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.

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.

Laser-assisted microdissection: a new tool for aquatic molecular parasitology.

Laser-assisted microdissection (LMD) has been developed to isolate distinct cell populations from heterogeneous tissue sections, cytological preparations, or live cell samples. Downstream applications typically include gene expression studies using real-time PCR and array platforms, diagnostic PCR, and protein expression studies. LMD techniques are now commonplace in mainstream biological research and clearly have suitable applications in the field of aquatic pathology and parasitology. The present study used LMD to isolate 2 dinoflagellate parasites (Hematodinium spp.) from formalin-fixed paraffin-embedded tissue sections from 2 crustacean hosts, Cancer pagurus and Portunus trituberculatus. DNA was isolated from LMD parasite preparations, and partial regions (up to 300 bp) of the small subunit and the first internal transcribed spacer region of the rRNA gene complex from the Hematodinium spp. were PCR amplified using diagnostic primers. The amplification products were sequenced to confirm the identity of the targeted regions. The techniques, applications, and limitations of LMD to address questions in aquatic molecular pathology and parasitology are discussed.

Enterospora canceri n. gen., n. sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer pagurus.

Only 1 genus (Nucleospora) within 1 family (Enterocytozoonidae) of the Microsporidia contains species that are parasitic within the nuclei of their host cells; to date, all described intranuclear Nucleospora spp. parasitise fish. This study describes the first intranuclear microsporidian parasite of an invertebrate, the European edible crab Cancer pagurus L. (Decapoda: Cancridae). Infected crabs displayed no obvious external signs, and maximum apparent prevalence of infection within a monthly sample was 3.45%. Infected hepatopancreatic tubules were characterised by varying numbers of hypertrophic and eosinophilic nuclei within epithelial cells. Parasite stages appeared as eosinophilic granular accumulations causing margination of host chromatin. In advanced cases, the tubule epithelia degenerated, with parasites and sloughed epithelial cells appearing in tubule lumens. All life stages of the parasite were observed within host nuclei. Uninucleate meronts were not detected, although binucleate stages were observed. Multinucleate plasmodia (sporogonal plasmodia) contained up to 22 nuclei in section, and late-stage plasmodia contained multiple copies of apparatus resembling the polar filament and anchoring disk, apparently associated with individual plasmodial nuclei. As such, aggregation and early assembly of sporoblast components took place within the intact sporogonial plasmodium, a feature unique to the Enterocytozoonidae. Liberation of sporoblasts from plasmodia or the presence of liberated sporoblasts was not observed in this study. However, large numbers of maturing and mature spores (measuring 1.3 +/- 0.02 x 0.7 +/- 0.01 microm) were frequently observed in direct contact with the host nucleoplasm. Considering the shared features of this parasite with microsporidians of the family Enterocytozoonidae, and the unique presence of this parasite within the nucleoplasm of decapod crustacean hepatopancreatocytes, this parasite (Enterospora canceri) is proposed as the type species of a new genus (Enterospora) of microsporidian. Molecular taxonomic work is now required, comparing Enterospora to Enterocytozoon and Nucleospora, the 2 other genera within the Enterocytozoonidae.

Enterospora sp., an intranuclear microsporidian infection of hermit crab Eupagurus bernhardus.

Recent work at our laboratory has led to the discovery of a new genus of microsporidian parasite residing in the family Enterocytozoonidae. The type species of this new genus, Enterospora canceri, is an intranuclear parasite infecting the hepatopancreatocytes of the decapod crustacean Cancer pagurus. Here we provide the second description of a parasite within the genus Enterospora, this time infecting the hermit crab Eupagurus bernhardus from U.K. waters. The pathological manifestation and ultrastructural features of the hermit crab parasite are very similar to those described for E. canceri. Further taxonomic comparisons based upon ultrastructural and molecular affinities of Enterospora are now required to define firmer links between this new genus within the Enterocytozoonidae and all other microsporidian families. The opportunistic nature of the discovery of a second intranuclear microsporidian within the Crustacea suggests that their presence may be more common than in higher animal groups.