniformes) and/or host geographic distribution (Atlantic coast of Portugal, Atlantic coast of the USA, Australian waters or Mediterranean Sea). The possible causes of these discrepancies are discussed, providing new insights into the phylogeny of the marine Chloromyxum clade.

An optimised multi-host trematode life cycle: fishery discards enhance trophic parasite transmission to scavenging birds.

Overlapping distributions of hosts and parasites are critical for successful completion of multi-host parasite life cycles and even small environmental changes can impact on the parasite's presence in a host or habitat. The generalist Cardiocephaloides longicollis was used as a model for multi-host trematode life cycles in marine habitats. This parasite was studied to quantify parasite dispersion and transmission dynamics, effects of biological changes and anthropogenic impacts on life cycle completion. We compiled the largest host dataset to date, by analysing 3351 molluscs (24 species), 2108 fish (25 species) and 154 birds (17 species) and analysed the resultant data based on a number of statistical models. We uncovered extremely low host specificity at the second intermediate host level and a preference of the free-swimming larvae for predominantly demersal but also benthic fish. The accumulation of encysted larvae in the brain with increasing fish size demonstrates that parasite numbers level off in fish larger than 140mm, consistent with parasite-induced mortality at these levels. The highest infection rates were detected in host species and sizes representing the largest fraction of Mediterranean fishery discards (up to 67% of the total catch), which are frequently consumed by seabirds. Significantly higher parasite densities were found in areas with extensive fishing activity than in those with medium and low activity, and in fish from shallow lagoons than in fish from other coastal areas. For the first time, C. longicollis was also detected in farmed fish in netpens. Fishing generally drives declines in parasite abundance, however, our study suggests an enhanced transmission of generalist parasites such as C. longicollis, an effect that is further amplified by the parasite's efficient host-finding mechanisms and its alteration of fish host behaviour by larvae encysted in the brain. The anthropogenic impact on the distribution of trophically-transmitted, highly prevalent parasites likely results in a strong effect on food web structure, thus making C. longicollis an ideal bioindicator to compare food webs in natural communities versus those impacted by fisheries and aquaculture.

Estimating trematode prevalence in snail hosts using a single-step duplex PCR: how badly does cercarial shedding underestimate infection rates?

BACKGROUND: Trematode communities often consist of different species exploiting the same host population, with two or more trematodes sometimes co-occuring in the same host. A commonly used diagnostic method to detect larval trematode infections in snails has been based on cercarial shedding, though it is often criticized as inaccurate. In the present study we compare infection prevalences determined by cercarial emission with those determined, for the first time, by molecular methods, allowing us to quantify the underestimation of single and double infections based on cercarial emission. We thus developed a duplex PCR for two host-parasite systems, to specifically differentiate between single and double infections. The Ebro samples include two morphologically similar opecoelids, whereas the Otago samples include two morphologically different larval trematodes. METHODS: Snails were screened for infections by incubating them individually to induce cercarial emission, thus determining infection following the "classical" detection method. Snail tissue was then removed and fixed for the duplex PCR. After obtaining ITS rDNA sequences, four species-specific primers were designed for each snail-trematode system, and duplex PCR prevalence was determined for each sample. Results from both methods were statistically compared using the McNemar's Chi-squared test and Cohen's Kappa Statistic for agreement between outcomes. RESULTS: Overall infection prevalences determined by duplex PCR were consistently and substantially higher than those based on cercarial shedding: among Ebro samples, between 17.9% and 60.1% more snails were found infected using the molecular method, whereas in the Otago samples, the difference was between 9.9% and 20.6%. Kappa values generally indicated a fair to substantial agreement between both detection methods, showing a lower agreement for the Ebro samples. CONCLUSIONS: We demonstrate that molecular detection of single and double infections by duplex PCR strongly outcompetes the classical method. Detection failure is most likely due to immature and covert infections, however, the higher incidence of misidentified double infections in the Ebro samples arises from morphological similarity of closely-related species. The higher accuracy of the duplex PCR method also adds to our understanding of community structure of larval trematodes in snail hosts, by providing a clearer assessment of the importance of interspecific interactions within the host.

Hidden diversity and evolutionary trends in malacosporean parasites (Cnidaria: Myxozoa) identified using molecular phylogenetics.

Malacosporeans represent a small fraction of myxozoan biodiversity with only two genera and three species described. They cycle between bryozoans and freshwater fish. In this study, we (i) microscopically examine and screen different freshwater/marine fish species from various geographic locations and habitats for the presence of malacosporeans using PCR; (ii) study the morphology, prevalence, host species/habitat preference and distribution of malacosporeans; (iii) perform small subunit/large subunit rDNA and Elongation factor 2 based phylogenetic analyses of newly gathered data, together with all available malacosporean data in GenBank; and (iv) investigate the evolutionary trends of malacosporeans by mapping the morphology of bryozoan-related stages, host species, habitat and geographic data on the small subunit rDNA-based phylogenetic tree. We reveal a high prevalence and diversity of malacosporeans in several fish hosts in European freshwater habitats by adding five new species of Buddenbrockia and Tetracapsuloides from cyprinid and perciform fishes. Comprehensive phylogenetic analyses revealed that, apart from Buddenbrockia and Tetracapsuloides clades, a novel malacosporean lineage (likely a new genus) exists. The fish host species spectrum was extended for Buddenbrockia plumatellae and Buddenbrockia sp. 2. Co-infections of up to three malacosporean species were found in individual fish. The significant increase in malacosporean species richness revealed in the present study points to a hidden biodiversity in this parasite group. This is most probably due to the cryptic nature of malacosporean sporogonic and presporogonic stages and mostly asymptomatic infections in the fish hosts. The potential existence of malacosporean life cycles in the marine environment as well as the evolution of worm- and sac-like morphology is discussed. This study improves the understanding of the biodiversity, prevalence, distribution, habitat and host preference of malacosporeans and unveils their evolutionary trends.

3D Morphology, ultrastructure and development of Ceratomyxa puntazzi stages: first insights into the mechanisms of motility and budding in the Myxozoa.

Free, amoeboid movement of organisms within media as well as substrate-dependent cellular crawling processes of cells and organisms require an actin cytoskeleton. This system is also involved in the cytokinetic processes of all eukaryotic cells. Myxozoan parasites are known for the disease they cause in economical important fishes. Usually, their pathology is related to rapid proliferation in the host. However, the sequences of their development are still poorly understood, especially with regard to pre-sporogonic proliferation mechanisms. The present work employs light microscopy (LM), electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) in combination with specific stains (Nile Red, DAPI, Phalloidin), to study the three-dimensional morphology, motility, ultrastructure and cellular composition of Ceratomyxa puntazzi, a myxozoan inhabiting the bile of the sharpsnout seabream.Our results demonstrate the occurrence of two C. puntazzi developmental cycles in the bile, i.e. pre-sporogonic proliferation including frequent budding as well as sporogony, resulting in the formation of durable spore stages and we provide unique details on the ultrastructure and the developmental sequence of bile inhabiting myxozoans. The present study describes, for the first time, the cellular components and mechanisms involved in the motility of myxozoan proliferative stages, and reveals how the same elements are implicated in the processes of budding and cytokinesis in the Myxozoa. We demonstrate that F-actin rich cytoskeletal elements polarize at one end of the parasites and in the filopodia which are rapidly de novo created and re-absorbed, thus facilitating unidirectional parasite motility in the bile. We furthermore discover the myxozoan mechanism of budding as an active, polarization process of cytokinesis, which is independent from a contractile ring and thus differs from the mechanism, generally observed in eurkaryotic cells. We hereby demonstrate that CLSM is a powerful tool for myxozoan research with a great potential for exploitation, and we strongly recommend its future use in combination with in vivo stains.

Molecular and morphological identification of larval opecoelids (Digenea: Opecoelidae) parasitising prosobranch snails in a Western Mediterranean lagoon.

In a study of the digeneans parasitising molluscs in the Els Alfacs lagoon (Ebro Delta, Western Mediterranean) we found heavy infections with sporocysts emitting two types of cotylocercous cercariae in the prosobranch trochid gastropod Gibbula adansonii and with metacercariae in the prosobranch nassariid gastropod Cyclope neritea. A comparative analysis using ITS ribosomal DNA sequences from these larval stages and published sequences of 17 larval and adult opecoelid stages allowed us to elucidate the life-cycle of Macvicaria obovata and to confirm the identification of Cainocreadium labracis based on cercarial morphology. We provide molecular evidence for the identification and the first detailed morphological descriptions of the intramolluscan larval stages of the two opecoelid species as well as partial 28S rDNA sequences to aid future studies on systematic relationships within the Opecoelidae.

Skoulekia meningialis n. gen., n. sp. (Digenea: Aporocotylidae Odhner, 1912) a parasite surrounding the brain of the Mediterranean common two-banded seabream Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) (Teleostei: Sparidae): description, molecular phylogeny, habitat and pathology.

This study describes a new aporocotylid genus and species, Skoulekia meningialis n. gen., n. sp. which was detected in the ectomeningeal veins surrounding the optic lobes of the brain of the common two-banded seabream Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) from the Gulf of Valencia (Mediterranean Sea). A detailed morphological description of S. meningialis is provided, including drawings, measurements and scanning electron microscopy images as well as a phylogenetic study of S. meningialis and closely related taxa using DNA sequence data obtained from whole ITS2 and partial 18S and 28S rDNA regions. Morphology as well as molecular phylogeny strongly support the erection of a new genus and demonstrate its close relationship with the genera Psettarium (Goto & Ozaki, 1930) and Pearsonellum Overstreet & Køie, 1989. Skoulekia is the second aporocotylid genus described in the Sparidae, a family including economical important fishes. In contrast to the majority of the aporocotylids, which inhabit the heart or the blood vessels of the gills, S. meningialis inhabits the ectomeningeal veins surrounding the optic lobes. Eggs were found trapped within the gill vessels. Normally, blood fluke pathology is almost exclusively related to the eggs. However, in the case of S. meningialis, main histopathological alterations were related to the adult blood flukes which were found to cause mild localised meningitis.

Zschokkella hildae Auerbach, 1910: phylogenetic position, morphology, and location in cultured Atlantic cod.

The myxozoan Zschokkella hildae Auerbach, 1910, was detected with a prevalence of 100% in cultured Atlantic cod, Gadus morhua L. aged 1+ from a culture facility on the west coast of Scotland. Sporogonic stages of Z. hildae, plasmodia producing 2-5 mature spores, were located predominantly in the collecting ducts and ureters of the kidney, and spores were present in the urine collected from the bladder. Less frequently, plasmodia were detected in the interstitial tissue of the kidney. The parasite prevalence in cultured fish was considerably higher than reported in wild fish but no obvious signs of pathology were detected. SSU rDNA sequencing and phylogenetic analysis showed that Z. hildae is closely related to a Sinuolinea sp. from the urinary system of turbot, Psetta maxima (L.), and that these two species, together with other myxozoans from the urinary system of marine fish cluster together in a sub-clade of the recognised marine clade of myxozoans. This sub-clade is characterised by a specific linear expansion segment, helix E23_15 in the secondary structure of variable region V4 of the SSU rDNA. Z. hildae and Sinuolinea sp. show extraordinary large linear expansion segment in both V4 and V7 and an important number of complementary base changes in the conservative regions of the SSU rDNA, indicating considerable evolutionary changes in the SSU rDNA of these species when compared with other myxozoans from the marine environment.