The oncomiracidium of Oculotrema hippopotami Stunkard, 1924 and relationships within the Polystomatidae (Monogenea).

Exceptional occurrences have fundamental interest in evolution relevant to understanding adaptations and origins. Monogeneans primarily infect aquatic lower vertebrates, i.e. fish, amphibians and chelonian reptiles, but there is a single instance of colonisation of a mammal: Oculotrema hippopotami Stunkard, 1924 infecting the eye of Hippopotamus amphibius Linnaeus. Its combination of systematic characters is amongst the most diverse in the Polystomatidae Gamble, 1896 and relationships are obscure. This study emphasises the primary significance of two features: the reinforcement of haptoral suckers with an internal skeleton and the pattern of ciliated cells on the oncomiracidium, especially the presence of conjoined cells. Closest relationships are with polystomatids infecting chelonians, specifically species of Polystomoides Ward, 1917 from the oral cavity/pharynx, or more likely (but with currently incomplete evidence) species of Neopolystoma Price, 1939 from the eye. Morphological characters of polystomoidines, all of which infect chelonians, appear to have remained relatively stable since at least the Jurassic (from zoogeographical evidence), but the highly derived characters of species of Oculotrema may have evolved during the comparatively short period (16 million years) since the Miocene origin of Hippopotaminae Gray. However, the initial host switch may plausibly have been to hippo ancestors, the anthracotheres, with similar semi-aquatic ecology and an Eocene origin (41 million years ago). Over the same time-scale, the oncomiracidial cell pattern remained closely comparable with that of presumed ancestors, emphasising its value in phylogenetic analyses.

Environmental constraints influencing survival of an African parasite in a north temperate habitat: effects of temperature on development within the host.

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.

Environmental constraints influencing survival of an African parasite in a north temperate habitat: effects of temperature on egg development.

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.

Evolutionary diversity in polystomatids infecting tetraploid and octoploid Xenopus in East African highlands: biological and molecular evidence.

Species of Protopolystoma are monogenean flukes that only infect allopolyploid hosts in the anuran genus Xenopus. Multivariate analyses of morphometric sclerite characters in the nominal species Protopolystoma simplicis suggest that morphologically distinguishable populations occur in the tetraploid host, Xenopus laevis victorianus, and in each of the octoploid hosts, X. vestitus and X. wittei. The species-level divergence of a lineage specific to X. laevis is supported by sequence variation in the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Protopolystoma simplicis from X. laevis is redesignated P. microsclera n. sp., with P. simplicis being retained for populations in octoploid hosts. This division is consistent with large differences in egg hatching schedule, fixed differences at the mannose-6-phosphate isomerase and fumarate hydratase loci, and host-specificity in experimental analyses. Although the respective P. simplicis populations in X. vestitus and X. wittei also show significant diversity in allozyme expression, morphometrics and egg hatching schedule, they are retained in the same species because their level of mitochondrial DNA divergence is similar to that found within other Protopolystoma species. The consequences of splitting P. simplicis for a recent interpretation of the origin of Protopolystoma faunas in octoploid Xenopus spp. is discussed.

Heterogeneous interspecific interactions in a host-parasite system.

Macroparasites of vertebrates usually occur in multi-species communities, producing infections whose outcome in individual hosts or host populations may depend on the dynamics of interactions amongst the different component species. Within a single co-infection, competition can occur between conspecific and heterospecific parasite individuals, either directly or via the host's physiological and immune responses. We studied a natural single-host, multi-parasite model infection system (polystomes in the anuran Xenopus laevis victorianus) in which the parasite species show total interspecific competitive exclusion as adults in host individuals. Multi-species infection experiments indicated that competitive outcomes were dependent on infection species composition and strongly influenced by the intraspecific genetic identity of the interacting organisms. Our results also demonstrate the special importance of temporal heterogeneity (the sequence of infection by different species) in competition and co-existence between parasite species and predict that developmental plasticity in inferior competitors, and the induction of species-specific host resistance, will partition the within-host-individual habitat over time. We emphasise that such local (within-host) context-dependent processes are likely to be a fundamental determinant of population dynamics in multi-species parasite assemblages.

Postlarval Protopolystoma spp. kidney infections in incompatible Xenopus spp. induce weak resistance to heterospecifics.

Protopolystoma xenopodis and Protopolystoma orientalis are polystomatid monogeneans respectively specific to the parapatric anurans Xenopus laevis and Xenopus muelleri. Parasite larval stages may invade the kidneys of foreign Xenopus spp. but die before migration to the definitive urinary bladder site. Laboratory experiments to assess the effect of a primary incompatible kidney infection on a secondary compatible infection found: (1) a small, significant decrease in the survivorship of P. xenopodis kidney stages (23-37 days p.i. at 25 degrees C) in X. laevis laevis previously challenged with P. orientalis; (2) a significant effect of prior P. orientalis challenge on P. xenopodis development and establishment in the urinary bladder of X. laevis 100 days p.i. (at 21 degrees C); (3) no effect of prior P. xenopodis challenge on adult P. orientalis establishment in X. muelleri (at 21 degrees C), but a significant negative influence on reproductive output (days 0-50 post-patency). Partial cross-resistance to heterospecifics may therefore be induced by Protopolystoma spp. infections in the kidneys of an incompatible host, demonstrating that at least some elements of the host response are non-species specific. The effects observed were weak compared to the strong host resistance known to be generated by an established compatible primary infection with respect to conspecifics. This difference suggests that strong acquired resistance to Protopolystoma species is species-specific and/or induced only by older stages surviving in compatible hosts.

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.

Pathological effects of Pseudodiplorchis americanus (Monogenea: Polystomatidae) on the lung epithelium of its host, Scaphiopus couchii.

Infection of the desert toad, Scaphiopus couchii, by the monogenean Pseudodiplorchis americanus involves 2 principal sites: post-invasion juveniles reside in the respiratory tract for 1 month before migrating to the urinary bladder where they reach sexual maturity and may live up to 4 years. While previous work has demonstrated the long-term impact on host condition of the blood-feeding adults, this study assesses pathological effects of the short-term pulmonary infection. Lung ultrastructure was compared in toads (i) maintained in captivity for 1 year without invasion, and (ii) experimentally infected with 50-300 juveniles/host, equivalent to burdens in the wild, and examined 23-44 days p.i. Typically, the alveolar lining of S. couchii is composed of a single cell type with characteristics of both Type I and Type II pneumocytes. However, infected lung tissue exhibited an inflammatory reaction with epithelial cell vacuolation, interstitial oedema, and an increase of alveolar exudate, leucocytes and fibrous tissue. Accompanying a post-infection increase in host immune cells in the lungs, there was evidence of reciprocal tegumental damage to the parasites. Lung epithelium of toads free of infection for 1 year exhibited scar tissue representing a residual effect of past infection. The pathological consequences of P. americanus infection therefore have 2 components. Acute lung infection coincides with the host's brief activity season: impaired respiratory function could compromise feeding and accumulation of reserves and hence ability to survive following a 10 month period of hibernation. Additionally, adult toads are normally exposed annually to re-infection and may accumulate chronic lung damage with extended effects on host survival.

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

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 Protopolystoma xenopodis (Monogenea) in compatible hosts ( Xenopus laevis: Anura) is strongly modulated by ambient temperature. Significantly fewer worms survived to the urinary bladder stage at 25 degrees C than at 15 degrees C (relatively high and low temperatures for this system in nature). Pre-infection exposure of X. laevis to cold (10 degrees C) did not significantly affect parasite establishment in the urinary bladder (at 15 or 25 degrees C p.i.), nor shorter-term postlarval survival in the kidneys (at 20 degrees 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 (Xenopus wittei). The link between thermal conditions and parasitic infection of ectotherms is discussed.

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 water's 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.

Host factors limiting monogenean infections: a case study.

Comprehensive field data on polystomatid monogeneans record low prevalence and intensity of infection and suggest that worm burdens in this group are strongly regulated: thus, in the majority of Polystoma species infecting anuran amphibians mean abundance is typically less than one parasite/host. There is circumstantial evidence that the dominant control is attributable to host factors which over-ride variations in transmission success. This review provides a brief summary of information on Pseudodiplorchis americanus, a parasite of the desert toad, Scaphiopus couchii, and then focuses in detail on the spectrum of factors regulating infrapopulations of Protopolystoma xenopodis, a parasite of the aquatic Xenopus laevis. Infection levels of adult worms and their contribution to transmission are regulated by external environmental factors (especially temperature), by host factors (including behaviour and population density), and by a range of parasite factors including intra- and inter-specific competitive interactions and variations in intrinsic characters, especially survivorship and reproductive output. In addition to these factors whose primary effect is to modulate transmission rates, there is a major attrition in parasite numbers between invasion and maturity (3 months post-infection). Long-term laboratory experiments on the Xenopus laevis/Protopolystoma xenopodis interaction demonstrate a powerful acquired immune response. Primary infection is characterised by a high prevalence of established adult worms but the success of subsequent challenge infection is greatly reduced, leading to low prevalence and extended pre-patent period. In the small proportion of hosts supporting a second infection of adult parasites, surviving burdens are small (one to two worms/host) and show reduced egg production. These results provide an explanation for the low burdens encountered in field studies: a majority of adult X. laevis in natural populations are likely to exhibit strong, relatively long-term, post-infection immunity after the loss of a previous infection.

Survival, feeding and embryo development of Gyrodactylus gasterostei (Monogenea: Gyrodactylidae).

Viviparous gyrodactylid monogeneans which are detached from their host may form an important source of infection in fish communities. This is particularly significant for these ectoparasites which have no specific free-living stage and where transmission usually occurs through transfer of adult parasites when fish come into close contact. In this study, in vitro survival of Gyrodactylus gasterostei was correlated with changes in tissue ultrastructure of parents and their embryos during detachment and following return to a host. At 10 degrees C, detached worms survived for up to 89 h (50%) survival = 20 h) with mortality influenced by both age-independent and age-dependent factors. The gastrodermis of attached, fed parasites is syncytial and contains a variety of vesicles and feeding vacuoles. However, following detachment and starvation, degenerative changes occur within the gastrodermis, eventually leading to marked damage to the embryo/s. When starved worms reattached to a fish, they began browsing on host epithelial cells within 1 min and ultrastructural evidence for phagocytic activity in the gastrodermis was detected after 5 min. Putative waste vacuoles increased in abundance after 5-30 min on the new host, coinciding with the reappearance of electron-dense vesicles and possibly indicating completion of the first intracellular cycle of digestion. Parental feeding directly influenced the normal cyclical maturation and regression of the uterine lining between births. In extreme cases, starvation led to detached parasites (6.1%) aborting their embryos. However, even short periods off the host influenced development and survival of embryos, suggesting that temporary interruption of nutrient flow to the embryo can significantly affect gyrodactylid reproductive rates.

Ultrastructure of spermiogenesis and spermatozoa of Discocotyle sagittata (Monogenea: Polyopisthocotylea: Discocotylinea).

Ultrastructural analysis revealed that the spermatozoon of Discocotyle sagittata (Leuckart, 1842) is composed of two parallel axonemes, mitochondrion, nucleus and cortical microtubules. The nucleus, which occupies a central/distal position and has an unusual crescent-shaped profile, is slightly shorter than the mitochondrial rod. The two axonemes, which are of unequal length, and the cortical microtubules (up to 68 forming a continuous ring in the principal region) extend almost the entire length of the spermatozoon. A fold of the plasma membrane creates a unilateral flange or undulating membrane. Epifluorescence microscopy indicated that spermatogenesis gives rise to clusters of 64 spermatids connected to a common cytophore. Spermiogenesis and the structure of the filiform sperm of D. sagittata conform to the typical polyopisthocotylean pattern.

Protopolystoma xenopodis (Monogenea) primary and secondary infections in Xenopus laevis.

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 degrees 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 15:1. Results suggest that primary infection with P. xenopodis can elicit strong, long-term protective immunity against re-infection in X. laevis.

A redescription of Chabaudus leberrei (Bain & Philippon, 1969) (Nematoda: Seuratoidea) from Xenopus spp. in Swaziland.

Chabaudus leberrei (Bain & Philippon, 1969) is redescribed from the pipid anurans Xenopus muelleri (Peters) and X. laevis laevis (Daudin) (new host records) in northern Swaziland, based on light and scanning electron microscope studies. The six anterior protuberances characteristic of the genus Chabaudus Inglis & Ogden, 1965, are, in C. leberrei, formed by bipartite lamellae associated with the internal margins of the three lips. Intraspecific variation in the number and disposition of male caudal papillae and in the development of the cephalic vesicle is documented.

Differentiation of two locally sympatric Protopolystoma (Monogenea: Polystomatidae) species by temperature-dependent larval development and survival.

The developmental response of egg stages to different environmental temperature regimes was studied in Protopolystoma xenopodis and Protopolystoma orientalis (Monogenea: Polystomatidae) isolates from southern Africa. Eggs failed to develop at 10 degrees C, whilst at 15 degrees C only P. xenopodis completed larval development, hatching 49--88 days post-collection. Respective hatching windows were 26--34 (P. xenopodis) and 37--49 (P. orientalis) days at 20 degrees C, and 18--26 and 27--37 days at 25 degrees C. Continuous maintenance at 30 degrees C was lethal for eggs of both species. There were no consistent interspecific differences in the response of egg stages to low and high temperature shocks during early embryonic development.

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.

Parasite adaptation to extreme conditions in a desert environment.

Deserts represent universally recognized extreme environments for animal life. This paper documents the highly specialized adaptations of Pseudodiplorchis americanus, a monogenean parasite of the desert toad, Scaphiopus couchii. Building on a long-term record of parasite population ecology (continuing since the early 1980s), field studies focus on the effects of severe drought in the Sonoran Desert, Arizona, in the mid 1990s. This provides a test of the ability of the host-parasite system to tolerate exceptional perturbation. The analysis provides new insight into parasite infection dynamics in a natural wildlife system through integration of host and parasite population age structure. The environmental check interrupted host recruitment in 1993-95 and parasite recruitment in 1995-97. This produced an imprint in age structure and infection levels recognizable over several years: parasite recruitment failure reduced transmission 2-3 years later. The host (maximum life span 17 years) tolerated the disruption but the impact was more serious for the parasite (life span 3 years) leading to extinction of some previously stable populations. Despite this demonstration of a rare event exacerbating external environmental constraints, experimental studies suggest that the internal (host) environment normally creates the most severe conditions affecting P. americanus. Only about 3% of parasites survive from invasion until first reproduction. Post-invasion factors including host immunity, characteristic of most parasite life cycles, constitute a greater constraint upon survival than external conditions, even in a desert environment.

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.