The monogenean Protopolystoma xenopodis has been established in Wales for >40 years following introduction with Xenopus laevis from South Africa. This provides an experimental system for determining constraints affecting introduced species in novel environments. Parasite development post-infection was followed at 15, 20 and 25°C for 15 weeks and at 10°C for ⩾1 year and correlated with temperatures recorded in Wales. Development was slowed/arrested at ⩽10°C which reflects habitat conditions for >6 months/year. There was wide variation in growth at constant temperature (body size differing by >10 times) potentially attributable in part to genotype-specific host-parasite interactions. Parasite density had no effect on size but host sex did: worms in males were 1·8 times larger than in females. Minimum time to patency was 51 days at 25°C and 73 days at 20°C although some infections were still not patent at both temperatures by 105 days p.i. In Wales, fastest developing infections may mature within one summer (about 12 weeks), possibly accelerated by movements of hosts into warmer surface waters. Otherwise, development slows/stops in October–April, delaying patency to about 1 year p.i., while wide variation in developmental rates may impose delays of 2 years in some primary infections and even longer in secondary infections.

Factors affecting survival of parasites introduced to new geographical regions include changes in environmental temperature. Protopolystoma xenopodis is a monogenean introduced with the amphibian Xenopus laevis from South Africa to Wales (probably in the 1960s) where low water temperatures impose major constraints on life-cycle processes. Effects were quantified by maintenance of eggs from infections in Wales under controlled conditions at 10, 12, 15, 18, 20 and 25°C. The threshold for egg viability/ development was 15°C. Mean times to hatching were 22 days at 25°C, 32 days at 20°C, extending to 66 days at 15°C. Field temperature records provided calibration of transmission schedules. Although egg production continues year-round, all eggs produced during >8 months/ year die without hatching. Output contributing significantly to transmission is restricted to 10 weeks (May–mid-July). Host infection, beginning after a time lag of 8 weeks for egg development, is also restricted to 10 weeks (July–September). Habitat temperatures (mean 15·5°C in summer 2008) allow only a narrow margin for life-cycle progress: even small temperature increases, predicted with ‘global warming’, enhance infection. This system provides empirical data on the metrics of transmission permitting long-term persistence of isolated parasite populations in limiting environments.

The survival of retinal ganglion cells (GCs) in the left eye was studied on retinal wholemounts from 2–33 weeks after the surgical removal of the right tectum in juvenile Xenopus. Two to five weeks after tectal removal, about 76% of neurons of the retinal ganglion cell (GC) layer showed signs of retrograde degeneration: swelling of their somata and chromatolysis. Neurons that were not affected by the operation were taken to be either displaced amacrine cells (DAs) or GCs not projecting to the tectum. A portion of GCs showing retrograde degeneration became pyknotic and died within the period of 2–16 weeks after operation. Counts of surviving GCs 20–33 weeks after tectal removal amounted to about 55% of the corresponding neuron number in the right intact retina of the same animal. No discernible GC loss was observed in animals where only the optic fibers were cut at their entry point to the tectum indicating that axotomy alone, followed by rapid regrowth to the target, does not adversely influence the survival of GCs. In long-surviving animals, the left optic nerve was exposed to cobaltic-lysine complex and the position of filled optic axons within the brain determined. Optic axons whose tectal target had been removed were seen to cross over to the left intact tectum via the posterior and pretectal commissures. Aberrant projections were detected to the ipsilateral tectum and the diencephalic periventricular grey in addition to an increased projection to the accessory optic nucleus. It is concluded that the removal of the tectum, the main target of optic fiber projection, induces a very substantial GC death. Since only a portion of optic fibers were able to grow to alternative targets, the surviving GCs may have also included those with main projection areas to the diencephalic visual centers.

A role for endogenous dopamine in the control of rod and contributions to a second-order retinal neuron, the horizontal cell (HC) was studied in the Xenopus retina. Relative rod and cone contributions were estimated from HC responses to scotopically balanced 491- and 650-nm flashes. In eyecups prepared in light then placed in darkness, cone input to the HC slowed and diminished on a time scale of hours. The decline in cone input was balanced by a slow growth of rod input to the HC. Administration of D-amphetamine, a dopamine releasing agent, restored the light-adapted waveform.

The kinetics of slow light adaptation were examined by recording HC responses from eyecups that had been dark-adapted previously for 11–14 h. When test flashes fell on a dark field, cone input to the HC grew for 2–4 h, reached a plateau, and later declined. If, however, flashes were superimposed on a weak background field, cone input to the HC continued to increase monotonically at about 10%/h. This increase was abolished by superfusion with a nonspecific dopamine receptor blocker, cis-flupenthixol (50 μM), resulting in the complete suppression of cone-to-horizontal cell synaptic transfer and the enhancement of rod-to-horizontal cell communication. Subcutaneous injection of reserpine, a drug that depletes dopamine stores (2 mg/kg on 1–4 successive days), or intraocular injection of the dopamine neurotoxin, 6-hydroxydopamine (10–30 μg) slowed and reduced the amplitude of cone input to the HC, even in completely light-adapted eyes. Subsequent treatment with D-amphetamine (5–50 μM) or dopamine (10 μM) partially restored the normal response.

Our experimental findings are consistent with the following hypothesis. Weak light is sufficient to stimulate dopamine release; dopamine augments cone-to-horizontal cell synaptic transfer and reduces rod-to-horizontal cell communication. The rapid kinetics of the fully light-adapted response depend on the presence of dopamine. Thus, dopamine appears to be an intraretinal signal for slow light adaptation.

We have recorded the electroretinogram (ERG) from the superfused eyecup of the Xenopus retina in order to assess the effects of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), and its agonists and antagonists, on individual ERG components. We found that GABA (0.5–10 mM) reduced the amplitudes of both the b- and d-waves of the Xenopus ERG. The GABA uptake blocker nipecotic acid (1 mM) had similar effects on b- and d-waves. GABA at 5 mM and 10 mM also caused an increase in the a-wave. The GABA antagonist picrotoxin (0.1–2 mM) and the GABA/a antagonist bicuculline (0.2 mM) both increased the amplitude of the b- and d-waves of the ERG. The GABA/b agonist baclofen (0.3 mM) reduced the amplitude of the ERG b-wave, enhanced the amplitude of the a-wave, and slightly reduced the amplitude and increased the peak time of the d-wave. The GABA/b antagonists phaclofen and saclofen had no reliable effects on the Xenopus ERG. Glutamate analogs known to affect specific types of retinal neurons were applied to modify the retinal circuitry and then the effects of GABA and its antagonists were examined under these modified conditions. 2-amino-4-phosphonobutyric acid (APB) increased the d-wave, and blocked the b-wave and the effect of GABA on the ERG, but not the antagonist-induced increase in the d-wave. KYN blocked the antagonist-induced increase in the b-wave, while GABA increases the amplitude of the b-wave if the d-wave has been removed by prior superfusion with kynurenic acid (KYN). N-methyl-DL-aspartate (NMDLA), which acts only in the proximal retina, reduced the amplitude of the ERG and blocked the effect of GABA and the antagonist-induced increase in ERG b- and d-waves amplitude. These results suggest that GABAergic mechanisms related to both A and B receptor types can influence the amplitude and light sensitivity of all the components of the Xenopus ERG. Since GABA is found in greatest abundance in the proximal retina, and B type of receptors are present almost exclusively there, the data suggests that most of the effects of GABA agonists and antagonists observed are dependent on proximal retinal mechanisms, and that there are separate mechanisms in the proximal retina related to the b- and the d-waves.

The aim of the present study was to determine the number of cone classes in the Xenopus retina. We examined the dimensions and staining properties of cones, utilizing two monoclonal antibodies, COS-1 and OS-2, developed by Szel and Rohlich (1985). Living cones also were reacted with the plant lectins peanut agglutinin (PNA) and wheat germ agglutinin (WGA) and with a fluorescent stilbene dye, DIDS, which binds selectively to red-sensitive cones (Kleinschmidt, 1991; Kleinschmidt & Harosi, 1992a, b). Three cone populations were distinguished based on differences in size and staining properties. Eighty-eight percent of all cones were stained strongly by COS-1, PNA, and DIDS, but weakly by OS-2. The group of cones stained by COS-1 had the largest mean dimensions of outer segment length, width, and oil droplet diameter. COS-1 negative cones were divisible into two groups: a subclass of miniature cones (approximately 4% total cones) was stained strongly by OS-2, PNA, and DIDS. The balance, constituting approximately 9% total cones, were of intermediate size, were not stained by PNA and reacted weakly to OS-2 and DIDS. WGA stained all cones. Large, COS-1 + cones appear to be red-sensitive and belong to the class of anion-tunable cone pigments. We suggest that the intermediate size, COS-1 negative cones are blue-sensitive based on the finding that blue-sensitive chromatic horizontal cells connect to them preferentially (Witkovsky et al., work in progress). The remaining class of miniature cones may be UV-sensitive, since another amphibian, the salamander, has been shown to possess red-, blue-, and UV-sensitive cones (Perry & McNaughton, 1991).

This review considers three case studies based on macroparasites of anurans: (a) natural infections in the permanently-aquatic Xenopus laevis which represent the worm burdens acquired, and the implications for pathology, when hosts are exposed to continuous, year-round, transmission; (b) the desert toad, Scaphiopus couchii, which experiences invasion very briefly each year and provides a simplified system involving only a single significant infection (Pseudodiplorchis americanus); (c) the mesic Bufo bufo which has been the subject of experimental laboratory studies designed to measure the effects of Rhabdias bufonis infection on host growth, physical performance and survival. Experimental manipulation of both Scaphiopus and Bufo provide quantitative data on disease effects of macroparasites, including precise measurements of parasite-induced host mortality. Field data for Xenopus and Scaphiopus show that, despite high initial worm burdens from efficient transmission, infection levels at parasite maturity are modulated below those leading to significant disease. Experimental data for Scaphiopus and Bufo have documented the time-course and magnitude of this decline in intensities, and there is circumstantial evidence for Scaphiopus that this regulation is host-mediated. Immunological studies on Xenopus show that disease effects of the pathogenic Pseudocapillaroides xenopodis are exacerbated in thymectomised hosts and reversed by implantation of thymuses from MHC-compatible donors. Thus, whilst factorial experiments can demonstrate the potential of helminths to cause significant disease and mortality in anuran host-macroparasite interactions, powerful post-invasion regulation of worm burdens appears to exert a strong control of parasite-induced disease in natural host populations.

We have isolated a cDNA (SKvl. 1) encoding a Shaker-related K+ channel from an adult cDNA library of the human parasitic trematode Schistosoma mansoni. The deduced amino acid sequence (512 aa, 56·5 kDa) contains 6 putative membrane-spanning domains (S1–S6) and a pore-forming domain (H5). SKv1.1 is grouped in the Shaker family, but forms a unique branch within this family, on the basis of dendrogram analysis. SKv1.1 shows significant sequence identity with most other Shaker channels, with 64—74% identity in the core region (S1–S6). It has the highest sequence identity with the K+ channel (Ak01a) from Aplysia. Northern blot analysis detected a single primary transcript of 2·8 kb. Southern blot analysis indicated that SKv1.1 is present as a single copy in the genomic DNA of S. mansoni. Expression of SKv1. 1 in Xenopus oocytes produced a rapidly activating and inactivating outward K+ current which is highly sensitive to 4-aminopyridine, but is insensitive to tetraethylammonium, mast cell degranulating peptide, dendrotoxin and charybdo-toxin. The presence of a Shaker homologue in Schistosoma suggests that Sh subfamilies may exist in other lower invertebrates as well as platyhelminths.

Dorsal–ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30° rotation of the subcortical yolk mass relative to the egg cortex. This rotation of symmetrisation is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15° from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90° from upright during maturation in vitro, the maturation spot developed 15° or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.

Xenopus ooctye cytoplasmic poly(A)+ RNA has been shown to include two major complex classes: mRNA and interspersed RNA. the former is defined by its translatalility, the latter consists of non–translatable repeat–containing transcripts with unknown functions. In this study we compared the accumulation patterns of total mRNA and a subfamily of interspersed RNA, the XR family (McGrew&Richter, 1989, Dev. Biot. 134, 267–70)

Microsomal fractions of Xenopus oocytes release preloaded 45Ca2+ when treated with inositol triphosphate (InsP3). The effective concentration of InsP3 required for half-maximal release (EC50) is 59 nM and maximal release occurs at ∼ 2 μM InsP3. Uptake and release of 45Ca2+ are not altered by the catalytic subunit of protein kinase A, dibutyrl cyclic adenosine monophosphate, protein kinase A peptide inhibitor or nocodazole. In contrast, taxol decreases the sensitivity of the microsomal fraction to InsP3, shifting the EC50 for InsP3-induced Ca2+ release from 59 to 259 nM. In lysates of oocytes, InsP3-induced Ca2+ release causes the tyrosine phorphorylation of a 42000 (Mr 42k) protein identified as 42k mitogen-activated protein (MAP) kinase. InsP3-induced tyrosine phosphorylation of MAP kinase is prevented by BAPTA and taxol, but not by nocodazole. Thus, microtubule polymerisation modifies InsP3-induced Ca2+ release, thereby inhibiting phosphorylation of MAP kinase.

Fresh cytosols extracted from unfertilised eggs of Xenopus laevis contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+ and is designated primary CSF (1°CSF). During storage of Ca2+-containing cytosols at 2°C, a stable CSF activity appears which is designated secondary CSF (2°CSF). In the present study, we report that 2°CSF activity can be induced in cytosols extracted from stage VI oocytes, unfertilised eggs, electrically activated eggs or blastulae, in the presence of Ca2+. Both the intensity and the rate of 2°CSF development are dependent on the concentration of Ca2+ ions added to the cytosol. At Ca2+ concentrations of 5–10 mM, 2°CSF activity reaches a maximum in about 7 days. Secondary CSF is relatively resistant to heat but loses all activity after 5 min at 70°C. When stored at −80°C, 2°CSF activity remains detectable for about 6 weeks. Cytological observations show that blastomeres arrested by microinjection of 2°CSF developed in cytosols of unfertilised eggs, activated eggs or blastulae contain metaphase chromosomes embedded in a bipolar spindle that has no asters developed at its poles. In contrast, blastomeres arrested by 2°CSF in cytosols of stage VI oocytes contain condensed chromosomes but no spindle is formed. The mechanisms of the development of 2°CSF in Ca2+-containing cytosols and its mode of action are discussed.

Interspersed RNA makes up two-thirds of cytoplasmic polyadenylated RNA in Xenopus and sea urchin eggs.Although it has no known function, previous work has suggested that at least one family of interspersed RNA, XR, binds Xenopus oocyte proteins, and can influence the rate of translation. We have used two Xenopus repeat families, Ocr and XR, to explore their protein binding abilities. Ocr RNA binds the same pattern of highly abundant oocyte proteins that XR RNA binds, which are believed to be messenger ribonucleoprotein (mRNP) particle proteins. In addition, we show that Ocr RNA binds the Oct-60 protein, a member of the POU-domain family of transcription factors found in Xenopus oocytes. Using a 32 base pair sequence from the XR repeat in a DNA affinity column two proteins were isolated, 66KDa and 92KDa, that together form a complex with XR DNA. One of these proteins (92KDa) also binds XR RNA. We suggest that the role of at least a subset of interspersed RNAs in development may be to bind, and sequester in the cytoplasm, DNA-binding proteins until the end of oogenesis

The reproductive kinetics of Protopolystoma xenopodis primary and secondary infections in Xenopus laevis were monitored in a 3-year study. Thirty-five naïve, lab-raised, full-sib X. laevis from 1 spawning were each exposed to 30 P. xenopodis eggs. The course of infections at 20 °C was monitored by screening isolated hosts for parasite egg production. Ninety-four percent of toads supported the development of gravid parasites. Infections became patent 9–19 weeks p.i., lasted 3–30 months and produced estimated totals of 1–7152 eggs/host. Variation in primary infection characters was discontinuous: a subgrouping of hosts (16%) was characterized by extended infection duration and low reproductive rate. In order to test the effect of long-term infection history on a subsequent challenge, each host was re-exposed to P. xenopodis infective stages (30 eggs/host) 6 months after the loss of its original infection. Establishment of patent infection was significantly lower (15%), and pre-patent period (12–28 weeks) longer, than in primary infections of the same hosts, and than in concurrently exposed naïve controls (contemporary full-sibs of the primary/secondary infection group, maintained in parallel; n = 28). There was no relationship between primary infection characteristics and secondary infection outcome. Overall reproductive output per initial infective stage for the primary exposure exceeded that for the secondary exposure by a ratio of 15[ratio ]1. Results suggest that primary infection with P. xenopodis can elicit strong, long-term protective immunity against re-infection in X. laevis.

The taxonomy and distribution of cephalochlamydid cestodes are reviewed. This group is primarily specific to pipid anurans and occurs naturally across sub-Saharan Africa. Paracephalochlamys gen. nov. has two large lateral testes (sometimes doubled or absent on one side) on the anterior margin of each segment, while Cephalochlamys Blanchard, 1908 has three to 23 small testes scattered in lateral fields. Paracephalochlamys papilionis sp. nov. infects Pseudhymenochirus merlini in Sierra Leone. The distribution of Cephalochlamys species is inferred from the previous literature and numerous new records. Cephalochlamys namaquensis (Cohn, 1906) occurs in Xenopus laevis laevis in Namibia, South Africa, Lesotho and Zimbabwe, X. l. poweri (new host record) in Zambia and the Democratic Republic of Congo (D.R.C.), X. l. victorianus in Kenya, Uganda, Rwanda and D.R.C., X. l. sudanensis (new host record) in Cameroon and Sudan, and the ranid anuran, Rana angolensis, in Zimbabwe. Anthropogenic dispersal of C. namaquensis, together with X. l. laevis, has occurred to localities in the south-western United States and Isle of Wight, U.K. Cephalochlamys compactus sp. nov., differentiated from C. namaquensis by a less well developed median ovarian cavity and more compact vitellarium, occurs in Xenopus muelleri in Togo, Ghana, Nigeria, Cameroon, D.R.C., Zimbabwe and South Africa, X. borealis in Kenya, X. clivii in Ethiopia, R. angolensis in Ethiopia and R. occipitalis in Nigeria. Cephalochlamys representatives of uncertain specific status have been recorded in X. gilli (new host record) in South Africa, X. pygmaeus and X. fraseri-like hosts (new host records) in the D.R.C., X. tropicalis (new host record) in Nigeria and R. occipitalis in Gabon. Despite the ability of some species to infect distantly related amphibians, the distribution of cephalochlamydid taxa amongst their principal hosts, pipids, can be related to the allopolyploid evolution of this anuran family.

The effects of kainate receptor-preferring glutamate ligands were tested on the electroretinogram (ERG) of the Xenopus retina. Kainate, domoic acid, and 5-iodowillardiine (20–100 μM) acted similarly in every respect. They increased peak amplitudes of the ERG a-, b-, and d-waves significantly over controls. The AMPA-specific antagonist, GYKI 53655, prevented a kainate-induced increase in ERG a- and d-waves, but was without effect on an increase in the b-wave. Once the effect of agonist on the b-wave had peaked, the ERG began to subside, leading to its nearly complete disappearance within 20 min. Prior exposure to GYKI followed by a combination of GYKI + agonist did not significantly slow the rate of b-wave disappearance. Our results indicate that (1) AMPA receptors contribute to ERG a- and d-waves. (2) The kainate-evoked increase in ERG a-, b-, and d-waves probably results, in part, from an excitotoxic swelling of inner retinal processes. (3) The inner retina has a population of GYKI-resistant, kainate-sensitive receptors which may contribute to b-wave generation.

The effects of the new cyclin-dependent kinase inhibitors, roscovitine and olomoucine, on oocytes and eggs of Xenopus laevis were investigated and compared with those of 6-dimethylamino purine (6-DMAP). The inhibitory properties of 6-DMAP, olomoucine and roscovitine towards p34cdc2-cyclin B isolated from Xenopus eggs revealed K-IC50 values of 300, 40 and 10 μM respectively. The three compounds inhibited progesterone-induced maturation with M-IC50 values of 200, 100 and 20 μM. These values were consistent with the K-IC50 values but the ratio M-IC50/K-IC50 was higher for roscovitine and olomoucine than for 6-DMAP. The disappearance of spindle and condensed chromosomes without pronucleus formation was observed when 1 mM 6-DMAP was applied for 4 h at germinal vesicle breakdown or at metaphase II, whereas no effect was observed using 1 mM olomoucine or 50 μM roscovitine. Changes in the electrophoretic mobility of p34cdc2 and erk2 were observed only in homogenates of matured oocytes or eggs exposed for 4 h to 1 mM 6-DMAP. When the drugs were microinjected into matured oocytes, olomoucine (100 μM) and roscovitine (50 μM) induced pronucleus formation more efficiently than did 6-DMAP (100 μM). Taken together, these results demonstrate that Xenopus oocytes possess a lower permeability to olomoucine and roscovitine and that these new compounds are suitable for in vivo studies after germinal vesicle breakdown provided they are microinjected.

Accurate and efficient processing of pre-rRNA is critical to the accumulation of mature functional ribosomal subunits for maintenance of cell growth. Processing requires numerous factors which act in trans as well as RNA sequence/structural elements which function in cis. To examine the latter, we have used directed mutagenesis and expression of mutated pre-rRNAs in yeast. Specifically, we tested requirements for formation of an ITS2-proximal stem on processing, a structure formed by an interaction between sequences corresponding to the 3′ end of 5.8S rRNA and the 5′ end of 25S. Pre-rRNA processing is inhibited in templates encoding mutations that prevent the formation of the ITS2-proximal stem. Compensatory, double mutations, which alter the sequence of this region but restore the structure of the stem, also restore processing, although at lower efficiency. This reduction in efficiency is reflected in decreased levels of mature 5.8S and 25S rRNA and increased levels of 35S pre-rRNA and certain processing intermediates. This phenotype is reminiscent of the biochemical depletion of U8 snoRNA in vertebrates for which the ITS2-proximal stem has been proposed as a potential site for interaction with U8 RNP. Thus, formation of the ITS2-proximal stem may be a requirement common to yeast and vertebrate pre-rRNA processing.

Adults of the monogenean genus Protopolystoma infecting Xenopus species occur in an extremely space-limited habitat, the urinary bladder. Xenopus wittei, from a population in Rwanda naturally infected with Protopolystoma fissilis and Protopolystoma simplicis, were exposed to reinfection in captivity (for 1–3 months post-capture) and then monitored in the laboratory for up to 5 months in transmission-free conditions. The two parasites co-occurred in individual bladders less frequently than expected if they were dispersed randomly. Distribution of bladder infections was significantly non-independent (n=157) and gravid worms of both species were never found in the same host. This pattern might be explained by interference competition between the parasites or by genetic differences in susceptibility within the host species, which is of allopolyploid origin. Other distributional data for sympatric polystomatid species pairs, including P. fissilis and P. ramulosus, show concurrent infections at frequencies consistent with random distributions (i.e. no evidence of interspecific competition or variability in species-specific susceptibility of the hosts). Interference between P. fissilis and P. simplicis (assuming host genetic factors are not involved) may therefore result from a mechanism specific to this species pair. Observations on infection turnover in captive hosts suggest that loss of adult worms may be related to the arrival of juveniles (of either species) in the urinary bladder. Ectopic infection of the host urinary ducts by adult and subadult P. fissilis was observed in some single-species infestations and may be density related. However, the use of an ectopic-site ‘refugium’ has never been observed in concurrent polystomatid infections.