The intestinal parasite Pomphorhynchus laevis (Acanthocephala) interferes with the uptake and accumulation of lead (210Pb) in its fish host chub (Leuciscus cephalus)

Uninfected chub as well as fish experimentally infected with the acanthocephalan parasite Pomphorhynchus laevis were exposed to ²¹⁰Pb²⁺ for up to 38 days and the uptake and distribution of lead within different fish organs and the parasites was determined at various time points. Highest metal concentrations were detected in the acanthocephalans, followed by intestine, bile, liver, gill and muscle of the fish host. Infected chub had significantly lower ²¹⁰Pb levels in the gills on day 17 (P≤0.01), in the bile on day 24 (P≤0.05) and in the gills as well as in the intestine on day 38 compared with uninfected fish. A subsequent polynomial regression revealed that lead levels for the infected fish ranged below the levels determined for uninfected fish during most of the exposure period. This is the first proof that P. laevis reduces lead levels in the bile thereby diminishing or even impeding the hepatic intestinal cycling of lead, which may reduce the amount of metals available for the fish organs. This is especially important for ecotoxicological research. For example, organisms used as accumulation indicators may erroneously indicate low levels of pollution if they are infected with parasites which alter their pollutant uptake mechanisms. Additionally, the results gave further experimental evidence for acanthocephalans as accumulation indicators for metals.     

Comparison between lead accumulation of Pomphorhynchus laevis (Palaeacanthocephala) in the intestine of chub (Leuciscus cephalus) and in the body cavity of goldfish (Carassius auratus auratus)

This experimental study assessed the role of the microhabitat in the uptake of metals by adult acanthocephalans. We examined the accumulation of lead by adult Pomphorhynchus laevis in the intestine of chub (Leuciscus cephalus) and compared it with that in goldfish, Carassius auratus auratus, in which the parasites penetrate the intestinal wall and enter the body cavity. Chub and goldfish experimentally infected with adult Pomphorhynchus laevis were exposed to 0.01 mg l(-1) Pb(2+) over 3 weeks. Lead was rapidly accumulated in the intestinal acanthocephalans reaching a mean concentration of 7.3 microg g(-1). This concentration was significantly greater than in the host muscle, liver and intestine and more than 730 times higher than the exposure concentration. Intraperitoneal P. laevis in goldfish exposed to lead did not accumulate the metal. Thus, it was conclusively shown that metal accumulation in acanthocephalans is associated with the intestinal location and does not occur in the body cavity.     

Larval morphology, genetic divergence, and contrasting levels of host manipulation between forms of Pomphorhynchus laevis (Acanthocephala)

Studies on parasite species with a wide geographic and ecological range may be confounded by still equivocal taxonomic identification. Here, we investigated genetic polymorphism and behavioural changes induced in a common intermediate host, in two different forms of Pomphorhynchus laevis based on the morphology of the larval infective stage (cystacanth). A 'smooth type' (S) and a 'wrinkled type' (W) of cystacanth were distinguished based on their surface and shape. We analysed sequence divergence at both nuclear (ribosomal gene 18S rDNA, and ribosomal internal transcribed spacers, ITS1/ITS2) and mitochondrial (cytochrome c oxidase subunit 1) genes of P. laevis cystacanths and adults at various geographical scales. A high level of sequence divergence at ITS1, ITS2 and cytochrome c oxidase subunit 1 (11, 8 and 20%, respectively) was found between these two forms. The divergence pattern consistently discriminated two groups independently of geographic origin or host, and was congruent with larval morphology. The two forms also strongly differed in the intensity of behavioural change induced in their common intermediate host, Gammarus pulex, with the S-type parasite inducing a positive phototactism, whereas W-type infected gammarids were as photophylic as uninfected ones. Overall, our data strongly support the specific status of these two forms. We suggest that smooth cystacanths correspond to P. laevis, whereas wrinkled cystacanths might correspond to the previously described and poorly documented, Pomphorhynchus tereticollis, considered a synonym of P. laevis. This study also confirms the value of a joint analysis of internal transcribed spacers and cytochrome c oxidase subunit 1 genes to biogeographic studies on these species. Finally, we emphasize the importance of linking morphological and biological characteristics of acanthocephalan cystacanths to molecular data, in the study of the evolutionary ecology and systematics of this group.     

Phylogeography and postglacial dispersion of the chub (Leuciscus cephalus) in Europe

A phylogeographic analysis of mitochondrial DNA variation was performed in order to test the hypothesis of a postglacial recolonization of mid- and north-European rivers from a Danubian refuge. Over 345 chub specimens from European rivers covering most of the species' native range were investigated using 600 bp of the cytochrome b gene. Chub in European rivers belong to four highly divergent mitochondrial groups (lineages) differing by mean divergence estimates from 5.2% to 7.89%. These four lineages have a largely allopatric distribution, implying four geographical sets: two Mediterranean, and two north-European sets. This pattern provided strong evidence for: (i) the eradication of this species from most of Europe during maximum ice extent; (ii) its survival in four refugia (Adriatic side of the Balkans, eastern Greece (Aegean Rivers), southern tributaries of the Danube, and periphery of Black and Caspian Seas); (iii) a differential postglacial recolonization of mid- and northern Europe from the last two refugia only; (iv) the occurrence of this recolonization in two steps for the Danubian (western) lineage that entered western Europe (Rhine-Rhone-Loire drainages) during the Riss-Würm interglacial period and survived the last glaciation there before colonizing Garonne, UK and German drainages up to the Elbe during the Holocene; and (v) the occurrence of this recolonization in a single step for the Ponto-Caspian (eastern) lineage that entered the Baltic area as far as the Oder in the Holocene. Both lineages came into contact in the River Elbe without evident mixing.     

Eukaryotic initiation factor 6 (eif6) overexpression affects eye development in Xenopus laevis

The translation initiation factor eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 serine 235 by PKC. Previous data showed that eif6 binds to the 60S ribosomal subunit when unphosphorylated, inhibiting assembly with the 40S subunit. Phosphorylation of Ser235 releases eif6 from the 60S subunit and allows assembly. eif6 acts as an anti-apoptotic factor via regulation of the bcl2/bax balance and acts selectively upstream of bcl2. This activity also depends upon phosphorylation of eif6 Ser235. One of the consequences of eif6 overexpression in Xenopus embryos is aberrant eye development. Here we evaluate the eye phenotype and show that it is transient. We show that the whole eye, particularly the retina layers, of the embryos injected with eif6-encoding mRNA recover by stage 42. Embryos over-expressing eif6 have normal expression of anterior- and brain-specific markers, indicating that outside the eye field, other neural regions appear unaffected by the eif6 injection. No eye defect was detected when morpholinos were used to reduce eif6 protein synthesis. We tested how two known pathways of eif6 function with respect to alteration of eye development. We found that injection of bcl2 did not produce the eye phenotype and eif6-bax co-injection did not rescue the eye defect, suggesting that the eye phenotype is not bearing on the anti-apoptotic role played by eif6 is not linked to its role as an anti-apoptotic factor. We also determined that PKC-dependant phosphorylation of Ser235 in eif6 is not required to produce defective eye development. These results indicate that the aberrant eye phenotype, produced by eif6 overexpression, is not directly linked to the PKC-regulated effects of eif6 on translation and ribosomal subunit interaction or on eif6 anti-apoptotic properties.     

Differential influence of Pomphorhynchus laevis (Acanthocephala) on brain serotonergic activity in two congeneric host species

The physiological mechanisms by which parasites with complex life cycles manipulate the behaviour of their intermediate hosts are still poorly understood. In Burgundy, eastern France, the acanthocephalan parasite Pomphorhynchus laevis inverses reaction to light in its amphipod host Gammarus pulex, but not in Gammarus roeseli, a recent invasive species. Here, we show that this difference in manipulation actually reflects a difference in the ability of the parasite to alter brain serotonergic (5-HT) activity of the two host species. Injection of 5-HT in uninfected individuals of both host species was sufficient to inverse reaction to light. However, a difference in brain 5-HT immunocytochemical staining levels between infected and uninfected individuals was observed only in G. pulex. Local adaptation of the parasite to the local host species might explain its inability to manipulate the behaviour and nervous system of the invasive species.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.     

Newbouldiosides A-C, phenylethanoid glycosides from the stem bark of Newbouldia laevis

From the stem bark of Newbouldia laevis three phenylethanoid glycosides, designated as newbouldioside A-C, were isolated together with a sodium salt of analogue B and the known compounds, verbascoside, 5-hydroxydehydro-iso-alpha-lapachone, 3,8-dihydroxydehydro-iso-alpha-lapachone, apigenin and luteolin. The structures of the phenylethanoid glycosides were elucidated by spectroscopic methods as beta-(3,4-dihydroxyphenyl)ethyl 5-O-syringoyl-beta-D-apiofuranosyloxy-(1-->2)-O-[alpha-L-rhamnopyranosyl-(1-->3)]-beta-D-glucopyranoside, ss-(3,4-dihydroxyphenyl)ethyl 5-O-syringoyl-beta-D-apiofuranosyloxy-(1-->2)-O-[alpha-L-rhamnopyranosyl-(1-->3)]-6-O-E-feruloyl-beta-D-glucopyranoside, and beta-(3,4-dihydroxyphenyl)ethyl 3-O-E-feruloyl-beta-D-apiofuranosyloxy-(1-->2)-O-alpha-L-rhamnopyranosyl-(1-->2)-6-O-E-sinapoyl-beta-D-glucopyranoside, respectively.     

Fluorescent labeling of endothelial cells allows in vivo,continuous characterization of the vascular development of Xenopus laevis

Appropriate blood supply and vascular development are necessary in development and in cancer, heart disease, and diabetes. Here, we report the use of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) to label endothelial cells and characterize the vasculature of live Xenopus embryos. The atlas we have created provides a detailed map of normal vascular development against which perturbations of normal patterning can be compared. By following the development of the intersomitic vessels in real-time, we show that, while rostrocaudal gradient of maturing intersomitic vessels occurs, it is not absolute. In addition, the comparative study of the ontogeny of nerve bundles from the spinal cord of transgenic Xenopus embryos expressing green fluorescent protein in the nervous system and blood vessels demonstrates a strong anatomical correlation in neurovascular development. These studies provide the basis for understanding how the vascular system forms and assumes its complicated stereotypical pattern in normal development and in disease.     

Nested clade and phylogeographic analyses of the chub, Leuciscus cephalus (Teleostei, cyprinidae), in Greece: implications for Balkan Peninsula biogeography

Phylogenetic relationships among Greek populations of the chub, Leuciscus cephalus, were investigated using 600 bp of the cytochrome b gene. The aim of this study was to test the assumption that the main difference in ichthyological composition between both sides of the Balkan Peninsula is directly linked to differences in the dispersion mechanisms used by fish in order to extend their distribution range. Phylogenetic and nested clade analyses clearly showed that populations in Greece are significantly differentiated. Greek populations were found to descend from three lineages in three geographical provinces: Western, Central, and Eastern Greece. The chub reached Western Greece at the beginning of the Pleistocene and Eastern Greece during the mid-Pleistocene. Chub dispersion occurred mainly by river confluence due to sea level lowering and river capture in Western Greece and sea dispersal with low-salinity conditions within the Aegean Sea in Eastern Greece. However, in Central Greece, the original mtDNA lineage has presumably been lost owing to a genetic introgression following a second invasion from the Danube during the final stage of the last glaciation. This study provides new elements for a better understanding of the composition of the contemporary ichthyofauna in Greece and highlights possible evolutionary mechanisms responsible for the high endemism rate in the Western Greek biogeographic province.     

Furanonaphthoquinones,atraric acid and a benzofuran from the stem barks of Newbouldia laevis

The series of naturally occurring furanonaphthoquinones is extended by identification of the derivatives 2-(1'-methylethenyl)-5-hydroxynaphtho[2,3-b]furan-4,9-dione and 2-(1'-methylethenyl)-7-hydroxynaphtho[2,3-b]furan-4,9-dione. They are accompanied in the stem barks of Newbouldia laevis by the known analogues 5-hydroxy-dehydro-iso-alpha-lapachone, 2-acetyl-5-hydroxynaphtho[2,3-b]furan-4,9-dione and 2-(1'-methylethenyl)naphtho[2,3-b]furan-4,9-dione along with the rare atraric acid and the new 2-(1'-methylethenyl)-6-hydroxy-2,3-dihydrobenzofuran. The structures of these compounds were established from spectroscopic studies.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

A role of unliganded thyroid hormone receptor in postembryonic development in Xenopus laevis

A fascinating feature of thyroid hormone (T3) receptors (TR) is that they constitutively bind to promoter regions of T3-response genes, providing dual functions. In the presence of T3, TR activates T3-inducible genes, while unliganded TR represses these same genes. Although this dual function model is well demonstrated at the molecular level, few studies have addressed the presence or the role of unliganded TR-induced repression in physiological settings. Here, we analyze the role of unliganded TR in Xenopus laevis development. The total dependence of amphibian metamorphosis upon T3 provides us a valuable opportunity for studying TR function in vivo. First, we designed a dominant negative form of TR-binding corepressor N-CoR (dnN-CoR) consisting of its receptor interacting domain. We confirmed its dominant negative activity by showing that dnN-CoR competes away the binding of endogenous N-CoR to unliganded TR and relieves unliganded TR-induced gene repression in frog oocytes. Next, we overexpressed dnN-CoR in tadpoles through transgenesis and analyzed its effect on gene expression and development. Quantitative RT-PCR revealed significant derepression of T3-response genes in transgenic animals. In addition, transgenic tadpoles developed faster than wild type siblings, with an acceleration of as much as 7 days out of the 30-day experiment. These data thus provide in vivo evidence for the presence and a role of unliganded TR-induced gene repression in physiological settings and strongly support our earlier model that unliganded TR represses T3-response genes in premetamorphic tadpoles to regulate the progress of development.     

Some effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes

A study was made of the effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes. 1 μg/ml of the venom made the resting plasma membrane potential more negative in cells voltage-clamped at −60 mV. The effect was potentially due to the closure of one or several conductances that were investigated further. Thus, we determined the effects of the venom on the following endogenous ionic-currents: (a) voltage-activated potassium currents, (b) voltage-activated chloride-currents, and (c) calcium-dependent chloride-currents (Tout). The results suggest that the venom exerts its action mainly on a transient outward potassium-current that is probably mediated by a Kv channel homologous to shaker. Consistent with the electrophysiological evidence we detected the expression of the mRNA coding for xKv1.1 in the oocytes.     

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.)

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.     

Distribution pattern of air pollution and epiphytic lichens in the Osaka Plain (Japan)

The geographic distributions of two common species of epiphytic lichens,Phaeophyscia limbata (Poelt) Kashiw. andLecanora pulverulenta Müll. Arg., on city park trees in the Osaka Plain and their relation to the distribution of SO₂ and NO₂ was examined. The concentrations of SO₂ and NO₂ are greater in the central area of this plain, and decrease with distance from the center. The frequency ofL. pulverulenta is lowest in the central area, and increases gradually toward the periphery. In contrast, the frequency ofP. limbata is highest in the intermediate area, and lower in the central and peripheral areas, i.e. at intermediate SO₂ and NO₂ concentrations. We conclude thatL. pulverulenta is more useful thanP. limbata as a bioindicator of air pollutants such as SO₂ and NO₂.     

Galiellalactone and its biogenetic precursors as chemotaxonomic markers of the Sarcosomataceae (Ascomycota)

(-)-Galiellalactone is a hexaketide metabolite with interesting pharmacological activities which was detected in four strains of Galiella rufa (Sarcosomataceae, Ascomycota) and in two unidentified fungi shown by their 18S rDNA sequences also to belong to the Sarcosomataceae. These were a wood-inhabiting apothecial species from Chile and an endophytic isolate from Cistus salviifolius (Sardinia). Other members of the family (Urnula helvelloides, one Strumella coryneoidea isolate) produced no galiellalactone but merely hexaketides structurally related to galiellalactone precursors, whereas a third group of species (Sarcosoma latahensis, Strumella griseola, one S. coryneoidea isolate) lacked hexaketide production altogether. Despite thorough screening programmes, galiellalactone and its precursors have not yet been found in any fungus outside the Sarcosomataceae and may thus be a chemotaxonomic marker of the family, supporting its current phylogenetic definition. Two pentaketide derivatives of the 6-pentyl-alpha-pyrone type were found in all G. rufa strains as well as in A111-95 and the hexaketide-producing S. coryneoidea isolate.     

Population dynamics of Barbus barbus plebejus (Valenciennes) and Leuciscus cephalus cabeda (Risso) in the middle River Po (Italy)

Scales from 427 Barbus barbus plebejus and 406 Leuciscus cephalus cabeda from the middle River Po were examined. The last annulus was formed between April and May 1977, except the 0–1 age-group of L.c. cabeda which developed its first year-ring between March and April 1977. The survival (S) and annual mortality (A) rates were equivalent in both fish populations; the same was true of both sexes of Leuciscus, but S in Barbus males was lower than in females. The sex ratio showed a preponderance of females in both species.Growth rate from June to September 1976 was higher than in the rest of the year. Annual growth in Barbus females was higher than in males, whereas both sexes in Leuciscus showed equivalent growth rates; growth rate was higher in Barbus than in Leuciscus. The P/B ratio of both species was higher in the young age-groups.Presented at the XLVII Congress of the Italian Zoological Union, Bergamo (Italy), September 1979.Presented at the XLVII Congress of the Italian Zoological Union, Bergamo (Italy), September 1979.