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Acoel flatworms have played a relevant role in classical (and current) discussions on the evolutionary origin of bilaterian animals. This is mostly derived from the apparent simplicity of their body architectures. This tenet has been challenged over the last couple of decades, mostly because detailed studies of their morphology and the introduction of multiple genomic technologies have unveiled a complexity of cell types, tissular arrangements and patterning mechanisms that were hidden below this 'superficial' simplicity. One tissue that has received a particular attention has been the nervous system (NS). The combination of ultrastructural and single cell methodologies has revealed unique cellular diversity and developmental trajectories for most of their neurons and associated sensory systems. Moreover, the great diversity in NS architectures shown by different acoels offers us with a unique group of animals where to study key aspects of neurogenesis and diversification od neural systems over evolutionary time.In this review we revisit some recent developments in the characterization of the acoel nervous system structure and the regulatory mechanisms that contribute to their embryological development. We end up by suggesting some promising avenues to better understand how this tissue is organized in its finest cellular details and how to achieve a deeper knowledge of the functional roles that genes and gene networks play in its construction., (© 2024. The Author(s).)

Single-cell RNA sequencing has become a standard technique to characterize tissue development. Hereby, cross-sectional snapshots of the diversity of cell transcriptomes were transformed into (pseudo-) longitudinal trajectories of cell differentiation using computational methods, which are based on similarity measures distinguishing cell phenotypes. Cell development is driven by alterations of transcriptional programs e.g., by differentiation from stem cells into various tissues or by adapting to micro-environmental requirements. We here complement developmental trajectories in cell-state space by trajectories in gene-state space to more clearly address this latter aspect. Such trajectories can be generated using self-organizing maps machine learning. The method transforms multidimensional gene expression patterns into two dimensional data landscapes, which resemble the metaphoric Waddington epigenetic landscape. Trajectories in this landscape visualize transcriptional programs passed by cells along their developmental paths from stem cells to differentiated tissues. In addition, we generated developmental "vector fields" using RNA-velocities to forecast changes of RNA abundance in the expression landscapes. We applied the method to tissue development of planarian as an illustrative example. Gene-state space trajectories complement our data portrayal approach by (pseudo-)temporal information about changing transcriptional programs of the cells. Future applications can be seen in the fields of tissue and cell differentiation, ageing and tumor progression and also, using other data types such as genome, methylome, and also clinical and epidemiological phenotype data.

Background: The genus Macrostomum consists of small free-living flatworms and contains Macrostomum lignano, which has been used in investigations of ageing, stem cell biology, bioadhesion, karyology, and sexual selection in hermaphrodites. Two types of mating behaviour occur within this genus. Some species, including M. lignano, mate via reciprocal copulation, where, in a single mating, both partners insert their male copulatory organ into the female storage organ and simultaneously donate and receive sperm. Other species mate via hypodermic insemination, where worms use a needle-like copulatory organ to inject sperm into the tissue of the partner. These contrasting mating behaviours are associated with striking differences in sperm and copulatory organ morphology. Here we expand the genomic resources within the genus to representatives of both behaviour types and investigate whether genes vary in their rate of evolution depending on their putative function., Results: We present de novo assembled transcriptomes of three Macrostomum species, namely M. hystrix, a close relative of M. lignano that mates via hypodermic insemination, M. spirale, a more distantly related species that mates via reciprocal copulation, and finally M. pusillum, which represents a clade that is only distantly related to the other three species and also mates via hypodermic insemination. We infer 23,764 sets of homologous genes and annotate them using experimental evidence from M. lignano. Across the genus, we identify 521 gene families with conserved patterns of differential expression between juvenile vs. adult worms and 185 gene families with a putative expression in the testes that are restricted to the two reciprocally mating species. Further, we show that homologs of putative reproduction-related genes have a higher protein divergence across the four species than genes lacking such annotations and that they are more difficult to identify across the four species, indicating that these genes evolve more rapidly, while genes involved in neoblast function are more conserved., Conclusions: This study improves the genus Macrostomum as a model system, by providing resources for the targeted investigation of gene function in a broad range of species. And we, for the first time, show that reproduction-related genes evolve at an accelerated rate in flatworms.

Positional information is fundamental to animal regeneration and tissue turnover. In planarians, muscle cells express signaling molecules to promote positional identity. At the ends of the anterior-posterior (AP) axis, positional identity is determined by anterior and posterior poles, which are putative organizers. We identified a gene, nr4A , that is required for anterior- and posterior-pole localization to axis extremes. nr4A encodes a nuclear receptor expressed predominantly in planarian muscle, including strongly at AP-axis ends and the poles. nr4A RNAi causes patterning gene expression domains to retract from head and tail tips, and ectopic anterior and posterior anatomy (e.g., eyes) to iteratively appear more internally. Our study reveals a novel patterning phenotype, in which pattern-organizing cells (poles) shift from their normal locations (axis extremes), triggering abnormal tissue pattern that fails to reach equilibrium. We propose that nr4A promotes pattern at planarian AP axis ends through restriction of patterning gene expression domains., Competing Interests: DL, CM, PR No competing interests declared, (© 2019, Li et al.)

We investigated feeding and reproductive performance of coexisting pelagic turbellarians from experiments on predation rates of Mesostoma ehrenbergii and M. craci as a function of food (Daphnia similis, three levels) and temperature (4 levels) during 10 days. Flatworms were collected from the pelagic of a subtropical lake in Brazil. Growth was more rapid at higher temperatures: more prey were consumed, and more eggs produced. M. craci and particularly M. ehrenbergii fitted a linear mixed-effects model and showed a type II functional response. M. craci was the more stenothermic of the two. Intrageneric predation also occurred: M. ehrenbergii fed on M. craci, but not vice versa. After a first clutch of subitaneous eggs, M. ehrenbergii produced resting eggs only. In M. craci an intermediate type of eggs hatched some time after release, survived passage through the gut of M. ehrenbergii, but did not resist drying. By primarily selecting cladoceran prey, M. ehrenbergii can make coexistence of both flatworms possible. As population density of M. ehrenbergii increases, it turns to producing resting and non-viable subitaneous eggs, thus limiting its population size. In nature, these processes structure the zooplankton community, while avoiding extinction of prey and predator.

Platyhelminthes (flatworms) have captivated the imagination of biologists for centuries. Indeed, planarian flatworms were used as experimental models decades before Caenorhabditis elegans became known as 'the worm'. Although planarians experienced a brief fall from grace, with the advent of molecular tools, planarians, such as Schmidtea mediterranea, have emerged in recent years as powerful models to study the basis of stem cell regulation and tissue regeneration. Flatworms are not just everyone's favorite experimental subjects from high school biology - they also include some of nature's most successful parasites, many of which have plagued humans throughout our history. This Primer will focus on several aspects of the remarkable biology found throughout the phylum Platyhelminthes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the context of global change, there is an urgent need for researchers in conservation physiology to understand the physiological mechanisms leading to the acquisition of stress acclimation phenotypes. Intertidal organisms continuously cope with drastic changes in their environmental conditions, making them outstanding models for the study of physiological acclimation. As the implementation of such processes usually comes at a high bioenergetic cost, a mitochondrial/oxidative stress approach emerges as the most relevant approach when seeking to analyze whole-animal responses. Here we use the intertidal flatworm Macrostomum lignano to analyze the bioenergetics of salinity acclimation and its consequences in terms of reactive oxygen/nitrogen species formation and physiological response to counteract redox imbalance. Measures of water fluxes and body volume suggest that M. lignano is a hyper-/iso-regulator. Higher salinities were revealed to be the most energetically expensive conditions, with an increase in mitochondrial density accompanied by increased respiration rates. Such modifications came at the price of enhanced superoxide anion production, likely associated with a high caspase 3 upregulation. These animals nevertheless managed to live at high levels of environmental salinity through the upregulation of several mitochondrial antioxidant enzymes such as superoxide dismutase. Contrarily, animals at low salinities decreased their respiration rates, reduced their activity and increased nitric oxide formation, suggesting a certain degree of metabolic arrest. A contradictory increase in dichlorofluorescein fluorescence and an upregulation of gluthathione-S-transferase pi 1 (GSTP1) expression were observed in these individuals. If animals at low salinity are indeed facing metabolic depression, the return to seawater may result in an oxidative burst. We hypothesize that this increase in GSTP1 could be a "preparation for oxidative stress", i.e. a mechanism to counteract the production of free radicals upon returning to seawater. The results of the present study shed new light on how tolerant organisms carry out subcellular adaptations to withstand environmental change., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Over the past decade, the free-living flatworm Macrostomum lignano has been successfully used in many areas of biology, including embryology, stem cells, sexual selection, bioadhesion and aging. The increased use of this powerful laboratory model, including the establishment of genomic resources and tools, makes it essential to have a detailed description of the chromosome organization of this species, previously suggested to have a karyotype with 2n = 8 and one pair of large and three pairs of small metacentric chromosomes. We performed cytogenetic analyses for chromosomes of one commonly used inbred line of M. lignano (called DV1) and uncovered unexpected chromosome number variation in the form of aneuploidies of the largest chromosomes. These results prompted us to perform karyotypic studies in individual specimens of this and other lines of M. lignano reared under laboratory conditions, as well as in freshly field-collected specimens from different natural populations. Our analyses revealed a high frequency of aneuploids and in some cases other numerical and structural chromosome abnormalities in laboratory-reared lines of M. lignano, and some cases of aneuploidy were also found in freshly field-collected specimens. Moreover, karyological analyses were performed in specimens of three further species: Macrostomum sp. 8 (a close relative of M. lignano), M. spirale and M. hystrix. Macrostomum sp. 8 showed a karyotype that was similar to that of M. lignano, with tetrasomy for its largest chromosome being the most common karyotype, while the other two species showed a simpler karyotype that is more typical of the genus Macrostomum. These findings suggest that M. lignano and Macrostomum sp. 8 can be used as new models for studying processes of partial genome duplication in genome evolution., Competing Interests: The authors have declared that no competing interests exist.

Background: Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory's own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri - a non-model animal., Results: Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope., Conclusions: We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions.

Phenotypic variation results from the balance between sources of variation and counteracting regulatory mechanisms. Canalization and developmental stability are two such mechanisms, acting at two different levels of regulation. The issue of whether or not they act concurrently as a common developmental buffering capacity has been subject to debate. We used geometric morphometrics to quantify the mechanisms that guarantee phenotypic constancy in the haptoral anchors of Ligophorus cephali. Canalization and developmental stability were appraised by estimating inter- and intra-individual variation, respectively, in size and shape of dorsal and ventral anchors. The latter variation was estimated as fluctuating asymmetry (FA) between anchor pairs. The general-buffering-capacity hypothesis was tested by two different methods based on correlations and Principal Components Analyses of the different components of size and shape variation. Evidence for FA in the dorsal and ventral anchors in both shape and size was found. Our analyses supported the hypothesis of a general developmental buffering capacity. The evidence was more compelling for shape than for size and, particularly, for the ventral anchors than for the dorsal ones. These results are in line with previous studies of dactylogyrids suggesting that ventral anchors secure a firmer, more permanent attachment, whereas dorsal anchors are more mobile. Because fixation to the host is crucial for survival in ectoparasites, we suggest that homeostatic development of the ventral anchors has been promoted to ensure the morphological constancy required for efficient attachment. Geometric morphometrics can be readily applied to other host-monogenean models, affording not only to disentangle the effects of canalization and developmental stability, as shown herein, but to further partition the environmental and genetic components of the former.

Helminth parasites of veterinary importance cause huge revenue losses to agrarian economy worldwide. With the emergence of drug resistance against the current formulations, there is a need to focus on the alternative approaches in order to control this menace. In the present study, biocompatible zinc oxide nanoparticles (ZnO NPs) were used to see their in vitro effect on the biliary amphistomes, Gigantocotyle explanatum, infecting Bubalus bubalis because these nanoparticles are involved in generation of free radicals that induce oxidative stress, resulting in disruption of cellular machinery. The ZnO NPs were synthesized by using egg albumin as a biotemplate and subsequently characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction and Spectrophotometrical, which showed that ZnO NPs were highly purified wurtzite type polycrystals, with a mean size of 16.7 nm. When the parasites were treated with lower concentrations (0.004% and 0.008%) of the ZnO NPs, the worms mounted a protective response by stimulating the antioxidant system but the treatment of G. explanatum with 0.012% ZnO NPs produced significant inhibition of the antioxidant enzymes like superoxide dismutase (SOD) (p< 0.05) and glutathione S- transferase (GST) (p<0.01), while the level of malondialdehyde (MDA), a lipid peroxidation marker, was significantly (p< 0.01) elevated. SEM and histopathology revealed pronounced tegumental damage showing the disruption of surface papillae and the annulations, particularly in the posterior region near acetabulum. The under expression of a number of polypeptides, loss of worm motility in a time dependent manner, further reflect strong anthelmintic potential of ZnO NPs. It can be concluded that the anthelmintic effect might be due to the production of reactive oxygen species that target a variety of macromolecules such as nucleic acid, protein and lipids which are involved in different cellular processes.

Background: The cutaneous monostome trematode Collyriclum faba (Bremser in Schmalz, 1831) is a bird parasite with a hitherto unknown life cycle and highly focal occurrence across the Holarctic and Neotropic ecozones., Methods: Representative specimens of benthic organisms were sampled at multiple sites and dates within the known foci of C. faba occurrence in Slovakia. A combined approach involving detailed morphological examination and sequencing of two independent DNA loci was used for their analysis., Results: We elucidated the complete life cycle of C. faba, which we determined to include the aquatic gastropod mollusk Bythinella austriaca (Frauenfeld, 1857) as the first intermediate host, the mayflies of the family Heptageniidae, Ecdyonurus venosus (Fabricius, 1775) and Rhithrogena picteti Sowa, 1971 x iridina (Kolenati, 1839), as the second intermediate hosts, and birds (primarily but not exclusively passeriform birds) as the definitive hosts. Bythinella austriaca occurs focally in the springs of tributaries of the Danube in the Alpine-Carpathian region. The restricted distribution of B. austriaca explains the highly focal distribution of C. faba noticed previously in spite of the broad distribution of its second intermediate and definitive host species. Utilization of both larval and adult Ephemeroptera spp. as the second intermediate hosts explains the known spectrum of the definitive host species, with the highest prevalence in species feeding on larvae of Ephemeroptera, such as Cinclus cinclus (Linnaeus, 1758) and Motacilla cinerea Tunstall, 1771, or adults of Ephemeroptera, such as Sylvia atricapilla (Linnaeus, 1758) and Regulus regulus (Linnaeus, 1758). In this study, we also determine the prevalence and DNA sequences of other immature trematode specimens found in the examined benthic organisms (particularly the families Microphallidae, Troglotrematidae and Nanophyetidae and Euryhelmis zelleri Grabda-Kazubska, 1980, Heterophyidae), and describe cercariae of C. faba., Conclusions: We determined the full life cycle of the Central European populations of C. faba. We speculate that other species of Bythinella and the closely related genus Amnicola may serve as first intermediate hosts in other parts of the distribution range of C. faba. Similarly, other Ephemeroptera of the family Heptageniidae may serve as the second intermediate hosts of C. faba in the Americas.

Neobenedenia melleni is a significant monogenean pathogen of fish in aquaculture facilities and public aquaria. Immunity after exposure to live N. melleni is well established, but the mechanisms of immunity remain unclear. In this study, tilapia (Oreochromis mossambicus) were continuously exposed to N. melleni over a four-month period and assessed for immunity as determined by a reduction in the number of parasites dislodged from the experimental animals during freshwater immersion. Specific mucosal and systemic antibody levels were by determined via enzyme-linked immunosorbent assay. At 45 days postexposure (DPE), fish displayed high parasite loads and baseline levels of mucosal antibodies. At 102 and 120 DPE parasite loads were significantly decreased, and antibody levels were significantly increased for mucus and plasma samples. The correlation between immunity (reduction in parasite load) and an increased humoral antibody response suggests a key role of antibody in the immune response. This is the first report of immunity against N. melleni that is associated with specific mucosal or systemic antibodies.

The African clawed frog Xenopus laevis (Anura: Pipidae) is host to more than 25 parasite genera encompassing most of the parasitic invertebrate groups. Protopolystoma xenopodis Price, 1943 (Monogenea: Polystomatidae) is one of two monogeneans infecting X. laevis. This study focussed on the external morphology of different developmental stages using scanning electron microscopy, histology and light microscopy. Eggs are released continuously and are washed out when the frog urinates. After successful development, an active swimming oncomiracidium leaves the egg capsule and locates a potential post-metamorphic clawed frog. The oncomiracidium migrates to the kidney where it attaches and starts to feed on blood. The parasite then migrates to the urinary bladder where it reaches maturity. Eggs are fusiform, about 300 μm long, with a smooth surface and are operculated. Oncomiracidia are elongated and cylindrical in shape, with an oval posterior cup-shaped haptor that bears a total of 20 sclerites; 16 marginal hooklets used for attachment to the kidney of the host and two pairs of hamulus primordia. Cilia from the 64 ciliated cells enable the oncomiracidium to swim for up to 24 h when the cilia subsequently curl up, become non-functional and are shed from the body. The tegument between the ciliated cells bears a series of sensory papillae. The body of the mature parasite is elongated and pyriform and possesses an opisthaptor armed with three pairs of suckers and two pairs of falciform hooks to ensure a firm grip on the flexible internal surface of the urinary bladder., (© M. Theunissen et al., published by EDP Sciences, 2014.)

Gyrodactylus salaris is a notifiable freshwater ectoparasite of salmonids. Its primary host is Atlantic salmon (Salmo salar), upon which infections can cause death, and have led to massive declines in salmon numbers in Norway, where the parasite is widespread. Different strains of S. salar vary in their susceptibility, with Atlantic strains (such as those found in Norway) exhibiting no resistance to the parasite, and Baltic strains demonstrating an innate resistance sufficient to regulate parasite numbers on the host causing it to either die out or persist at a low level. In this study, Leslie matrix and compartmental models were used to generate data that demonstrated the population growth of G. salaris on an individual host is dependent on the total number of offspring per parasite, its longevity and the timing of its births. The data demonstrated that the key factor determining the rate of G. salaris population growth is the time at which the parasite first gives birth, with rapid birth rate giving rise to large population size. Furthermore, it was shown that though the parasite can give birth up to four times, only two births are required for the population to persist as long as the first birth occurs before a parasite is three days old. As temperature is known to influence the timing of the parasite's first birth, greater impact may be predicted if introduced to countries with warmer climates than Norway, such as the UK and Ireland which are currently recognised to be free of G. salaris. However, the outputs from the models developed in this study suggest that temperature induced trade-offs between the total number of offspring the parasite gives birth to and the first birth timing may prevent increased population growth rates over those observed in Norway.

Acoelomorphs are bilaterally symmetric small marine worms that lack a coelom and possess a digestive system with a single opening. Two alternative phylogenetic positions of this group within the animal tree are currently debated. In one view, Acoelomorpha is the sister group to all remaining Bilateria and as such, is a morphologically simple stepping stone in bilaterian evolution. In the other, the group is a lineage within the Deuterostomia, and therefore, has derived a simple morphology from a more complex ancestor. Acoels and the closely related Nemertodermatida and Xenoturbellida, which together form the Acoelomorpha, possess a very limited number of cell types. To further investigate the diversity and origin of mesodermal cell types we describe the expression pattern of 12 orthologs of bilaterian mesodermal markers including Six1/2, Twist, FoxC, GATA4/5/6, in the acoel Isodiametra pulchra. All the genes are expressed in stem cells (neoblasts), gonads, and at least subsets of the acoel musculature. Most are expressed in endomesodermal compartments of I. pulchra developing embryos similar to what has been described in cnidarians. Our molecular evidence indicates a very limited number of mesodermal cell types and suggests an endomesodermal origin of the gonads and the stem cell system. We discuss our results in light of the two prevailing phylogenetic positions of Acoelomorpha.

Gyrodactylid population growth may depend on abiotic variables such as temperature. We tested the survival and reproductive rate of Gyrodactylus mexicanus, a parasite infecting fins of Girardinichthys multiradiatus, at 3 different water temperatures, 10-13, 19-22, and 24 C. The temporal sequence of birth and age at death of each parasite isolated from the hosts was recorded through at least 8 generations. Our results showed that the average number of offspring per parasite was 2.0 when averaged across all temperatures. However, the generation time was negatively correlated with temperature. The innate capacity for increase (r(m)) was positively correlated with water temperature: from 0.29 parasite/day at 13 C to 0.48 parasite/day at 24 C. These data confirm that water temperature has a direct influence on parasite population dynamics. The current study represents the first contribution to understanding the population ecology of the monogenean G. mexicanus in central Mexico.

A filtration-aided acid cleaning method was used to collect biosilica structures from a diatom culture medium, natural seawater, or water bloom. Cell extraction, acid cleaning, and acid removal were all performed on a polytetrafluoroethylene (PTFE) filter cloth, significantly improving the treatment capacity and efficiency of the traditional acid wash method. Five typical diatoms were cultivated in the laboratory for acid cleaning. Different growth speeds were introduced, and different process parameters for acid cleaning were utilized. After the acid cleaning, biosilica structures were collected from the frustules of diatoms using different methods. Girdle bands and valves of Coscinodiscus sp. were separated by floating of the valves. Central spines of Ditylum brightwellii and valves of Skeletonema costatum were separately collected by settling or filtration. Rod-like frustules, such as those of Bacillaris paradoxa, are not suitable for large quantities of acid wash. The silica structures were observed and tested using an AFM-calibrated glass needle to determine their elasticity. Elasticity tests showed that ringent girdle bands are more flexible than complete ones (Coscinodiscus sp.) and that both long-chain clusters of Nitzschia palea and central spines of D. brightwellii have certain elasticities. The required pressure for deforming or breaking the biosilica structures of diatoms was also determined.

Theory predicts that sexual reproduction promotes disease invasion by increasing the evolutionary potential of the parasite, whereas asexual reproduction tends to enhance establishment success and population growth rate. Gyrodactylid monogeneans are ubiquitous ectoparasites of teleost fish, and the evolutionary success of the specious Gyrodactylus genus is thought to be partly due to their use of various modes of reproduction. Gyrodactylus turnbulli is a natural parasite of the guppy (Poecilia reticulata), a small, tropical fish used as a model for behavioural, ecological and evolutionary studies. Using experimental infections and a recently developed microsatellite marker, we conclusively show that monogenean parasites reproduce sexually. Conservatively, we estimate that sexual recombination occurs and that between 3.7-10.9% of the parasites in our experimental crosses are hybrid genotypes with ancestors from different laboratory strains of G. turnbulli. We also provide evidence of hybrid vigour and/or inter-strain competition, which appeared to lead to a higher maximum parasite load in mixed infections. Finally, we demonstrate inbreeding avoidance for the first time in platyhelminths which may influence the distribution of parasites within a host and their subsequent exposure to the host's localized immune response. Combined reproductive modes and inbreeding avoidance may explain the extreme evolutionary diversification success of parasites such as Gyrodactylus, where host-parasite coevolution is punctuated by relatively frequent host switching.

As a consequence of anthropogenic CO₂ emissions, oceans are becoming more acidic, a phenomenon known as ocean acidification. Many marine species predicted to be sensitive to this stressor are photosymbiotic, including corals and foraminifera. However, the direct impact of ocean acidification on the relationship between the photosynthetic and nonphotosynthetic organism remains unclear and is complicated by other physiological processes known to be sensitive to ocean acidification (e.g. calcification and feeding). We have studied the impact of extreme pH decrease/pCO₂ increase on the complete life cycle of the photosymbiotic, non-calcifying and pure autotrophic acoel worm, Symsagittifera roscoffensis. Our results show that this species is resistant to high pCO₂ with no negative or even positive effects on fitness (survival, growth, fertility) and/or photosymbiotic relationship till pCO₂ up to 54 K µatm. Some sub-lethal bleaching is only observed at pCO₂ up to 270 K µatm when seawater is saturated by CO₂. This indicates that photosymbiosis can be resistant to high pCO₂. If such a finding would be confirmed in other photosymbiotic species, we could then hypothesize that negative impact of high pCO₂ observed on other photosymbiotic species such as corals and foraminifera could occur through indirect impacts at other levels (calcification, feeding).

Parasites and parasitism is common. Worm macroparasites have evolved life-history traits that allow them to successfully transmit between spatially and temporally separated patches of host resource and to survive within these environments. Macroparasites have common life-history strategies to achieve this, but these general themes are modified in a myriad of ways related to the specific biology of their hosts. Parasite life histories are also dynamic, responding to conditions inside and outside of hosts, and they continue to evolve, especially in response to our attempts to control them and the harm that they cause., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Many coastal areas have served as repositories of different anthropogenic and naturally induced organic material and nutrients. The major sources thereof are riverine inputs which strongly influence the spatial and temporal distribution of benthic communities. In this study, the benthic foraminiferal, meiofaunal, and macrofaunal colonies in front of three rivers in a poorly known, but environmentally valuable, area of the Central Adriatic Sea have been examined concurrently. The physico-chemical parameters of bottom water and sediment characteristics were determined in order to characterize both the sediment-water interface and the benthic environments. Although changes in the biota are neither univocal nor unidirectional, a moderate influence of riverine input on the different communities' components can be inferred. The most affected taxa are foraminifera and copepods and, to a lesser extent, meiofaunal polychaetes and platyhelminthes. These results are also tested by the ABC curves, which reveal that the macrofaunal communities closest to the river mouths are moderately disturbed. This integrated investigation documents, for the first time, how benthic communities can be used as an early warning indicator with which to monitor the health quality of a coastal ecosystem.

The gill monogene communities of Pimephales promelas (fathead minnow) in 3 distinct sites on converging streams were investigated from 2004 to 2006 in 3 different seasons. Thirty collections of P. promelas were made in southeastern Nebraska along 3 converging tributaries: Elk Creek (40.88534°N, 96.83366°W), West Oak Creek (40.9082°N, 96.81432°W), and Oak Creek (40.91402°N, 96.770583°W), Lancaster County, Nebraska. In all, 103 P. promelas were collected from Elk Creek, 115 from West Oak Creek, and 78 from Oak Creek and examined for gill monogenes. Among the P. promelas collected, 93.5% were infected with up to 3 species of Dactylogyrus, including Dactylogyrus simplex Mizelle, 1937, Dactylogyrus bychowskyi Mizelle, 1937, and Dactylogyrus pectenatus Mayes, 1977. Mean intensities at Elk Creek, West Oak Creek, and Oak Creek were 17.6, 22.8, and 25.1, and prevalences 88, 95, and 97%, respectively. At these 3 sites: (1) P. promelas does not share Dactylogyrus species with Semotilus atromaculatus (Creek chub) or Notropis stramineus (Sand shiner); (2) fish size and sex are not predictive of Dactylogyrus infection; (3) Dactylogyrus spp. vary (not always predictably) in their seasonal occurrence; (4) populations of Dactylogyrus spp. respond to environmental differences among sites; and (5) the community structure of Dactylogyrus spp. (order of abundance) is independent of environment.

One previously described species, Haliotrema striatohamus Zhukov, 1981, found off the coast of Cuba and Campeche (Gulf of Mexico), is redescribed and transferred to Haliotrematoides Kritsky, Yang and Sun, 2009, as Haliotrematoides striatohamus ( Zhukov, 1981) n. comb. This transfer is based on the study of new specimens of this species found in the gill lamellae of grunts, i.e., Haemulon aurolineatum Cuvier, Haemulon plumierii (Lacepède), and Haemulon sciurus (Shaw) (Haemulidae), all from the southern coast of Quintana Roo, Mexico. In addition, another previously described species, Haliotrema brevispirocirrus Zhukov, 1981 from Haemulon striatum (Linnaeus) from Havana, Cuba, is also transferred to Haliotrematoides based on the comparative morphology of its original description. Data are provided for prevalence and mean intensity as well as for the morphologic and biometric variability of H. striatohamus from different hosts. The new replacement of H. striatohamus ( Zhukov, 1981) n. comb. and H. brevispirocirrus (Zhukov, 1981) n. comb. infecting these taxa represents the first known species of Haliotrematoides on these fishes and new geographical records for these dactylogyrid monogenoids.

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.

We quantified Neobenedenia melleni from the skin of Caribbean surgeonfishes (Acanthuridae) from June through October 2005 and 2007. Prevalence, or mean intensity of infection, or both, varied significantly among the 3 species, and among sites and between years for the most heavily infected species, blue tang (Acanthurus coeruleus). Among 6 sites sampled, no more than 12% of ocean surgeonfish (Acanthurus bahianus) were infected, compared with 10 to 100% of A. coeruleus. The prevalence of infection among doctorfish (Acanthurus chirurgus), collected at only 1 of the sites, was intermediate between the other 2 species (46%). Mean intensity (range) of infection for the few infected A. bahianus was 1 (1) to 3 (1-8), compared with 1.3 (1-2) to 14.3 (1-59) for A. coeruleus, and 2.5 (1-8) for A. chirurgus. Expected abundance of N. melleni on A. coeruleus from shallow bay sites was greater than for those from non-bay sites. Higher infections on A. coeruleus may be attributable to differences in habitat use, or susceptibility to infection, or both, compared to other species. Among site and between-year differences may be associated with differences in benthic habitat, or water conditions, or both. This system seems ideal for future comparative studies on the relationship between environmental variables and parasites on Caribbean coral reefs.

The monogene Dactylogyrus eucalius Mizelle and Regensberger, 1945 and its ability to maintain a population from year to year on the annual fish Culaea inconstans Kirkland was examined in a small lake in central Ontario. Fish were sampled toward the end of their annual breeding season, at a time when the host population consisted of 2 cohorts, i.e., young-of-the-year (0+) and mature adults (1+). Prevalence of infection was 94%, with a mean intensity of 8.8 +/- 9.6; neither measure varied significantly with host length or between cohorts (P > 0.05). At necropsy, parasites were characterized as juveniles that included postoncomiracidia (immature, with a ventrally directed haptor) as well as developing protandrous males (body with a near-complete haptor and with little or no pigmented vitellaria), or as adults (with testis, ovarium, darkened vitellaria, and occasionally bearing a tanned egg). The proportion of juvenile to adult parasites differed significantly between cohorts (P < 0.05), with 0+ fish infected with a mixture of juveniles and adults, whereas 1+ fish had almost exclusively adult parasites. Since adult (1+) brook stickleback typically die after spawning, the increased frequency of juvenile parasites exploiting juvenile hosts may represent an evolutionary adaptation, maximizing the chances of parasites infecting hosts that will enter winter. It is suspected that 0+ fish can be infected in the nest within 2 wk of hatching and persist by effectively infecting new host recruits when they are sympatric with their parents.

Molecular approaches to the study of development and evolution have had profound effects on our understanding of the nature of the evolutionary process. Developmental biologists became intoxicated with fanciful notions of reconstructing genetic pathways of morphogenesis while evolutionary biologists were sobered by the fallacy of reconstructing organismal relationships along increasing grades of morphological complexity. Increased taxon sampling and improvements in analytical techniques are providing a new approach and are forcing biologists to move past historical biases to allow more accurate mapping of morphological and developmental characters through evolutionary time. Here, we discuss the possible developmental and morphological features of the 'urbilaterian', the triploblastic animal with anterior-posterior and dorsoventral axes and predecessor of the protostome-deuterostome ancestor. We argue that this animal, with features resembling acoelomorph flatworms, was far simpler morphologically than the protostome-deuterostome ancestor despite possessing a nearly complete eubilaterian genome. We show that the deployment of some genes expected to pattern the protostome-deuterostome ancestor is not deployed in acoels in the predicted manner and thus might have been co-opted after the evolution of the urbilaterian. We also identify the developmental changes related to gastrulation that gave rise to the urbilaterian from a simpler cnidarian-like ancestor.

Over the past 40 years, much has been published on the ultrastructure and cellular development of embryonic structures in a wide range of cestodes. However, the literature contains many discrepancies in both terminology and interpretations because of the facts that these organisms are phylogenetically diverse within their respective orders and families, the habitats that affect embryonic envelope structure are diverse, and the work has been done in various laboratories around the world. This review and synthesis was initiated by a working group of biologists from around the world convened at the Fifth International Workshop on Cestode Systematics and Phylogeny in Ceské Budejovice, at the Institute of Parasitology of the Biology Centre, Academy of Sciences of the Czech Republic. It brings together the data from published work and establishes a uniform terminology and interpretation based on the data as they are presented. A consensus was reached for standardised definitions of the oncosphere, hexacanth, coracidium, embryonic envelopes, outer envelope, inner envelope, embryophore, vitelline capsule, shell, and outer coat. All of these are defined as components of the embryo or its vitellocyte-derived or uterine-derived coatings.

Background: The parasitic Platyhelminthes (Neodermata) contains three parasitic groups of flatworms, each having a unique morphology, and life style: Monogenea (primarily ectoparasitic), Trematoda (endoparasitic flukes), and Cestoda (endoparasitic tapeworms). The evolutionary origin of complex life cyles (multiple obligate hosts, as found in Trematoda and Cestoda) and of endo-/ecto-parasitism in these groups is still under debate and these questions can be resolved, only if the phylogenetic position of the Monogenea within the Neodermata clade is correctly estimated., Results: To test the interrelationships of the major parasitic flatworm groups, we estimated the phylogeny of the Neodermata using complete available mitochondrial genome sequences and a newly characterized sequence of a polyopisthocotylean monogenean Microcotyle sebastis. Comparisons of inferred amino acid sequences and gene arrangement patterns with other published flatworm mtDNAs indicate Monogenea are sister group to a clade of Trematoda+Cestoda., Conclusion: Results confirm that vertebrates were the first host for stem group neodermatans and that the addition of a second, invertebrate, host was a single event occurring in the Trematoda+Cestoda lineage. In other words, the move from direct life cycles with one host to complex life cycles with multiple hosts was a single evolutionary event. In association with the evolution of life cycle patterns, our result supports the hypothesis that the most recent common ancestor of the Neodermata giving rise to the Monogenea adopted vertebrate ectoparasitism as its initial life cycle pattern and that the intermediate hosts of the Trematoda (molluscs) and Cestoda (crustaceans) were subsequently added into the endoparasitic life cycles of the Trematoda+Cestoda clade after the common ancestor of these branched off from the monogenean lineage. Complex life cycles, involving one or more intermediate hosts, arose through the addition of intermediate hosts and not the addition of a vertebrate definitive host. Additional evidence is required from monopisthocotylean monogeneans in order to confirm the monophyly of the group.

This study investigates the ecology of monogenean gill parasites of Aslyanax altiparanae Garutti & Britski, 2000 and Rhamdia quelen (Quoy & Gaimard, 1824) in a stretch of the Sao Francisco Verdadeiro River, Parana, Brazil. Statistical and ecological indices were used to examine observed levels of parasitism in relation to host and environmental characteristics. A. altiparance and R. quelen had infestation intensities of 2.8 and 23.1 parasites per fish, respectively. The only significant environmental influence was observed at the upstream station for R. quelen. For both host species, parasitized and non-parasitized individuals presented similar weight-ength relationships. Parasitized individuals had dispersed K,, values indicating abnormal conditions. The low levels of parasitism observed in this study suggest that the environment is relatively undisturbed. Additional studies should compare these two species and their respective parasites following completion of the hydroelectric headquarters planned for construction in this stretch of the Sao Francisco Verdoadeiro River.

We report the development of an Expressed Sequence Tag (EST) resource for the flatworm Macrostomum lignano. This taxon is of interest due to its basal placement within the flatworms. As such, it provides a useful comparative model for understanding the development of neural and sensory organization. It was anticipated on the basis of previous studies [e.g., Sánchez-Alvarado et al., Development, 129:5659-5665, (2002)] that a wide range of developmental markers would be expressed in later-stage macrostomids, and this proved to be the case, permitting recovery of a range of gene sequences important in development. To this end, an adult Macrostomum cDNA library was generated and 7,680 Macrostomum ESTs were sequenced from the 5' end. In addition, 1,536 of these aforementioned sequences were sequenced from the 3' end. Of the roughly 5,416 non-redundant sequences identified, 68% are similar to previously reported genes of known function. In addition, nearly 100 specific clones were obtained with potential neural and sensory function. From these data, an annotated searchable database of the Macrostomum EST collection has been made available on the web. A major objective was to obtain genes that would allow reconstruction of embryogenesis, and in particular neurogenesis, in a basal platyhelminth. The sequences recovered will serve as probes with which the origin and morphogenesis of lineages and tissues can be followed. To this end, we demonstrate a protocol for combined immunohistochemistry and in situ hybridization labeling in juvenile Macrostomum, employing homologs of lin11/lim1 and six3/optix. Expression of these genes is shown in the context of the neuropile/muscle system.

Two monogeneans species, Polylabris mamaevi Ogawa & Egusa, 1980 and Tetrancistrum nebulosi Young, 1967, are sympatric on the gills of Siganus fuscescens (Houttugn, 1782) in the South China Sea. An investigation into their distribution on the host's gills based on monthly samples from both wild and caged populations revealed that these 2 species co-occurred on the same gill arches, but they were segregated. P. mamaevi attached to the distal half of the gill filaments, whereas T. nebulosi attached to the proximal half. This difference may partly be attributed to microhabitat specialization. Microhabitat distributions were intensity dependent for both species. As intensity increased, monogeneans were increasingly found on the second, third, and even fourth gill arches, and niche breadths increased. The changes in distribution and niche breadths at the host gill arch level in relation to differences in host size and sampling period were considered to represent a functional response to increased intensity. Differences in host resources, in either net-caged or wild populations, did not significantly influence the parasite distributions at the host gill arch level. Simultaneous infections of these 2 species were common, but there was no evidence of negative interspecific interactions.

In this study, metazoan parasites of bream (Abramis brama Linnaeus, 1758) in the Lake Durusu (Terkos) were investigated between June 2002 and May 2003. During this study, a total of 67 bream were examined for the presence of metazoan parasites. Ten species of parasites were found on 64 of the 67 fish examined. These parasites are: Dactylogyrus sphyrna (Linstow, 1878) and D. distinguendus (Nybelin, 1936) Monogenoidea, Caryophyllaeus laticeps (Pallas, 1781) Cestoidea, Tetracotyle sp, Diplostomum sp. and Tylodelphys clavata (Nordmann, 1832) metacercaria Trematoda, Eustrongylides excisus (Jagerskiöld, 1909) Nematoda, Piscicola geometra (Linnaeus, 1758) Hirudinea, glochidia of mollusk, Bivalvia, Argulus foliaceus (L., 1758) Crustacea. Diplostomum sp., Dactylogyrus sphyrna and D. distinguendus were found to be the dominant parasites of A. brama. Both the prevalence and intensity of other parasites were not found to be high. All identified parasites are a new finding for A. brama in the Lake Durusu. This is the first time that D. distinguendus has been identified in Turkey.

Philureter trigoniopsis parasitizes the ureters and urinary bladder of Galaxias maculatus in Patagonian Andean lakes. To investigate factors associated with variation in the prevalence and intensity of this monogenean, fish were sampled periodically over 2 yr in Lake Gutiérrez. Prevalence and mean intensity are higher in smaller fishes than in larger ones. A seasonal pattern was observed, with peak recruitment and peak mean intensity occurring in early spring (September), followed by lows in late summer (January-February). Galaxias maculatus length classes are spatially segregated due to seasonal migrations, so the annual infection cycle is characterized by higher prevalence and intensity from late winter to early summer in the smaller fish from the deep zone of the lake.

Compared to other monogenoidean groups, viviparous gyrodactylids exhibit extraordinary species diversity and broad host range. It has been suggested that this evolutionary success is associated with a suite of morphological and life-history traits that include, in part, continuous transmission (i.e., ability to infect new hosts throughout the gyrodactylid life cycle). Experiments were conducted to explore the putative adaptive advantage of continuous transmission within viviparous gyrodactylids during colonization of new host resources. Differences in infrapopulation growth, such as abundance, prevalence, and duration of the infection, of Gyrodactylus anisopharynx on 3 species of fish--Corydoras paleatus and Corydoras ehrhardti (both natural hosts) as well as Corydoras schwartzi (a host not known to harbor G. anisopharynx)--held under isolated and grouped conditions were determined. Results showed that infrapopulations of G. anisopharynx on C. paleatus and C. schwartzi had higher growth when the parasite had the opportunity for host transfer (grouped hosts). Infrapopulations of G. anisopharynx on isolated and grouped C. ehrhardti showed an opposite trend, although differences in mean duration and maximum abundance were not statistically different. Results obtained from experiments with C. paleatus and C. schwartzi support the hypothesis that continuous transmission in viviparous gyrodactylids enhances colonization success, probably by allowing initial avoidance of Red Queen dynamics. The absence of statistical differences between infrapopulations on isolated and grouped C. ehrhardti suggests that parasite dynamics may be influenced by factors other than continuous transmission in this host.

The susceptibility of a Baltic salmon stock Salmo salar (Indalsälv, central Sweden) to Norwegian Gyrodactylus salaris (Figga strain, central Norway) was experimentally tested and compared with previously obtained results on East Atlantic salmon (Lierelva, SE Norway). Contrary to expectation, the Baltic salmon, which had no prior exposure to this parasite strain, appeared almost as susceptible as the Norwegian salmon parr that naturally experience G. salaris-induced mortality. Individually isolated salmon of both stocks sustained G. salaris infections with little evidence of innate resistance. A few individuals of the Indalsälv stock controlled their infection from the beginning, but overall there was considerable heterogeneity in the course of infection in both stocks. On individual hosts, G. salaris growth rates declined steadily throughout the infection, a trend which was particularly marked amongst the Lierelva stock. On shoaling Lierelva fish, there was some evidence of reduced parasite population growth towards the end of the infection; this was not apparent in Indalsälv fishes. These results reflect a growing awareness that not all Baltic salmon may be resistant to Norwegian G. salaris, and that Norwegian and Baltic G. salaris strains may differ in virulence. Consequently, management decisions concerning this parasite-host system should be based upon the actual, and tested, susceptibility of stocks under consideration and not upon identification of stocks as either Atlantic or Baltic.

The susceptibility of various races of salmonids towards infections with the skin parasitic monogenean Gyrodactylus salaris Malmberg, 1957, differs markedly. Norwegian and Scottish salmon strains are known as extremely susceptible to infection, whereas Baltic salmon races such as the Neva strain (Russian origin) and the Indals river (Swedish origin) salmon have been characterized as relatively resistant. However, the status of the many other Baltic strains has remained unknown. The present study reports on the susceptibility of the Baltic salmon from the Swedish river Lule. It was shown that this strain is susceptible to infection but to a lesser extent than the Scottish salmon. Further studies showed that injection of immuno-suppressants (dexamethasone) greatly increased population growth of G. salaris on Scottish salmon but not on the Baltic salmon. Mucous cell density on fins differed between strains, and a general trend to decreased cell density on infected fish 8 wk post-infection, compared to uninfected fish, was observed. The largest decrease in mucous cell density following infection was seen in the most resistant fish. After administration of immuno-suppressants, this decrease in mucous cell density was inhibited in the Scottish salmon but not in the Baltic salmon. Thus, there seems to be a relationship between the fishes' ability to discard mucous cells and the ability to resist infections with Gyrodactylus salaris. Although the Lule salmon seems more susceptible to infection compared to previous reports on the Neva salmon, the results support the notion that Baltic salmon strains are generally more resistant than East Atlantic salmon.

Planarians are the free-living members (order Tricladida) of the phylum Platyhelminthes. They are triploblastic, acoelomate, unsegmented and located at the base of the Lophotrochozoa clade. Besides their huge regenerative capacity, planarians have simple eyes, considered similar to the prototypic eye suggested by Charles Darwin in his book 'On the Origin of Species'. The conserved genetic network that determines the initial steps of eye development across metazoans supports a monophyletic origin of the various eye types present in the animal kingdom. Here we summarise the pattern of expression of certain genes involved in the eye network that have been isolated in planarians, such as Otx, Pax-6, Six, Rax and opsin. We describe the effects of RNA interference-mediated loss of function on eye regeneration. Finally, we discuss the relevance of these findings for the evolution of the eye gene network.

Dendromonocotyle colorni sp. n. (Monogenea: Monocotylidae) is described from the dorsal skin surface of two specimens of Himantura uarnak (Forsskål) kept at the Eilat Underwater Observatory in Israel. Dendromonocotyle colorni is distinguished from the other eight species in the genus by the morphology of the terminal papillar sclerite on the haptor, the distal portion of the male copulatory organ and the morphology of the vagina. The development of the male copulatory organ is detailed for D. colorni and the adaptations of species of Dendromonocotyle to life on the dorsal skin surface of rays are discussed. Dendromonocotyle octodiscus Hargis, 1955 was identified from the dorsal skin surface of the southern stingray Dasyatis americana Hildebrand et Schroeder off Bimini, Bahamas and represents a new host record.

1. The phenomenon of asexual multiplication is rare in the animal kingdom. It occurs, however, in all main flatworm taxa. Flatworms are characterized by an extensive versatility, ranging from the different types of asexual multiplication to the different orthogonal plans for the nervous system. The role of the nervous system in the asexual multiplication taking place in flatworms is pointed out and discussed. 2. Immunocytochemical studies of the changes in the flatworm neuroanatomy show that the nervous system, particularly the main never cords, has a central role during asexual development. 3. Antibodies to different neuronal substances yield different immunoreactivity patterns and develop according to different time schedules. Serotoninergic nervous elements seem to have a leading role. 4. Substances produced by the nervous system influence fissioning and subsequent regeneration in free-living flatworms in the following ways. (a) A function as a wound hormone has been suggested for the neuropeptide RF-amide. (b) Mitogenic effects have been shown for several biogenic amines and neuropeptides. (c) Inhibitory roles are suggested for somatostatin and melatonin in connection with cell proliferation respective fissioning. 5. Growth factors have been observed both in free-living and parasitic flatworms. 6. Cells reactive to antibodies against epidermal growth factor increase in number in parallel with increases in mitotic activity in the gull tapeworm and occur in regions with high mitotic activity. A correlation between these two phenomena is suggested.

We analyzed the effects of infection with Trichobilharzia ocellata on hemocytes of its snail host, Lymnaea stagnalis, and correlated them with successive stages of parasite development. Circulating hemocytes were studied at 0, 2, 4, 6, and 8 weeks post exposure (p.e.) with respect to cell number, distribution of subpopulations (as characterized by morphology, determinants recognized by either of two lectins and a monoclonal antibody) and to proliferative, phagocytic and endogenous peroxidase activity. Infection results in a net elevated level of activity of circulating hemocytes at 2 weeks p.e., when mother sporocysts are present in the head-foot-mantle region, as well as at 4 weeks p.e., when daughter sporocysts are migrating to and growing in the digestive gland region. A lower level of activity was observed at 6 weeks p.e., when cercariae are differentiating within daughter sporocysts. A net activation was again found at 8 weeks p.e., when cercariae are escaping. So, infection with T. ocellata results in a net general activation of the internal defense system of L. stagnalis, during several stages of development of the parasite.