Parasite infectivity to hybridising host species: a link between hybrid resistance and allopolyploid speciation?

Variation in host-specific infectivity was studied in monogenean polystome parasites (Protopolystoma spp.) of the interfertile, parapatric anurans Xenopus laevis laevis and Xenopus muelleri. Laboratory-raised host F1 hybrids were resistant to parasites respectively specific to each parent taxon in nature. This resistance occurred against parasite isolates from both inside and outside a host hybrid/sympatric zone (and no isolate was compatible with the foreign host species under experimental conditions). Geographical Protopolystoma xenopodis isolates showed variable infectivity to a single full-sib group of their usual host, X. l. laevis, and strains with high or low infectivity to these sibs co-occurred in spatially distant local areas (separated by 1,700 km). The host compatibility of P. xenopodis was also subject to host genotypexparasite genotype interactions. Refractoriness to some parasites or pathogens, as a consequence of hybridisation, may have conferred a selective advantage on the allopolyploid pathway by which most Xenopus spp. are believed to have evolved.     

Speciation of Protopolystoma Bychowsky, 1957 (Monogenea: Polystomatidae) in hosts of the genus shape Xenopus (Anura: Pipidae)

The taxonomy, geographical distribution and hostrange of the polystomatid genus ProtopolystomaBychowsky, 1957 are reviewed. P. xenopodis(Price, 1943) and five new species are recognised,which occur in clawed toads ( Xenopus spp.)throughout subsaharan Africa. Of the two clawed toadsubgenera, Xenopus and Silurana, only theformer is infected. Protopolystoma spp. aredifferentiated by morphological variation of the gut,large hamulus and penis armature. P.xenopodis is found in Xenopus laevis subspeciesin South Africa, Transkei, Zimbabwe, DemocraticRepublic of Congo (D.R.C.), Rwanda, Uganda, Kenya andCameroon ( X. l. poweri and X. l.sudanensis are new host records). It also occurs inintroduced populations of X. l. laevis in theUnited States (southern California) and United Kingdom(South Wales). In subsaharan Africa the speciesdisplays significant, but continuous, geographicalvariation of penis spine size between southernpopulations in X. l. laevis and those in morenortherly host subspecies. Data on the natural hostrange of this parasite were complemented by anexperimental study of host-specificity in the southernform. This can produce patent infections in X.l. victorianus and X. gilli, but not X.wittei nor X. (Silurana) tropicalis. P.simplicis n. sp. is endemic to central and eastAfrican areas, infecting X. laevis subspecies ineastern D.R.C., Rwanda, Uganda and western Kenya, X. wittei-like hosts in eastern D.R.C., westernUganda, Rwanda and Burundi, X. vestitus inwestern Uganda and Xenopus sp. at Nairobi,Kenya. P. ramulosus n. sp. occurs in X.fraseri-like toads in eastern D.R.C. (Gabon andCameroon are also possible literature records), and P. fissilis n. sp. is found in X. fraseri-and X. wittei-like species in Cameroon andeastern D.R.C., and in southern Rwanda, respectively. Two Protopolystoma taxa are found in X.muelleri populations now suspected to representdistinct species: P. occidentalis n. sp. occursin X. muelleri (western form) in Ghana, Togo,Nigeria and Cameroon, while P. orientalis n. sp.is found in X. muelleri (eastern form) in SouthAfrica, Zimbabwe and Tanzania. The allopatricallydistributed species P. ramulosus, P.simplicis, P. occidentalis and P.orientalis form a relatively homogenous grouping withsome interspecific morphological overlap. These taxaare distinguished from P. xenopodis by penisspine morphology and from P. fissilis by hamulusroot form and aspects of gut morphology. Unidentified Protopolystoma sp. have been recorded in X. clivii in Ethiopia, X. fraseri aff. inCameroon and Xenopus sp. in Kenya and Tanzania. At some localities, single host species were infectedby two representatives of Protopolystoma. P. fissilis was recorded in eastern D.R.C. with P. ramulosus, with Protopolystoma sp. inCameroon in X. fraseri-like hosts and with P. simplicis in X. wittei-like hosts in Rwanda. P. xenopodis co-occurred with P. simplicisin X. laevis subspecies through central and eastAfrica.     

Paramphistome digeneans from shape Xenopus species (Pipidae) in Africa: taxonomy,host-specificity and biogeography

The taxonomy, host range and geographical distribution of paramphistome digeneans from Xenopus spp. in sub-Saharan Africa are reviewed. Two representatives of Progonimodiscus Vercammen-Grandjean, 1960 are recognised, both of which are narrowly or primarily specific to Xenopus. An analysis of morphometric and meristic characters indicated geographical variation in Progonimodiscus doyeri (Ortlepp, 1926), with two allopatric forms showing significant, but continuous, variation in testis size and vitelline follicle number. P. colubrifer n. sp. is distinguished from P. doyeri by the form of muscular elevations on the acetabulum accessory peduncle. It infects Xenopus ( Silurana) tropicalis-like toads from lowland tropical rain forest zones in Nigeria, Togo and the Ivory Coast, while P. doyeri occurs in hosts of the subgenus Xenopus from a wide variety of biotypes. Previous literature records indicate the presence of the southern P. doyeri morphological variant in X. laevis laevis in South Africa and Zimbabwe and the northern variant in X. l. victorianus, X. fraseri aff. and X. muelleri in the Democratic Republic of Congo, X. wittei in Uganda and the Democratic Republic of Congo, and the ranid Conraua crassipes in Cameroon (the only record of Progonimodiscus from a non-pipid host). New host and/or geographical records for this species are of the northern form in X. l. victorianus, X. l. bunyoniensis and X. vestitus in Uganda, X. l. sudanensis in Cameroon, X. borealis in Kenya, X. pygmaeus in the Democratic Republic of Congo, X. fraseri aff. in Cameroon, X. wittei aff. in the Democratic Republic of Congo and X. muelleri in Cameroon and Nigeria. While the geographical limits of the two P. doyeri variants are not known with precision, existing data are consistent with a turnover in the region of 15°S, where a notable discontinuity occurs in the distributions of other Xenopus parasites. Species of Diplodiscus Diesing, 1836 in Xenopus hosts are rare. Diplodiscus peregrinator n. sp. was recovered from X. tropicalis at a single locality in the Ivory Coast and distinguished by a combination of body size, egg size, genital pore position and acetabulum morphology. D. fischthalicus Meskal, 1970 was not found during the present study.     

The taxonomic status,host range and geographical distribution of Dollfuschella Vercammen-Grandjean, 1960 (Digenea: Halipeginae) from Xenopus spp. (Anura: Pipidae)

The taxonomy, host range and geographical distribution of halipegine digeneans from Xenopus spp. are reviewed. Dollfuschella Vercammen-Grandjean, 1960 is reinstated from synonymy with Halipegus Looss, 1899 because of the presence of a sinus-sac and weakly developed permanent sinus-organ. Only one representative of the genus, D. rodhaini Vercammen-Grandjean, 1960, is recognised, of which H. rhodesiensis Beverley-Burton, 1963 is considered a synonym. Based on previous literature records this parasite occurs in Xenopus laevis laevis from South Africa and Zimbabwe, X. l. victorianus from Zaire and Uganda, X. l. bunyoniensis from Rwanda, X. wittei from Zaire and Uganda, X. vestitus from Uganda and X. muelleri from Zaire (in some cases host identification has been revised). New host and/or locality records are from X. l. poweri in Zambia, X. l. victorianus in Rwanda, X. l. bunyoniensis in Uganda and X. clivii in Ethiopia. All known hosts belong to a clade characterised by multiples of 2n = 36 chromosomes. There are no records of halipegines from the other major lineage within Xenopus, X. tropicalis-like species with multiples of 2n = 20 chromosomes. This latter group occurs in lowland tropical rain forest from west Africa in contrast to the hosts of D. rodhaini which are found typically in grassland and wooded savanna and in montane forest biotypes. The distribution of D. rodhaini might, therefore, be limited by phylogenetic specificity to the definitive host group or by other ecological factors (e.g., availability of suitable molluscan hosts). Its wide geographical and host range, in common with some other parasite species from the 2n = 36 Xenopus lineage, may result from the lack of ecological or geographical barriers between different definitive host species and subspecies. However, significant geographical variation in egg-size occurs between northern D. rodhaini populations (north of about 15° S) and those from X. l. laevis in southern Africa. This is not considered sufficient for taxonomic recognition but it could reflect the operation of some isolating factor: parasite divergence concurs with evidence that X. l. laevis is evolutionarily relatively distant from the other (more northerly) members of the laevis Rassenkreis.     

Widespread historical presence of Batrachochytrium dendrobatidis in African pipid frogs

Aim Amphibian chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is associated with global amphibian population declines and species extinctions. Current evidence indicates that the pathogen has recently spread globally from an enzootic focus, with Xenopus spp. (family Pipidae) in South Africa having been identified as a likely source. The aim of this study was to investigate further the likelihood of African Xenopus spp. as the original source of Bd. Location We examined 665 museum specimens of 20 species of African and South American pipid frogs collected between 1844 and 1994 and held in the collection of the Natural History Museum, London. Methods Skin brushings taken from adult amphibians and brushings from the mouthparts, lips and developing hind limbs of larval pipid frogs were examined for the presence of Bd using real‐time PCR. Results We found six cases of Bd infection in three Xenopus spp. (from Africa), but none of the South American pipids was positive, although only 45 South American frogs were available for examination. The earliest case of Bd infection was in a specimen of Xenopus fraseri collected from Cameroon in 1933. A consistently low prevalence of infection over time indicates that a historical equilibrium existed between Xenopus spp. and Bd infection in Africa. Main conclusions Our results suggest that Bd infection was present in Xenopus spp. across sub‐Saharan Africa by the 1930s, providing additional support for the ‘out of Africa’ hypothesis. If this hypothesis is correct, it strengthens the argument for stringent control of human‐assisted movements of amphibians and other wildlife world‐wide to minimize the likelihood of pathogen introduction and disease emergence that can threaten species globally. Our findings help inform species selection for conservation in the face of the current Bd pandemic and also guide future research directions for selecting Bd isolates for sequencing and virulence testing.     

Oocyte and somatic 5S ribosomal RNA and 5S RNA encoding genes in Xenopus tropicalis.

We have investigated the structure of oocyte and somatic 5S ribosomal RNA and of 5S RNA encoding genes in Xenopus tropicalis. The sequences of the two 5S RNA families differ in four positions, but only one of these substitutions, a C to U transition in position 79 within the internal control region of the corresponding 5S RNA encoding genes, is a distinguishing characteristic of all Xenopus somatic and oocyte 5S RNAs characterized to date, including those from Xenopus laevis and Xenopus borealis. 5S RNA genes in Xenopus tropicalis are organized in clusters of multiple repeats of a 264 base pair unit; the structural and functional organization of the Xenopus tropicalis oocyte 5S gene is similar to the somatic but distinct from the oocyte 5S DNA in Xenopus laevis and Xenopus borealis. A comparative sequence analysis reveals the presence of a strictly conserved pentamer motif AAAGT in the 5'-flanking region of Xenopus 5S genes which we demonstrate in a separate communication to serve as a binding signal for an upstream stimulatory factor.     

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.     

Reprint of: Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

Screening for small molecule inhibitors of embryonic pathways: sometimes you gotta crack a few eggs

Extract prepared from Xenopus eggs represents a cell-free system that has been shown to recapitulate a multitude of cellular processes, including cell cycle regulation, DNA replication/repair, and cytoskeletal dynamics. In addition, this system has been used to successfully reconstitute the Wnt pathway. Xenopus egg extract, which can be biochemically manipulated, offers an ideal medium in which small molecule screening can be performed in near native milieu. Thus, the use of Xenopus egg extract for small molecule screening represents an ideal bridge between targeted and phenotypic screening approaches. This review focuses on the use of this system for small molecules modulators of major signal transduction pathways (Notch, Hedgehog, and Wnt) that are critical for the development of the early Xenopus embryo. We describe the properties of Xenopus egg extract and our own high throughput screen for small molecules that modulate the Wnt pathway using this cell-free system. We propose that Xenopus egg extract could similarly be adapted for screening for modulators of the Notch and Hedgehog pathways.     

非洲爪蟾PAPC多克隆抗体的制备及其特异性鉴定

非洲爪蟾ParaxialProtocadherin(PAPC)是一个在爪蟾Spemann组织者特异表达的膜蛋白.它在爪蟾原肠运动阶段的汇聚延伸运动和体节发生阶段的体节边界形成,以及早期听泡的形态发生和细胞特化过程中都有重要的作用.为了研究PAPC基因在早期胚胎发育过程中的表达及其生物学功能,需要制备PAPC抗体.应用谷胱甘肽S-转移酶(glutathioneStransferase,GST)表达系统表达GST-PAPC融合蛋白,亲和纯化后用以免疫新西兰大白兔,获得PAPC多克隆抗体.免疫印迹分析发现,以1∶3000稀释的该多克隆抗体为一抗时,能够在转染了全长PAPC质粒的HEK293T细胞的蛋白质抽提物中,特异地识别出150ku的印迹条带.同时,GST-PAPC融合蛋白可以竞争性抑制该抗体对全长PAPC质粒转染细胞的蛋白质抽提物的特异性条带.用1∶500稀释的该抗体为一抗进行免疫荧光分析时,发现,PAPC多克隆抗体能够识别在HEK293T细胞中过表达以及爪蟾动物极细胞中过表达的PAPC蛋白,荧光信号定位在细胞膜上.免疫印迹分析证明,PAPC抗体能够识别爪蟾胚胎中内源表达的PAPC蛋白.     

Xenopus as a model system for vertebrate heart development

The African clawed frog, Xenopus laevis, is a valuable model system for studies of vertebrate heart development. In the following review, we describe a range of embryological and molecular methodologies that are used in Xenopus research and discuss key discoveries relating to heart development that have been made using this model system. We also discuss how the sequence of the Xenopus tropicalis genome provides a valuable tool for identification of orthologous genes and for identification of evolutionarily conserved promoter elements. Finally, both forward and reverse genetic approaches are currently being applied to Xenopus for the study of vertebrate heart development.     

Teleost Fh14-3-3a protein protects Xenopus oocytes from hyperosmolality

We have previously cloned and characterized a novel 14-3-3 gene from the euryhaline telost Fundulus heteroclitus, Fh14-3-3a (Kültz et al., 2001). The corresponding gene product is osmoregulated and most highly expressed in gill epithelium of this fish. In the present study we have expressed Fh14-3-3a cRNA in Xenopus laevis oocytes and investigated the survival and electrophysiological parameters of Xenopus oocytes in isosmotic and various hyperosmotic media. Xenopus oocytes expressing Fh14-3-3a show no mortality after a 16 hour exposure to hyperosmolality in the form of elevating medium K(+), Na(+), polyethylene glycol, or sorbitol concentrations up to 444 mosmol/kg. In contrast, 16 hours of the same hyperosmolality caused 100% mortality in control Xenopus oocytes injected with water. As a result of hyperosmolality the Xenopus oocyte membrane potential decreased between 10 and 70% in oocytes expressing Fh14-3-3a whereas it was completely abolished in control oocytes. We report that one potential cause for the osmoprotective effect of Fh14-3-3a on Xenopus oocytes could be its inhibition of an endogenous chloride current. Hyperosmotic urea was not as harmful to Xenopus oocytes as hypertonicity and maybe acting through a different mechanism. Coexpression of Fh14-3-3a with a human calcium channel in Xenopus oocytes did not affect the electrophysiological properties of this exogenous channel. Thus, the osmoprotective effect of Fh14-3-3a may prove a valuable tool for the characterization of exogenous ion channels in Xenopus oocytes exposed to hyperosmotic conditions.     

Postoperative analgesics in South African Clawed frogs (Xenopus laevis) after surgical harvest of oocytes

Heightened awareness for the welfare of earlier-evolved laboratory species has prompted increasing inquiries by institutional animal care committees, investigators, and laboratory animal veterinarians regarding the need for post-surgical analgesics in laboratory Xenopus. Basic research into the mechanisms and regulation of pain in Rana pipiens has demonstrated the clinical potential of opioid, alpha2-adrenergic, and non-opioid analgesic agents in amphibians. However, clinical studies using objectively established indices of amphibian pain, or pharmacological studies in either Rana pipiens or laboratory Xenopus have not been conducted. As discussed above, comparison of limited lethality data suggests that the safety index for these agents is quite narrow in Rana pipiens. Analgesic use in laboratory Xenopus has the added risk of drowning due to over sedation. Drug doses extrapolated from such studies and intended to provide pain relief in Xenopus should therefore be considered very carefully. An additional concern for laboratory Xenopus is that the effects of these agents on amphibian oogenesis, oocyte quality, and embryogenesis are unknown. As the numbers of laboratory Xenopus used in basic and biomedical research continues to increase, clinical studies that address all of these issues cannot come too soon.