R. C. Tinsley

Combined ribosomal DNA and morphological analysis of individual gyrodactylid monogeneans

A method is presented for the isolation and analysis of hamuli, marginal hooks, and bars from individual gyrodactylid monogeneans using scanning electron microscopy (SEM), while simultaneously processing parasites for rDNA analysis using the polymerase chain reaction (PCR). The haptors of ethanol-fixed gyrodactylids were protease digested to liberate hooks for SEM, whereas DNA extracted from the bodies was used for PCR. The method resulted in hooks and hamuli being prepared from more than 90% of Gyrodactylus turnbulli individuals, a significant improvement on previously published digestion-based SEM techniques. PCR on the same parasites was less successful, but sequence data were obtained from 50% of individuals. Amplification of rDNA internal-transcribed spacer regions from individual worms used for SEM gave PCR products consistent with those predicted from our previous sequence analysis. This method allows the correlation of morphology and DNA sequence from the same individual and can be applied to ethanol-fixed material, such as field collected and museum specimens.

Behavior Favoring Transmission in the Viviparous Monogenean Gyrodactylus turnbulli

Transmission by Gyrodactylus turnbulli occurs most frequently when its hosts (Poecilia reticulata) come into close contact. This study is the first description of a specific migratory behavior that facilitates transmission of a gyrodactylid from dead hosts. Recently-dead guppies typically float at the waters surface; G. turnbulli moves off these fish into the water film, hanging motionless with the haptor held by surface tension. Because guppies are surface feeders, detached parasites in the water film are more likely to contact a new host.

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.

Parasitic disease in amphibians: control by the regulation of worm burdens.

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.

Environmental effects on transmission of Discocotyle sagittata (Monogenea): egg production and development.

Egg production by the freshwater gill monogenean Discocotyle sagittata infecting rainbow trout (Oncorhynchus mykiss) was monitored at 5, 13 and 18 degrees C; eggs were incubated at 6, 10, 13, 15, 16, 18, 22 and 26 degrees C and the hatching times determined. Mean per capita output at 13 degrees C ranged from 0.8 eggs/worm/day (e/w/d) to 18.3 e/w/d for 25 hosts (burdens 1-17 worms/host). Average egg production rates for worms in single burdens varied from 3 e/d to 14.6 e/d at 13 degrees C, mean 7.6 e/d (+/- 0.63) (n = 8). Egg production was temperature dependent, increasing from a mean of 1.5 e/w/d at 5 degrees C to 12.0 e/w/d at 18 degrees C. Developmental rate was also temperature dependent: larvae began hatching after 84 days at 6 degrees C, 28 days at 13 degrees C, and 20 days at 18 degrees C. Eggs laid within the same 24 h period hatched over 3-7 days at 13-18 degrees C; this range was extended at decreasing temperatures. Egg viability was generally high: between 13 and 18 degrees C a mean of 92% of larvae successfully emerged. However, viability declined at the highest and lowest temperatures (23% hatching at 6 degrees C, 55% at 8 degrees C and 47% at 26 degrees C). The implications for seasonal parasite transmission are discussed.

Effects of environmental temperature on the susceptibility of Xenopus laevis and X. wittei (Anura) to Protopolystoma xenopodis (Monogenea)

Abstract. Immune responses in ectothermic vertebrates and the life-processes of their parasites are profoundly linked to ambient temperature, but the functional effect of thermal environment on infectivity and host-specificity in helminths from ectotherms is poorly known. Primary infection establishment of Protopolystomaxenopodis (Monogenea) in compatible hosts (Xenopuslaevis: Anura) is strongly modulated by ambient temperature. Significantly fewer worms survived to the urinary bladder stage at 25°C than at 15°C (relatively high and low temperatures for this system in nature). Pre-infection exposure of X. laevis to cold (10°C) did not significantly affect parasite establishment in the urinary bladder (at 15 or 25°C p.i.), nor shorter-term postlarval survival in the kidneys (at 20°C p.i.), suggesting that residual immunosuppressive effects on host susceptibility are not important. Low temperatures had no permissive effect on the establishment of P. xenopodis in incompatible hosts (Xenopuswittei). The link between thermal conditions and parasitic infection of ectotherms is discussed.

Ultrastructural adaptations for viviparity in the female reproductive system of gyrodactylid monogeneans

The female reproductive system of viviparous monogeneans (Gyrodactylus and Macrogyrodactylus) has been examined using fluorescence microscopy and transmission electron microscopy. The female system is tubular, made up of a thin-walled proximal seminal receptacle/ootype and a distal uterus, separated by a complex cellular region. Both parts have a continuous syncytial cytoplasmic lining. Maturing oocytes in the seminal receptacle/ootype are in intimate contact with the receptacle lining. The uterus cytoplasmic lining completely surrounds the developing embryo, and is continuous with anterior and posterior cell bodies which fluoresce strongly when stained with bisBenzimide. This lining is most extensive around small embryos, when it contains specialised organelles including star-shaped configurations of electron-dense membranes and multilamellate bodies. Pits in the uterus wall bridged by membranous structures connect the cytoplasmic lining to parenchyma or digestive cells. The cytoplasmic lining regresses as the embryo develops, but remains continuous and in intimate contact with the embryonic tegument (at least until the near-term embryo begins independent movement). Numerous ribosomes, membranes and mitochondria in the uterine cytoplasmic layer indicate a high metabolic rate, and exo/endocytotic vesicles in the F1 tegument suggest transfer of materials occurs between parent and embryo. Putative vitelline cells in the posterior of the body contain abundant RNA, ribosomes and membrane-bound secretory bodies, and are filled with an electron-lucent secretion. However, there are no ducts associated with these cells, and their function remains unknown. The cytoplasmic lining of both the seminal receptacle/ootype and the uterus appears to regulate oocyte/embryo nutrition. Similar syncytial layers occur in rotifers, but are unlike the nutritive epithelia of most other viviparous organisms.

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.

Larval survival characteristics and behaviour of the gill monogenean Discocotyle sagittata

The survival characteristics of Discocotyle sagittata larvae are both age- and temperature-dependent. In laboratory studies at constant temperatures of 6, 10, 13, 18 and 22 degrees C, oncomiracidia had a maximum life-span of 96 h at 6 degrees C, declining with increasing temperature to 26 h at 22 degrees C. Larval swimming activity was also age dependent, and continued for a large proportion of survival time. The relationship between time at which larvae stop swimming as a proportion of total survival time was statistically significant between temperatures. Proportionally, oncomiracidia swam for longer periods at lower temperatures. As oncomiracidia age, they become progressively less active, spending less time in the water column. In contrast to observations recorded in the literature, D. sagittata larvae are infective soon after emergence (within the minimum of 2 h studied), and feed on blood from the gill capillaries within 2 h post-exposure. The temperature-dependent characteristics of the larval stage are likely to have important implications for seasonal changes in parasite transmission.

The Rift Valley is a major barrier to dispersal of African clawed frogs (Xenopus) in Ethiopia

The Ethiopian highlands – home to striking species diversity and endemism – are bisected by the Rift Valley, a zone of tectonic divergence. Using molecular data we examined the evolutionary history of two co‐distributed species of African clawed frog (Xenopus clivii and X. largeni) that are endemic to this region. Our field collections substantially extend the known distribution of X. largeni, a species formerly known from highlands southeast of the Rift, but that also occurs to the northwest. In both species, analysis of mitochondrial DNA and 19 autosomal loci identifies significant population structure, suggests little or no recent migration across the Rift Valley, and provides divergence time estimates across the Rift of ∼1–3.5 million years. These results indicate that the Ethiopian Rift Valley is a major obstacle to dispersal of highland‐adapted amphibians.