Disentangling the determinants of symbiotic species richness in native and invasive gammarids (Crustacea, Amphipoda) of the Baltic region.

Dispersal of alien species is a global problem threatening native biodiversity. Co-introduction of non-native parasites and pathogens adds to the severity of this threat, but this indirect impact has received less attention. To shed light on the key factors determining the richness of microorganisms in native and invasive host species, we compared symbiotic (parasitic and epibiotic) communities of gammarids across different habitats and localities along the Baltic coast of Poland. Seven gammarid species, two native and five invasive, were sampled from 16 freshwater and brackish localities. Sixty symbiotic species of microorganisms of nine phyla were identified. This taxonomically diverse species assemblage of symbionts allowed us to assess the effect of host translocation and regional ecological determinants driving assembly richness in the gammarid hosts. Our results revealed that (i) the current assemblages of symbionts of gammarid hosts in the Baltic region are formed by native and co-introduced species; (ii) species richness of the symbiotic community was higher in the native Gammarus pulex than in the invasive hosts, probably reflecting a process of species loss by invasive gammarids in the new area and the distinct habitat conditions occupied by G. pulex and invasive hosts; (iii) both host species and locality were key drivers shaping assembly composition of symbionts, whereas habitat condition (freshwater versus brackish) was a stronger determinant of communities than geographic distance; (iv) the dispersion patterns of the individual species richness of symbiotic communities were best described by Poisson distributions; in the case of an invasive host, the dispersion of the rich species diversity may switch to a right-skewed negative binomial distribution, suggesting a host-mediated regulation process. We believe this is the first analysis of the symbiotic species richness in native and invasive gammarid hosts in European waters based on original field data and a broad range of taxonomic groups including Microsporidia, Choanozoa, Ciliophora, Apicomplexa, Platyhelminthes, Nematoda, Nematomorha, Acanthocephala and Rotifera, to document the patterns of species composition and distribution.

Molecular characterization and transcriptional regulation of two types of H+-pyrophosphatases in the scuticociliate parasite Philasterides dicentrarchi.

Proton-translocating inorganic pyrophosphatases (H+-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H+ translocation across membranes. In this study, we conducted a molecular characterization of two isoenzymes (PdVP1 and PdVP2) located in respectively the alveolar sacs and in the membranes of the intracellular vacuoles of a scuticociliate parasite (Philasterides dicentrarchi) of farmed turbot. We analyzed the genetic expression of the isoenzymes after administration of antiparasitic drugs and after infection in the host. PdVP1 and PdVP2 are encoded by two genes of 2485 and 3069 bp, which respectively contain 3 and 11 exons and express proteins of 746 and 810 aa of molecular mass 78.9 and 87.6 kDa. Topological predictions from isoenzyme sequences indicate the formation of thirteen transmembrane regions (TMRs) for PdVP1 and seventeen TMRs for PdVP2. Protein structure modelling indicated that both isoenzymes are homodimeric, with three Mg2+ binding sites and an additional K+ binding site in PdVP2. The levels of identity and similarity between the isoenzyme sequences are respectively 33.5 and 51.2%. The molecular weights of the native proteins are 158 kDa (PdVP1) and 178 kDa (PdVP2). The isoenzyme sequences are derived from paralogous genes that form a monophyletic grouping with other ciliate species. Genetic expression of the isoenzymes is closely related to the acidification of alveolar sacs (PdVP1) and intracellular vacuoles (PdVP2): antiparasitic drugs inhibit transcription, while infection increases transcription of both isoenzymes. The study findings show that P. dicentrarchi possesses two isoenzymes with H+-PPase activity which are located in acidophilic cell compartment membranes and which are activated during infection in the host and are sensitive to antiparasitic drugs. The findings open the way to using molecular modelling to design drugs for the treatment of scuticociliatosis.

Antibody responses to chimeric peptides derived from parasite antigens in mice and other animal species.

Peptide vaccines constitute an interesting alternative to classical vaccines due to the possibility of selecting specific epitopes, easy of production and safety. However, an inadequate design may render these peptides poorly immunogenic or lead to undesirable outcomes (e.g., formation of B neoepitopes). As an approach to vaccine development, we evaluated the antibody response to chimeras composed of two or three known B epitopes from Trichinella and Fasciola, and several linkers (GSGSG, GPGPG and KK) in species as different as mice, sheep and turbot. All these species could mount an effective immune response to the short chimeric peptides. Nevertheless, this response depended on several factors including a favorable orientation of B-cell epitopes, adequateness of linkers and/or probability of formation of T neoepitopes. We also observed that, at least in mice, the inclusion of a decoy epitope may have favorable consequences on the antibody response to other epitopes in the chimera.

Combined antiparasitic and anti-inflammatory effects of the natural polyphenol curcumin on turbot scuticociliatosis.

The histiophagous scuticociliate Philasterides dicentrarchi is the aetiological agent of scuticociliatosis, a parasitic disease of farmed turbot. Curcumin, a polyphenol from Curcuma longa (turmeric), is known to have antioxidant and anti-inflammatory properties. We investigated the in vitro effects of curcumin on the growth of P. dicentrarchi and on the production of pro-inflammatory cytokines in turbot leucocytes activated by parasite cysteine proteases. At 100 µm, curcumin had a cytotoxic effect and completely inhibited the growth of the parasite. At 50 µm, curcumin inhibited the protease activity of the parasite and expression of genes encoding two virulence-associated proteases: leishmanolysin-like peptidase and cathepsin L-like. At concentrations between 25 and 50 µm, curcumin inhibited the expression of S-adenosyl-L-homocysteine hydrolase, an enzyme involved in the biosynthesis of the amino acids methionine and cysteine. At 100 µm, curcumin inhibited the expression of the cytokines tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) produced in turbot leucocytes activated by parasite proteases. Results show that curcumin has a dual effect on scuticociliatosis: an antiparasitic effect on the catabolism and anabolism of ciliate proteins, and an anti-inflammatory effect that inhibits the production of proinflammatory cytokines in the host. The present findings suggest the potential usefulness of this polyphenol in treating scuticociliatosis.

Role of scuticociliate proteinases in infection success in turbot, Psetta maxima (L.).

Scuticociliatosis caused by Philasterides dicentrarchi is one of the most severe diseases of farmed turbot, Psetta maxima (L.). Immunized fish showed elevated levels of specific antibodies (Ab), which caused the destruction of parasites through the activation of complement by the alternative and classical pathways. By using affinity chromatography on bacitracin-sepharose columns, we demonstrated the existence of high levels of parasite proteinases in the serum and, to a lesser extent, in the ascitic fluid of experimentally infected fish, and the absence of such proteinases in the serum of uninfected fish. Serum from uninfected fish displayed haemolytic activity against sheep red blood cells. However, incubation of this serum with parasite proteinases led to a decrease in serum haemolytic activity, suggesting that proteinases are able to destroy fish complement. Proteinases isolated from serum or ascitic fluid of infected fish were also able to degrade turbot Ab. Preincubation of turbot serum containing specific Ab for P. dicentrarchi with the proteinases led to a significant decrease in the killing activity of the serum. The results confirm that P. dicentrarchi proteinases in serum from infected fish may provide a mechanism for circumventing normal host immunity by inactivating the Ab and complement factors required for complement activation.

Macroparasites of five species of ray (genus Raja) on the northwest coast of Spain.

A parasitological study of rays captured on the Atlantic continental shelf off the estuary Muros-Noia in NW Spain (42 degrees 35' to 42 degrees 41' N, 9 degrees 2' to 9 degrees 10' W; mean capture depth 11.6 +/- 4.1 m) was performed. A total of 128 rays were examined: 52 specimens of Raja microocellata, 60 of R. brachyura, 6 of R. montagui, 3 of R. undulata and 7 of an unidentified Raja species, known locally as 'fancheca'. A total of 23 macroparasite species were detected: 5 monogeneans (Acanthocotyle sp., Calicotyle kroyeri, Empruthotrema raiae, Merizocotyle undulata, Rajonchocotyle emarginata), 11 cestodes (Acanthobothrium sp., Crossobothrium sp., Echeneibothrium sp., Echinobothrium brachysoma, Grillotia erinaceus, Grillotia sp., Lecanicephalum sp., Nybelinia lingualis, Onchobothrium uncinatum, Phyllobothrium lactuca, Tritaphros retzii), 6 nematodes (Anisakis simplex, Hysterothylacium sp., Histodytes microocellatus, Piscicapillaria freemani, Proleptus sp., Pseudanisakis baylisi) and a copepod (Holobomolochus sp.). All parasite species were present in several ray species, except for Acanthocotyle sp. and G. erinaceus (detected only in R. brachyura), H. microocellatus (detected only in R. microocellata) and T. retzii (detected only in R. montagui). Three species (C. kroyeri, M. undulata, E. brachysoma) have not been reported previously from Spain. The host with the highest parasite species richness was R. brachyura (18 species), followed by R. microocellata (17) and the unidentified Raja species (14). The parasite with the highest prevalence in R. microocellata was M. undulata, followed by R. emarginata, Acanthobothrium sp. and Echeneibothrium sp. The species with the highest prevalence in R. brachyura was R. emarginata, followed by C. kroyeri and P. baylisi. Some differences in parasite prevalence were detected between sexes and among size classes in both R. brachyura and R. microocellata.

Influence of host age and sex on the helminth fauna of the yellow-legged gull (Larus michahellis) in Galicia (northwestern Spain).

We studied the influence of host age and sex on the helminth fauna of 324 Larus michahellis captured in different locations in the region of Galicia (northwestern Spain). Gulls were grouped into prefledglings, first-year immature birds, second- and third-year immature birds, and adults. Second-year, third-year, and adult birds were grouped by sex. Thirty-six helminth species were recorded. Total species richness and mean infracommunity species richness were both significantly lower for pre-fledglings than for the other age groups. Prevalence increased significantly with age for Brachylecithum microtesticulatum, probably reflecting changing feeding habits. Likewise, 8 species (Cardiocephaloides longicollis, Microphallus similis, Maritrema gratiosum, Gynaecotyla longiintestinata, Brachylecithum microtesticulatum, Himasthla elongata, Parorchis acanthus, and Renicola sp.) were absent or had very low prevalence in prefledglings. At least 5 of these 8 species are transmitted to gulls through ingestion of molluscs or crustaceans, which suggests that these types of prey are seldom fed to prefledglings. In Gymnophallus deliciosus, G. longiintestinata, and Cosmocephalus obvelatus, mean intensity, and in the latter case prevalence, declined with age, suggesting that protective immunity against these species increase with age. Only G. deliciosus, Microphallus similis, and G. longiintestinata presented significant differences between the sexes.

In vitro efficacy of new antiprotozoals against Philasterides dicentrarchi (Ciliophora, Scuticociliatida).

Philasterides dicentrarchi is a histiophagous ciliate that causes severe losses in turbot and sea bass farming. This study investigated the in vitro efficacy against P. dicentrarchi of 85 newly synthesized compounds and 12 commercial compounds, of which 2 are fluoroquinolones (norfloxacine and lomefloxacine) with known antibacterial activity. Seventeen of the newly synthesized compounds (2 naphthyridines, 2 pyridothienodiazines and 13 pyridothienotriazines) and the fluoroquinolone norfloxacin showed good activity. The most promising compound was the pyridothienotriazine 12k, with activity similar to that of the salicylanilides niclosamide and oxiclozanide (MLC 0.8 mg l(-1) in PBS, 1.5 mg l(-1) in seawater; MLC = minimum 24 h lethal concentration).

Characterization by enzyme electrophoresis of specimens of the genus Helicometra (Trematoda, Opecoelidae) from fish caught off the coast of northwest Spain.

Species within the genus Helicometra are difficult to distinguish on morphological grounds alone, and are best discriminated with the aid of biochemical techniques. In the work reported here, the electrophoretic mobility of malate dehydrogenase isoenzyme of the Mdh-1 locus was used to characterize 375 individuals of Helicometra obtained from various teleost species (Anguilla anguilla, Conger conger, Gobius niger or Ciliata mustela) caught off the coast of northwest Spain. The results suggest that all specimens belong to only one species, probably H. fasciata. Observed genotype frequencies did not differ from those expected under the assumptions of Hardy-Weinberg equilibrium, even when the genic frequencies differ considerably respecting a Mediterranean population.