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 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.

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.

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.

Immunocytochemical evidence for the involvement of an FMRFamide-related peptide in egg production in the flatworm parasite Polystoma nearcticum.

The monogenean flatworm Polystoma nearcticum exhibits reproductive synchrony with its treefrog host, Hyla versicolor, and becomes reproductively active only during the short period of host sexual activity at spawning. In this way, it provides a useful model system for exploring factors that may influence egg production in flatworm parasites. One such factor is the peptidergic innervation of the egg chamber or ootype. By using immunocytochemical techniques, the occurrence and distribution of GYIRFamide-like immunoreactivity, an authentic flatworm FMRFamide-related peptide (FaRP), have been monitored in the cells and fibres innervating the reproductive apparatus of worms collected at different stages of host sexual activity. Serotonin (5-HT) immunoreactivity in the worm was mapped for comparison. Extensive immunostaining for the FaRP and 5-HT was obtained throughout both the central and the peripheral nervous systems of worms, which were recovered from reproductively active frogs. In contrast, the innervation of the ootype of worms that were determined to be sexually inactive, including those recovered from frogs postspawning, showed little or no immunoreactivity for the FaRP; immunostaining for 5-HT in the ootype was unaffected by the reproductive state of the worm. These results indicate that FaRP expression in the neurons of the ootype innervation of P. nearcticum coincides with the parasite's brief period of egg production and, thus, provides evidence that regulatory peptides may be involved in the egg-assembly mechanism in flatworm parasites.

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.

Correlation of parasite speciation and specificity with host evolutionary relationships.

Protopolystoma (Monogenea, Polystomatidae) is strictly specific to the anuran amphibian genus Xenopus. The host group is characterised by a polyploid series in which chromosome numbers reflect diploid, tetraploid, octoploid and dodecaploid constitutions; the series is considered to have evolved through interspecies hybridisation and genome duplication. This study correlates information on host evolutionary relationships with patterns of parasite speciation and host specificity. Protopolystoma is restricted to one subgenus (Xenopus) with multiples of 36 chromosomes, and is absent from the subgenus Silurana (with multiples of 20 chromosomes). Molecular, biochemical and karyotype evidence distinguishes three subgroups within Xenopus. Representative species from each subgroup, Xenopus muelleri, Xenopus fraseri and Xenopus laevis, have been examined for polystomatid infection. Two species of Protopolystoma occur in each of these host species. In X. muelleri, the two Protopolystoma species reflect parasite co-speciation corresponding with the divergence of two sibling host species. Xenopus fraseri and X. laevis (both with 2n = 36 chromosomes) are implicated in the hybrid origin of two octoploid species, Xenopus wittei and Xenopus vestitus (both 2n = 72). The relationships of the Protopolystoma species in these Xenopus taxa reflect this presumed ancestry. Xenopus wittei carries two species of Protopolystoma, one shared with X. fraseri and the other shared with X. laevis. Xenopus vestitus carries a single species of Protopolystoma which is shared with X. laevis but there is no "heirloom" which reflects its hybrid origin involving X. fraseri. In addition to these shared parasite species which may reflect shared host genes, X. fraseri and X. laevis each carry separate species-specific Protopolystoma which do not occur in other Xenopus species even where there is evidence of common genetic information (as in the allopolyploid wittei and vestitus). This case study may be interpreted as indicating a powerful influence of host genetic factors on susceptibility to infection, host-specificity, and parasite speciation.

Reproductive interference in concurrent infections of two Protopolystoma species (Monogenea: Polystomatidae).

The prevention of interspecific reproductive interference is one possible explanation for spatial niche divergence between congeneric monogeneans. However, there is little direct evidence that reproductive interactions with other species are potentially deleterious to the majority of parasitic platyhelminths. Xenopus fraseri-like clawed toads from lowland rainforest in eastern Democratic Republic of Congo are infected by two species of polystomatid monogenean, Protopolystoma fissilis and Protopolystoma ramulosus. Both occur as adults in the host urinary bladder, and exhibit identical copulatory structures and similar body sizes. The small area of the habitat in relation to parasite body size makes close proximity inevitable in concurrent infections. Eggs were collected from five naturally infected hosts: two of these harboured concurrent infections, and three were infected with P. fissilis only. Eggs from concurrent infections showed reduced viability (57.6% embryonation, n = 413) compared with those from P. fissilis-only infections (85.2%, n = 439). This effect may be due to some form of reproductive interference, possibly failure to develop following interspecific cross-fertilisation.

Incompatibility of Protopolystoma xenopodis (Monogenea: Polystomatidae) with an octoploid Xenopus species from southern Rwanda.

Protopolystoma xenopodis is an oviparous monogenean occurring as an adult in the urinary bladder of the clawed toad Xenopus laevis. Oncomiracidia invade the host's kidneys where juveniles develop, subsequently migrating to the definitive site. In central Africa, the tetraploid X. laevis occurs in sympatry with octoploid congeners, including Xenopus wittei, believed to be the hybrid derivatives of X. laevis- and Xenopus fraseri-like parental lineages. Twenty laboratory-raised, naive specimens of an X. wittei-like species from southern Rwanda were each exposed to 30 embryonated P. xenopodis eggs (at 20 degrees C) and screened for parasite egg production until 9 months post-exposure. These toads failed to support the development of gravid parasites (comparable experimental procedures produce at least 35% prevalence of patent infection in the natural host X. laevis). Further X. wittei aff. (n = 26) and X. laevis (n = 17) were exposed to P. xenopodis oncomiracidia and dissected at variable times post-exposure: larvae were able to invade the kidneys of X. wittei aff. and began feeding and morphological development. Severe mortality of juveniles occurred in both natural and unnatural hosts between invasion and 39 days post-exposure. However, while small numbers of parasites persisted in X. laevis, no stages were found in X. wittei aff. beyond 39 days. Present data demonstrate the incompatibility of P. xenopodis with X. wittei aff. and are consistent with a hypothesis that specificity in Protopolystoma-Xenopus systems is determined primarily by the ability of juveniles to complete development in the host's kidneys.

Effects of temperature on oviposition rate in Protopolystoma xenopodis (Monogenea: Polystomatidae).

Protopolystoma xenopodis is an oviparous monogenean occurring as an adult in the urinary bladder of the African clawed toad, Xenopus laevis. Egg production was monitored in two groups of infected hosts exposed to decreasing environmental temperatures. Despite possible upward trends in reproductive capacity with parasite age, oviposition rate was severely depressed at lower temperatures. In one experiment, egg production was monitored in a sample of seven hosts at 2 degrees C intervals between 20 and 8 degrees C. Overall mean egg production rate showed a consistent decline from 11.9 eggs/worm/day (e/w/d) at 20 degrees C (days 1-10) to 0.9 e/w/d at 8 degrees C (days 115-124). In a second experiment (n = 11 hosts) oviposition rate was recorded at the same intervals between 20 and 6 degrees C and then hosts were returned to 20 degrees C. Overall mean egg production rate decreased consistently from 4.1 e/w/d at 20 degrees C (days 1-10) to 0.2 e/w/d at 6 degrees C (days 124-131), at which temperature all infections continued to produce eggs. When parasites were then returned to 20 degrees C (141-150 days), mean oviposition rate at this temperature (12.7 e/w/d) was found to have increased by 310% from the start of the experiment (130 days earlier) and by 6350% from production at 6 degrees C (9 days earlier). P. xenopodis has been introduced to South Wales, U.K. and present results show that it could produce eggs here through almost all of the year. However, a massive annual reduction in the reproductive output of this parasite in the U.K., compared with that in natural sites, is predicted.

Histological analysis of the egg capsule of the ovoviviparous polystomatid monogenean, Pseudodiplorchis americanus.

Transmission of Pseudodiplorchis americanus is restricted to the brief period when its host, a desert toad, enters water to spawn. The parasite accumulates its entire annual reproductive output within an elongated uterus during the 10-11-month period of host hibernation. Embryos of P. americanus, at all stages of development, are retained within the uterus which eventually becomes packed with around 150 encapsulated infective larvae. Recently formed eggs, which comprise a fertilized ovum and 2-3 vitelline cells, are closely surrounded by a primary eggshell which stains positively for acidic proteins and keratin. Initially, during passage along the proximal uterus, the egg capsule is only 60 microns in diameter, but as it passes to the distal uterus it expands to 800 microns in diameter to accommodate the growing larva. Due to chemical alterations or complete replacement of the shell, the final (secondary) egg capsule is a large sac-like structure composed of elastin. The flexible nature of this shell maximizes the numbers of infective larvae which can be stored in utero. TEM studies have revealed this capsule to be composed of multi-laminate membranes with a specialized cytoplasmic lining involved in a unique mechanism for embryo nutrition. This is the first report of an elastin-type eggshell within the Monogenea.

Melanin deposition in the gut of the monogenean Macrogyrodactylus polypteri Malmberg 1957.

Macrogyrodactylus polypteri, a skin parasite of the African freshwater fish Polypterus senegalus, has a black pigmented gut. Unusually, the gut is striped; 4 pigmented transverse bands become apparent within 2 h of birth and intensify in colour as the parasite ages. Ultrastructurally, the gut is a syncytium, similar to that of other gyrodactylids. Within the pigmented bands, electron-dense ovoid (1.0 micron x 0.25 micron) granules accumulate in feeding vacuoles in the gut syncytium, and the same granules are abundant in the gut lumen. These granules have the histochemical characteristics of melanosomes, and were also found in the epidermis of the host fish. It is concluded, therefore, that the black coloration is due to melanin derived from host epithelial cells in the diet, and that this parasite, in common with most other monopisthocotylean monogeneans, is an epithelial browser. The function of the gut banding is unknown, but may create a disruptive colour pattern, more difficult to see against the pigmented scales of the host than a uniformly dark gut.

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.

Cholinergic, serotoninergic and peptidergic components of the nervous system of Discocotyle sagittata (Monogenea:Polyopisthocotylea).

Cholinergic, serotoninergic (5-HT) and peptidergic neuronal pathways have been demonstrated in both central and peripheral nervous systems of adult Discocotyle sagittata, using enzyme histochemistry and indirect immunocytochemistry in conjunction with confocal scanning laser microscopy. Antisera to 2 native flatworm neuropeptides, neuropeptide F and the FMRFamide-related peptide (FaRP), GNFFRFamide, were employed to detect peptide immunoreactivity. The CNS is composed of paired cerebral ganglia and connecting dorsal commissure, together with several paired longitudinal nerve cords. The main longitudinal nerve cords (lateral, ventral and dorsal) are interconnected at intervals by a series of annular cross-connectives, producing a ladder-like arrangement typical of the platyhelminth nervous system. At the level of the haptor, the ventral cords provide nerve roots which innervate each of the 9 clamps. Cholinergic and peptidergic neuronal organisation was similar, but distinct from that of the serotoninergic components. The PNS and reproductive system are predominantly innervated by peptidergic neurones.

Life history specializations of monogenean flatworms: a review of experimental and microscopical studies.

This review illustrates the use of experimental approaches combined with microscopy to study the biology of monogenean parasites. Studies of feeding, development, reproduction, and systematics have been based on gyrodactylids, flatworms infecting teleost fishes. In a contrasting system involving an amphibian host in a desert environment, analysis of adaptations to extreme conditions has focused on Pseudodiplorchis americanus. The unusual reproductive strategies, particularly the interactions between mother and offspring, are highlighted for both monogeneans. Species of Gyrodactylus are viviparous, maintaining up to three generations of embryos simultaneously in utero, and many of their reproductive specializations are related to progenesis. Embryo nutrition takes place via a metabolically-active syncytial uterine lining that has close association with the parental gut. Microscopy has also proved an essential adjunct to molecular studies of speciation and host specificity. P. americanus is ovoviviparous and the adaptations for embryo maintenance are unique. The primary keratin-type eggshell is replaced by a flexible secondary elastin capsule produced by the uterus; parental nutrients are transferred through cytoplasmic connections to the developing embryo. TEM has demonstrated unique adaptations of P. americanus to its micro-environments, including secretion of tegumental vesicles that provide protection from digestive enzymes during migration through the host gut. This paper highlights the potential of monogeneans for studies of fundamental biological principles.

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.

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 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.

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.

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.