Transcriptome analysis of pacific white shrimp (Penaeus vannamei) intestines and hepatopancreas in response to Enterocytozoon hepatopenaei (EHP) infection.

Penaeus vannamei is the most economically important species of shrimp cultured worldwide. Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that severely affects the growth and development of shrimps. In this study, the transcriptome differences between EHP-infected and uninfected shrimp were investigated through next-generation sequencing. The unigenes were assembled with the reads from all the four libraries. The differentially expressed genes (DEGs) of intestines and hepatopancreas were analyzed. There were 2,884 DEGs in the intestines and 2,096 DEGs in the hepatopancreas. The GO and KEGG enrichment analysis indicated that DEGs were significantly enriched in signaling pathways associated with nutritional energy metabolism and mobilizing autoimmunity. Moreover, the results suggested the downregulation of key genes in energy synthesis pathways contributed greatly to shrimp growth retardation; the upregulation of immune-related genes enhanced the resistance of shrimp against EHP infection. This study provided identified genes and pathways associated with EHP infection revealing the molecular mechanisms of growth retardation.

The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control.

Disease is a major limiting factor in the global production of cultivated shrimp. The microsporidian parasite Enterocytozoon hepatopenaei (EHP) was formally characterized in 2009 as a rare infection of the black tiger shrimp Penaeus monodon. It remained relatively unstudied until mid-2010, after which infection with EHP became increasingly common in the Pacific whiteleg shrimp Penaeus vannamei, by then the most common shrimp species farmed in Asia. EHP infects the hepatopancreas of its host, causing hepatopancreatic microsporidiosis (HPM), a condition that has been associated with slow growth of the host in aquaculture settings. Unlike other infectious disease agents that have caused economic losses in global shrimp aquaculture, EHP has proven more challenging because too little is still known about its environmental reservoirs and modes of transmission during the industrial shrimp production process. This review summarizes our current knowledge of the EHP life cycle and the molecular strategies that it employs as an obligate intracellular parasite. It also provides an analysis of available and new methodologies for diagnosis since most of the current literature on EHP focuses on that topic. We summarize current knowledge of EHP infection and transmission dynamics and currently recommended, practical control measures that are being applied to limit its negative impact on shrimp cultivation. We also point out the major gaps in knowledge that urgently need to be bridged in order to improve control measures.

Diagnosis and prevalence of two new species of haplosporidians infecting shore crabs Carcinus maenas: Haplosporidium carcini n. sp., and H. cranc n. sp.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

Cepaea spp. as a source of Brachylaima mesostoma (Digenea: Brachylaimidae) and Brachylecithum sp. (Digenea: Dicrocoeliidae) larvae in Poland.

Parasite diagnostics were carried out on 11 Polish populations of Cepaea spp. In three of them, coming from the roadside ditches of a village (Rytel, northern Poland), very high (up to 60%) prevalence of Brachylaima mesostoma was observed. This study provides the first molecular evidence of the presence of B. mesostoma inside Cepaea spp. in Europe. In a few snails from a population found in a private garden in a small town (Chelmza, northern Poland), larvae of Brachylecithum sp. were present. Cercariae and/or metacercariae of B. mesostoma were observed in both species of Cepaea: C. hortensis and C. nemoralis, whereas larvae of Brachylecithum sp. were found only in C. nemoralis. Both species of parasites inhabited snail hepatopancreas whose structure was significantly damaged by larvae. There was no significant connection between parasite invasion and snail host morphotype. The research did not allow the reasons for the high prevalence of B. mesostoma in Cepaea spp. to be explained, and also did not explicitly indicate how the parasite invaded Cepaea spp. individuals making them, at the same time a second intermediate host. However, it poses important questions about the life cycle of the parasite that may threaten extensively kept small-size farms of poultry.

Proteomic analysis highlights the immune responses of the hepatopancreas against Hematodinium infection in Portunus trituberculatus.

The parasitic dinoflagellate Hematodinium is considered an important pathogen of economically important marine crustaceans and has been reported from many wild and cultured species. While limited studies have been conducted to reveal the host-parasite interaction in crustaceans, the underlying molecular mechanisms between Hematodinium and its crustacean hosts are scarcely known. We conducted a comprehensive study to investigate the proteomic responses to Hematodinium infection in the hepatopancreas of Portunus trituberculatus using an iTRAQ-based quantitative proteomic technology. A total of 905 identified proteins including 392 differentially expressed proteins (DEPs) were subjected to GO, COG and KEGG-pathway enrichment analysis, with sixteen DEPs further validated by quantitative real-time PCR. Hematodinium parasites resulted in immune-suppressive and adverse effects on affected hosts, thorough inhibition of the important pattern recognition receptors (C-lectin, SR class B, and Toll)-mediated immune responses, regulation of the complement and coagulation pathway, dysregulation of important cell adhesion molecules and extracellular matrix, and imbalance of the cellular redox homeostasis in the hepatopancreas of affected crabs. Moreover, the lysosomes pathway was dysregulated seriously in the hepatopancreas of P. trituberculatus post Hematodinium challenge. The results provided evidences on how the Hematodinium parasite overcame the innate immunity of P. trituberculatus and caused pathological alteration in affected tissues BIOLOGICAL SIGNIFICANCE: The manuscript presented the first iTRAQ-based proteomic study of the host-parasite interaction between an important marine crustacean and the parasitic dinoflagellate Hematodinium. The manuscript reported the key pathways and proteins involved in the host-parasite interactions. The major findings will contribute to the better understanding of the molecular mechanism of the particular host-parasite interaction, as wells as the pathogenic process in susceptible tissues of affected crustacean hosts.

Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP).

BACKGROUND: Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. It is probably endemic in Australasia and was first characterized and named from the giant or black tiger shrimp Penaeus monodon from Thailand in 2009. Later, it was also found to infect exotic Penaeus vannamei imported for cultivation in Asia. HPM is not normally associated with shrimp mortality, but information from shrimp farmers indicates that it is associated with significant growth retardation that is not clearly noticeable until 2-3 months of cultivation. In order to study modes of HPM transmission and to test possible control measures, a laboratory challenge model was needed that would mimic the mode of infection in shrimp ponds. RESULTS: We describe successful transmission in a cohabitation model with natural E. hepatopenaei (EHP)-infected shrimp in closed, perforated plastic containers placed in aquaria together with free-swimming, uninfected shrimp. After a period of 14 days all the free-swimming shrimp tested positive by PCR (approximately 60% with heavy infections evident by 1-step PCR positive test results) and gave positive histological and in situ hybridization results for E. hepatopenaei (EHP) in the hepatopancreas. CONCLUSIONS: A laboratory cohabitation model for studying E. hepatopenaei (EHP) has been developed and used to confirm that E. hepatopenaei (EHP) can be directly transmitted horizontally among shrimp via water. The model will facilitate studies on methods to prevent the E. hepatopenaei (EHP) transmission.

Expression patterns of two heat-shock cognate 70 genes during immune responses and larval development of the Chinese mitten crab Eriocheir sinensis.

Two heat-shock protein (HSP) 70 family transcripts, heat-shock protein 70 cognate 5 and heat-shock protein 70 cognate 3 (designated as EsHSC70-5 and EsHSC70-3, respectively), were isolated from the Chinese mitten crab Eriocheir sinensis and their expression profiles were evaluated for their responsiveness to larval development and immune challenge in adult crabs. The HSPs exhibited 45-89% identity with other heat-shock proteins, and they shared similar structural features. EsHSC70 mRNA expression was detected not only during infection but also during the developmental larval stages. The EsHSC70s were enriched, and their expression fluctuated during early development. EsHSC70 mRNA expression was significantly induced by Vibrio parahaemolyticus challenge in all of the tissues studied (P < 0.05). Expression of EsHSC70 mRNA in the hepatopancreas and at the early zoeal stages was particularly pronounced, and the two EsHSC70s exhibited differential expression patterns both chronologically and spatially. The EsHSC70-5 mRNA level was significantly downregulated in the intestine and gills compared to that in controls at nearly all time points, and was expressed at a lower level after the bacterial challenge, indicating that EsHSC70-5 and EsHSC70-3 respond to immune challenges. The stage-specific enrichment of EsHSC70 transcripts in crabs suggests that these stress proteins play an essential role during brachyurization events.

Infections of Larval Stages of Dicrocoelium dendriticum and Brachylaima sp. in Brown Garden Snail, Helix aspersa, in Turkey.

The aim of this study was to determine the presence and prevalence of larval stages of Dicrocoelium dendriticum and Brachylaima sp. in the first intermediate host, a species of land snail, Helix aspersa, in Turkey. A total of 211 snails were collected in April-May 2014 from pastures in Mersin District. Larval stages of D. dendriticum were identified under a light microscope. Hepatopancreas from naturally infected H. aspersa snails were examined histologically. The prevalence of larval stages of D. dendriticum and Brachylaima sp. in H. aspersa snails was found to be 2.4% and 1.9%, respectively, in Mersin, Turkey. Cercariae were not matured in sporocysts at the beginning of April; however, it was observed that cercariae matured and started to leave sporocysts by early-May. Thus, it was concluded that H. aspersa acts as an intermediate host to D. dendriticumin and Brachylaima sp. in Mersin, Turkey. A digenean trematode Brachylaima sp. was seen for the first time in Turkey.

Early transcriptional response to the parasitic dinoflagellate Hematodinium in hepatopancreas of Portunus trituberculatus.

The swimming crab Portunus trituberculatus supports a large proportion of crab aquaculture in China. In the last decade, the sustainable culture of this crab was threatened by the parasitic dinoflagellate Hematodinuim, resulting in massive mortality (up to 95%) in severely impacted culture ponds. Previous studies of Hematodinium were mainly focused on histology, molecular characterization, epizootiology, etc., with limited studies conducted to explore this specific host-parasite interaction. Thus, to give a primary insight into the anti-parasitic immune response at the critical stage of infection, the expression levels of 8 immune-related genes together with enzyme activities of phenoloxidase (PO), alkaline phosphatase (AKP), and acid phosphatase (ACP), were evaluated in hepatopancreas during 3-192h (h) post inoculation. Hematoxylin-eosin (H&E) staining showed noticeable pathological changes in hepatopancreas. The enzyme activities of PO, AKP, and ACP were significantly induced after inoculation. The changes of the prophenoloxidase (proPO) transcripts and the constantly enhanced PO activity reflected the critical function of the proPO system in resisting against the parasites. The decreased expression levels of LGBP and PPAF implied an immunosuppressive mechanism of the parasites against the host proPO system. And the significant variations in transcriptional levels of two important proteinase inhibitors (serpin, α2m) and three P. trituberculatus clip-domain serine proteinases (PTcSPs) suggested that the parasites could affect proteinase cascade reactions associated with immune response by destroying the balance between serine proteinases and the inhibitors. Moreover, the results indicated that the hepatopancreas of P. trituberculatus was significantly affected by invasion of the parasite, and hepatopancreas played important roles in the crustacean innate immunity against the parasitic infection.

Characterization of α-L-fucosidase and other digestive hydrolases from Biomphalaria glabrata.

Schistosoma mansoni is one of the major agents of the disease Schistosomiasis, which is one of the major global public health concerns. Biomphalaria glabrata is an obligate intermediate mollusc host of S. mansoni. Although the development of S. mansoni occurs in the snail hepatopancreas, studies that focus on this organ remain limited. In this study, we biochemically identified five distinct carbohydrases (amylase, maltase, α-glucosidase, trehalase, and α-L-fucosidase), lipases, and peptidases in the B. glabrata hepatopancreas and focused on the isolation and characterization of the activity of α-L-fucosidase. The isolated α-L-fucosidase has a molecular mass of 141 kDa, an optimum pH of 5.8, and is inhibited by Tris, fucose, and 1-deoxyfuconojirimycin. B. glabrata α-L-fucosidase is an exoglycosidase that can hydrolyze the natural substrate fucoidan to fucose residues. It presented Km values of 48.4 µM to 4-Methylumbelliferyl α-L-fucopyranoside and 0.55 mM to p-nitrophenyl-α-L-fucopyranoside. Thus, α-L-fucosidase has a high activity in the hepatopancreas of B. glabrata, and the differential expression of this enzyme between susceptible and resistant strains indicates that besides its digestive role, α-L-fucosidase may also be important in host/parasite interactions.

Infections of Larval Stages of Dicrocoelium dendriticum and Brachylaima sp. in Brown Garden Snail, Helix aspersa, in Turkey

The aim of this study was to determine the presence and prevalence of larval stages of Dicrocoelium dendriticum and Brachylaima sp. in the first intermediate host, a species of land snail, Helix aspersa, in Turkey. A total of 211 snails were collected in April-May 2014 from pastures in Mersin District. Larval stages of D. dendriticum were identified under a light microscope. Hepatopancreas from naturally infected H. aspersa snails were examined histologically. The prevalence of larval stages of D. dendriticum and Brachylaima sp. in H. aspersa snails was found to be 2.4% and 1.9%, respectively, in Mersin, Turkey. Cercariae were not matured in sporocysts at the beginning of April; however, it was observed that cercariae matured and started to leave sporocysts by early-May. Thus, it was concluded that H. aspersa acts as an intermediate host to D. dendriticumin and Brachylaima sp. in Mersin, Turkey. A digenean trematode Brachylaima sp. was seen for the first time in Turkey.

White feces syndrome of shrimp arises from transformation, sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies superficially resembling gregarines.

Accompanying acute hepatopancreatic necrosis disease (AHPND) in cultivated Asian shrimp has been an increasing prevalence of vermiform, gregarine-like bodies within the shrimp hepatopancreas (HP) and midgut. In high quantity they result in white fecal strings and a phenomenon called white feces syndrome (WFS). Light microscopy (LM) of squash mounts and stained smears from fresh HP tissue revealed that the vermiform bodies are almost transparent with widths and diameters proportional to the HP tubule lumens in which they occur. Despite vermiform appearance, they show no cellular structure. At high magnification (LM with 40-100x objectives), they appear to consist of a thin, outer membrane enclosing a complex of thicker, inter-folded membranes. Transmission electron microscopy (TEM) revealed that the outer non-laminar membrane of the vermiform bodies bore no resemblance to a plasma membrane or to the outer layer of any known gregarine, other protozoan or metazoan. Sub-cellular organelles such as mitochondria, nuclei, endoplasmic reticulum and ribosomes were absent. The internal membranes had a tubular sub-structure and occasionally enclosed whole B-cells, sloughed from the HP tubule epithelium. These internal membranes were shown to arise from transformed microvilli that peeled away from HP tubule epithelial cells and then aggregated in the tubule lumen. Stripped of microvilli, the originating cells underwent lysis. By contrast, B-cells remained intact or were sloughed independently and whole from the tubule epithelium. When sometimes engulfed by the aggregated, transformed microvilli (ATM) they could be misinterpreted as cyst-like structures by light microscopy, contributing to gregarine-like appearance. The cause of ATM is currently unknown, but formation by loss of microvilli and subsequent cell lysis indicate that their formation is a pathological process. If sufficiently severe, they may retard shrimp growth and may predispose shrimp to opportunistic pathogens. Thus, the cause of ATM and their relationship (if any) to AHPND should be determined.

[The prevalence of Dicrocoeliid (Trematoda: Digenea) larval stages in Helix lucorum Linneus 1758 (Mollusca: Pulmonata) in the vicinity of Kastamonu]. / Kastamonu civarinda dagilis gösteren Helix lucorum Linnaeus, 1758 (Mollusca: Pulmonata)'da Dicrocoeliid (Trematoda: Digenea) larval safhalarinin yayginligi.

OBJECTIVE: The aim of this study was to investigate the prevalence of Dicrocoeliid larval stages in Helix lucorum Linnaeus, 1758 (Mollusca: Pulmonata) living in the vicinity of Kastamonu. METHODS: Snails were collected in the vicinity of Tasköprü, Kastamonu in March, April, May 2013. They were dissected while alive and their hepatopankreas were removed and placed on clean glass slide with a drop of 0.6% NaCl solution. The hepatopankreas were incised with a mounted needle and a thin film of the hepatopankreas fluid was drown out on a slide for examination of live parasites. After examination, larval stages fixed in %70 alcohol, formaldehyde-ethyl alcohol, 10% formalin and bouin. RESULTS: H. lucorum which is a land snail species has been reported for the first time being an intermediate host in the life cycle of Dicrocoeliid (Trematoda: Digenea) species with the prevalence of 27.6% from our country. Morphological and histological features of larval stages were determined. CONCLUSION: H. lucorum is an intermediate host in the life cycle of Dicrocoeliid species. The prevalence of infection was highest in April. This land snail species is important for the health of animal and human.

[The evaluation of snail host-trematode parasite trophic relationships using stable isotope analysis].

Stable isotope ratios of carbon (13C/12C, δ13C) and nitrogen (15N/14N, δ15N) in snail-host tissue (the foot and hepatopancreas) and trematode parasites on two stages of their life cycle were analyzed. Trophic structure in co-occurring trematode larvae was examined in the following species: five species of cercariae (Echinoparyphium recurvatum, Hypodereum conoideum, Plagiorchis mutationis, Diplostomum chromatophorum and D. volvens) and two species of metacercariae (Cotylurus cornutus and Echinoparyphium recurvatum) within two closely related snail hosts Lymnaea stagnalis and L. tumida using stable isotope analyses. Snail and parasite sampling was conducted in a riverine portion of the Kargat River of the Lake Chany basin, in the south of Western Siberia (54 degrees 37'76"N, 78 degrees 13'07" E), in August 2009. Four out of five studied cercariae species were depleted in 15N as well as in 13C relatively to snail hosts tissues (foot and hepatopancreas), supporting our previously published data for Plagiorchis mutationis and Echinoparyphium recurvatum cercariae. Such fractionation of cercariae is untypical of the commonly observed relationship between consumers and their food. D. chromatophorum cercariae had demonstrated an insufficient enrichment in δ15N volume in relation to the hepatopancreas. Both two species of metacercariae (C. cornutus and E. recurvatum) showed a significant enrichment in δ15N volume relatively to the host tissue consumed (fractionation values ranging from 1.5 to 4 per thousand depending on the species). The differences in δ15N and δ13C volume between cercariae and metacercariae observed in this study illustrate the complexity of the host--parasite trophic relationships. Such isotopic differences between cercariae and metacercariae can probably be explained by selective consumption of specific amino acids or lipids or by changes in metabolism associated with the life cycle of the parasite. The present study represents the first comparative analysis of trophic relationships between the host and the endoparasite at different life cycle stages. It demonstrates the potency of the stable isotope analyses for understanding trophic relationships in multispecies parasite communities.

Morphology and histopathology of Calyptospora sp. (Apicomplexa: Calyptosporidae) in speckled peacock bass, Cichla temensis Humboldt, 1821 (Perciformes: Cichlidae), from the Marajó-Açu River, Marajó Island, Brazil.

Several species of coccidia are protozoan parasites that cause infection in a wide variety of animal groups. Calyptospora is an important genus of protozoan, which infests both freshwater and marine fish. The hepatopancreases of 150 speckled peacock bass captured on Marajó Island, Brazil were studied macro- and microscopically. Oocysts were found in 84 (56%) of the specimens in both the examination of the fresh material by compression and the analysis of histological sections stained with hematoxylin-eosin. Small, circular, homogeneous forms in negative contrast had a mean diameter of 21.2 µm, frequently with pyriform sporocysts, with a mean length of 9.2 µm and width of 3.1 µm, and a thin-walled capsule, were observed in both the hepatic and the pancreatic parenchyma, but were completely devoid of any inflammatory reaction. Calyptospora infections are documented for the first time in the Marajó-Açu River.

Three goose-type lysozymes in the gastropod Oncomelania hupensis: cDNA sequences and lytic activity of recombinant proteins.

Three goose-type (g-type) lysozymes, designated as OHLysG1, OHLysG2 and OHLysG3 were identified from expressed sequence tags (ESTs) of a gastropod Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The full cDNA sequences of OHLysG1, OHLysG2 and OHLysG3 consisted of 735, 909 and 808 nucleotides, with an open reading frame of 198, 214 and 249 codons containing a 21, 7 and 8 amino acid (aa) signal peptide at the N-terminus, respectively. The three g-type lysozymes shared conserved features with other g-type lysozymes, such as the substrate binding sites, the catalytic residues critical for the fundamental structure and function of g-type lysozymes. It seems possible that g-type lysozymes in molluscs shared one conserved cysteine with those in birds and mammals, and six conserved cysteines were observed for mollusc g-type lysozymes, with two unique cysteines present in the g-type lysozymes of O. hupensis. The three lysozyme genes were expressed mainly in hepatopancreas, with relatively low expression level observed in head-foot muscle and intestine. When comparing S. japonicum-infected and uninfected snails, significant increase (P<0.05) was observed for all the three lysozymes in infected snails, with the highest increase detected in hepatopancreas, and lowest in intestine, implying their defensive role in the host-parasite, i.e. snail-trematode system. The three recombinant lysozymes expressed in Escherichia coli strain M15 showed lytic activity against Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus. In conclusion, the finding of three g-type lysozymes in O. hupensis provides structural and functional evidence of multiple g-type lysozymes in gastropod, which may have evolutional implication in the snail-trematode system.

Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe.

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.

Larval digenean community parasitizing the freshwater snail, Biomphalaria peregrina (Pulmonata: Planorbidae), from a temporary pond in Patagonia, Argentina.

Knowledge of population dynamics of parasites in freshwater snails from South America is scarce. The objective of the present study was to describe the infection dynamics of larval digeneans in the planorbid snail, Biomphalaria peregrina , during 2 sampling periods in a Patagonian temporary pond. In total, 1,003 snails were examined. Rediae of Notocotylus biomphalariae and Echinoparyphium sp., sporocysts of Cotylurus sp., and metacercariae of the 2 latter species were found. The overall prevalence was significantly higher in the second sampling period, always as single-species infections in the hepatopancreas. The presence of larvae in the first sampled snails of the second hydroperiod indicated that parasitized snails survive drought. Both species exhibited different seasonal prevalence patterns, with Echinoparyphium sp. present in all sampling months. Metacercariae of Echinoparyphium sp. occurred in the heart and kidney, and those of Cotylurus sp. between organs. No significant differences in overall prevalence of metacercariae were found, and a progressive rise in prevalence from spring to summer for both species was observed. Almost all size classes of B. peregrina were infected with metacercariae of both species, but rediae and sporocysts were present only in snails larger than 3.1 mm. The predictability of the hydroperiod year after year, the tolerance of B. peregrina to drought, and the survival of infected specimens allows the parasite community to show a similar pattern of infection over time. This is the first study in Argentina analyzing the infection dynamics of digeneans of a pulmonate snail from a temporary pond.

Utilization of tissue habitats by Myxobolus wulii Landsberg & Lom, 1991 in different carp hosts and disease resistance in allogynogenetic gibel carp: redescription of M. wulii from China and Japan.

Myxobolus wulii (=Myxosoma magna) was first described from the gills of goldfish, Carassius auratus auratus, in China. Subsequently, a myxosporean infecting the hepatopancreas of allogynogenetic gibel carp, C. auratus gibelio, was designated as a different species, Myxobolus guanqiaoensis, although the morphological features were almost identical to those of M. wulii. In Japan, an unidentified Myxobolus sp. was found in the gills and hepatopancreas of goldfish. Morphological and molecular analyses in the present study identified these myxosporeans as M. wulii, which was thus shown to use different habitats in the host fish. Phylogenetic analyses of small subunit ribosomal RNA gene sequences showed that M. wulii is closely related to two gill-infecting Myxobolus species, M. ampullicapsulatus and M. longisporus. Fish infected with M. wulii in the hepatopancreas exhibit swollen abdomens and chronic mortality. Hepatopancreas tissues are virtually destroyed and replaced with plasmodia of M. wulii. A remarkable difference in susceptibility to M. wulii between two clones of allogynogenetic gibel carp was observed, suggesting that resistance to the myxosporean infection was established in a clone of fish bred by allogynogenesis.

Superoxide dismutase activity in juvenile Litopenaeus vannamei and Nodipecten subnodosus exposed to the toxic dinoflagellate Prorocentrum lima.

The toxic effect of the dinoflagellate Prorocentrum lima on juvenile American whiteleg shrimp Litopenaeus vannamei and giant lion-paw scallop Nodipecten subnodosus was evaluated. Organisms were exposed to three densities (500, 2000, or 5000 cells mL(-1)), superoxide dismutase activity and soluble protein in the hepatopancreas and muscle were determined at 1, 6, 24 and 48 h after challenge. Shrimp exposed at 5000 cells mL(-1) significantly increased SOD activity in the hepatopancreas at 1 h post-challenge, whereas enzymatic activity in muscle significantly increased at 24 h at all densities. Scallops exposed to 500 and 2000 cells mL(-1) showed significant SOD activity increase in hepatopancreas at 24 and 12 h, respectively. Mortality at 48 h was 100% in scallops exposed to 5000 cells mL(-1). Shrimp showed higher levels of SOD activity than scallops. Soluble protein content in the shrimp hepatopancreas was significantly higher at densities of 500 and 2000 cells mL(-1) at 6 and 1 h, respectively. Soluble protein content in the scallop hepatopancreas was higher than control values at 1 h after challenge. In this study, 500 cells mL(-1) was enough to trigger SOD activity in two benthic species exposed to the toxic dinoflagellate P. lima.