TREMATODE SPECIES DETECTION AND QUANTIFICATION BY ENVIRONMENTAL DNA-qPCR ASSAY IN LAKE CHANY, RUSSIA.

Environmental DNA (eDNA) surveys promise to be a sensitive and powerful tool for the detection of trematodes. This can contribute to the limited studies on trematode ecology, specifically in aquatic ecosystems. Here, we developed species-specific primer and probe sets for Moliniella anceps, Opisthioglyphe ranae, and Plagiorchis multiglandularis cercariae and applied a novel eDNA qPCR assay to detect larval trematodes quantitatively. We evaluated the effectiveness of the assays using filtered lake water samples collected from different sites of Lake Fadikha and Kargat River Estuary in Lake Chany, Russia, showing high species specificity and sensitivity in all 3 assays. Further, all 3 assays had high efficiencies ranging from 94.9 to 105.8%. Moliniella anceps, O. ranae, and P. multiglandularis were detected in the environmental water samples through real-time PCR. Thus, we anticipate that our approach will be beneficial for biomonitoring, measuring, and managing ecological systems.

Infection with Clonorchis sinensis (Cobbold, 1875) Metacercariae in Fish from the East Lake of Wuhan: Freshwater Fish in Urban Lakes May Act as Infection Sources of Liver Fluke.

The liver fluke disease caused by Clonorchis sinensis is one of the most serious food-borne parasitic diseases in China. Many freshwater fish and shrimps can be infected with C. sinensis metacercariae as the second intermediate hosts in endemic regions. Owing to the lack of infected humans and the good administration of pet dogs and cats in cities of non-endemic regions, few fish are expected to be infected with C. sinensis metacercariae in urban lakes. To determine the infection of C. sinensis metacercariae in freshwater fish and shrimps in urban lakes, a total of 18 fish species and one shrimp species were investigated in the East Lake of Wuhan City. Metacercariae were isolated by artificial digestive juice and identified using morphology and rDNA-ITS2 sequences. Five species of fish, Pseudorasbora parva, Ctenogobius giurinus, Squalidus argentatus, Hemiculter leuciclus, and Rhodeus spp., were infected with C. sinensis metacercariae. The overall prevalence of C. sinensis was 32.5%. The highest prevalence was found in P. parva with 57.9%, while S. argentatus exhibited the highest mean abundance (13.9). Apart from the C. sinensis metacercariae, four species of other trematode metacercariae were also identified across twelve fish species in total. Owing to the consumption of undercooked fish and feeding cats with small fish caught by anglers, there is a potential risk that the small fish infected with C. sinensis metacercariae may act as an infection source to spread liver fluke. Given the complete life cycle of C. sinensis, stray cats and rats were inferred to act as the important final hosts of C. sinensis in urban lakes in non-endemic areas.

Immunology and pathology of echinostomes and other intestinal trematodes.

Intestinal trematodes constitute a major group of helminths that parasitize humans and animals with relevant morbidity and mortality. Despite the importance of the intestinal trematodes in medical and veterinary sciences, immunology and pathology of these helminth infections have been neglected for years. Apart from the work focused on the members of the family Echnistomatidae, there are only very isolated and sporadic studies on the representatives of other families of digeneans, which makes a compilation of all these studies necessary. In the present review, the most salient literature on the immunology and pathology of intestinal trematodes in their definitive hosts in examined. Emphasis will be placed on members of the echinostomatidae family, since it is the group in which the most work has been carried out. However, we also review the information on selected species of the families Brachylaimidae, Diplostomidae, Gymnophallidae, and Heterophyidae. For most of these families, coverage is considered under the following headings: (i) Background; (ii) Pathology of the infection; (iii) Immunology of the infection; and (iv) Human infections.

Cumulative assessment of Diplectanum spp. occurrence, prevalence, and pathological impact in Dicentrarchus labrax from varied Egyptian fish farms.

Monogenean trematodes, particularly those belonging to the Diplectanidae family, are significant metazoan parasites with substantial implications for aquaculture expansion. This study, investigatied the occurrence, prevalence, and pathological impact of Diplectanum spp. in European seabass (Dicentrarchus labrax) across three distinct Egyptian fish farms. During 2021-2022, we sampled 1800 European seabass (Dicentrarchus labrax) from three Egyptian fish farms (600 fish per farm). Farms 1 and 2 used semi-intensive earthen pond systems, while Farm 3 utilized an intensive floating cage system. Employing Clinical, post-mortem, parasitological, and molecular examination technique. Pathological lesions were identified, including skin and gill discoloration, emaciation, and internal organ abnormalities. Seasonal prevalence exhibited significant variations between farms, with highest rates observed in spring and Farm 3 reached an overall peak prevalence of 84.67 %. Parasitological examination distinguished two Diplectanum species morphologically, while molecular techniques exhibited limited specificity. Histopathology unveiled damage to gill, liver, spleen, kidney, and intestine, attributed to Diplectanum haptors including inflammation and internal bleeding, potentially leading to secondary infections. Molecular identification via PCR targeting ITS and 28SrDNA genes, revealing similar band sizes for the two Diplectanum species, indicating limited intraspecific genetic diversity. The study emphasizes investigating parasitic infections' prevalence and impact in aquaculture, necessitating robust molecular techniques for species differentiation. This study underscores the importance of investigating the prevalence and impact of parasitic infections in aquaculture. It highlights the need for robust molecular techniques to differentiate species. By focusing on Diplectanum spp. infections in D. labrax, the study offers valuable insights into managing parasites in aquaculture effectively.

Next-generation sequencing technologies for helminth diagnostics and surveillance in ruminants: shifting diagnostic barriers.

Helminth infections in grazing ruminants are a major issue for livestock farming globally, but are unavoidable in outdoor grazing systems and must be effectively managed to avoid deleterious effects to animal health, and productivity. Next-generation sequencing (NGS) technologies are transforming our understanding of the genetic basis of anthelmintic resistance (AR) and epidemiological studies of ruminant gastrointestinal parasites. They also have the potential to not only help develop and validate molecular diagnostic tests but to be directly used in routine diagnostics integrating species-specific identification and AR into a single test. Here, we review how these developments have opened the pathway for the development of multi-AR and multispecies identification in a single test, with widespread implications for sustainable livestock farming for the future.

Thermal tolerance and survival are modulated by a natural gradient of infection in differentially acclimated hosts.

Wild ectotherms are exposed to multiple stressors, including parasites, that can affect their responses to environmental change. Simultaneously, unprecedented warm temperatures are being recorded worldwide, increasing both the average and maximum temperatures experienced in nature. Understanding how ectotherms, such as fishes, will react to the combined stress of parasites and higher average temperatures can help predict the impact of extreme events such as heat waves on populations. The critical thermal method (CTM), which assesses upper (CTmax) and lower (CTmin) thermal tolerance, is often used in acclimated ectotherms to help predict their tolerance to various temperature scenarios. Despite the widespread use of the CTM across taxa, few studies have characterized the response of naturally infected fish to extreme temperature events or how acute thermal stress affects subsequent survival. We acclimated naturally infected pumpkinseed sunfish (Lepomis gibbosus) to four ecologically relevant temperatures (10, 15, 20 and 25°C) and one future warming scenario (30°C) for 3 weeks before measuring CTmax and CTmin. We also assessed individual survival the week following CTmax. Parasites were counted and identified following trials to relate infection intensity to thermal tolerance and survival. Interestingly, trematode parasites causing black spot disease were negatively related to CTmax, suggesting that heavily infected fish are less tolerant to acute warming. Moreover, fish infected with yellow grub parasites showed decreased survival in the days following CTmax implying that the infection load has negative survival consequences on sunfish during extreme warming events. Our findings indicate that, when combined, parasite infection and high prolonged average temperatures can affect fish thermal tolerance and survival, emphasizing the need to better understand the concomitant effects of stressors on health outcomes in wild populations. This is especially true given that some parasite species are expected to thrive in warming waters making host fish species especially at risk.

Large-scale spatial drivers of avian schistosomes in Northern Michigan inland lakes.

Avian schistosomes are snail-borne trematode parasites (Trichobilharzia spp.) that can cause a nasty skin rash in humans when their cercariae mistake us for their normal bird hosts. We sought to investigate drivers of the spatial distribution of Trichobilharzia cercaria abundance throughout Northern Michigan lakes. For 38 sites on 16 lakes, we assessed several dozen potential environmental predictors that we hypothesized might have direct or indirect effects on overall cercaria abundance, based on known relationships between abiotic and biotic factors in wetland ecosystems. We included variables quantifying local densities of intermediate hosts, temperature, periphyton growth rates, human land use and hydrology. We also measured daily abundance of schistosome cercariae in the water over a 5-week period, supported by community scientists who collected and preserved filtered water samples for qPCR. The strongest predictor of cercaria abundance was Lymnaea host snail density. Lymnaea density was higher in deeper lakes and at sites with more deciduous tree cover, consistent with their association with cool temperature habitats. Contrary to past studies of human schistosomes, we also found a significant negative relationship between cercaria abundance and submerged aquatic vegetation, possibly due to vegetation blocking cercaria movement from offshore snail beds. If future work shows that these effects are indeed causal, then these results suggest possible new approaches to managing swimmer's itch risk in northern MI lakes, such as modifying tree cover and shallow-water vegetation at local sites.

Integrative taxonomy of metazoan parasites of the bluntnose sixgill shark Hexanchus griseus (Bonnaterre, 1788) in the Mediterranean Sea, with the resurrection of Grillotia acanthoscolex Rees, 1944 (Cestoda: Trypanorhyncha).

Appropriate diagnoses of parasites of apex marine predators are crucial to understand their biodiversity, host specificity, biogeography, and life cycles. Such diagnoses are also informative of ecological and biological characteristics of both host and environment in which the hosts and their parasites live. We here (i) investigate the parasite fauna of a bluntnose sixgill shark Hexanchus griseus (Bonnaterre, 1788) obtained from the Gulf of Naples (Tyrrhenian Sea), (ii) characterize molecularly all its metazoan parasites, and (iii) resurrect and report the main morphological features and phylogenetic position of Grillotia acanthoscolex, a cestode species previously synonymized with Grillotia adenoplusia. A rich parasite fauna represented by eight different taxa was found, including two monogeneans (Protocotyle grisea and Protocotyle taschenbergi), one digenean (Otodistomum veliporum), four cestodes (Crossobothrium dohrnii, Clistobothrium sp., G. acanthoscolex, and G. adenoplusia), and one copepod (Protodactylina pamelae). Sequencing of these samples accounts for an important molecular baseline to widen the knowledge on the parasitic fauna of bluntnose sixgill sharks worldwide and to reconstruct their correct food chains. The bluntnose sixgill shark was found to be a definitive host for all endoparasites found here, confirming that it occupies an apex trophic level in the Mediterranean Sea. The taxa composition of the trophic parasite fauna confirms that the bluntnose sixgill shark mostly feeds on teleost fish species. However, the occurrence of two phillobothrid cestodes (C. dohrnii and Clistobothrium sp.) suggests that it also feeds on squids. Finally, we emphasize the importance of using integrative taxonomic approaches in the study of parasites from definitive and intermediate hosts to elucidate biology and ecology of taxa generally understudied in the Mediterranean Sea.

Idiopyrgus souleyetianus (Gastropoda: Tomichiidae) as an intermediate host of the liver fluke Amphimerus sp. (Trematoda: Opisthorchiidae) in Brazil.

Despite the importance of fish-borne trematodes of the family Opisthorchiidae as causative agents of human liver fluke disease, studies on these parasites outside Asia are relativally scarce. In South America, human focus of amphimerosis is known in Ecuador since the mid-20th century, and Amphimerus spp. have also been reported in wild and domestic mammals. Nevertheless, the knowledge on the snails that act as the first intermediate host of these potentially zoonotic parasites are scarce. Herein, a new cercaria of the pleurolophocercous morphotype found in the freshwater snail Idiopyrgus souleyetianus from Brazil was subjected to morphological and molecular studies. Multigene phylogenetic analyses based on 28S, 5.8S-ITS-2 and Cox-1 sequences enabled the identification of Amphimerus sp., a species distinct from that reported in humans from Ecuador. This cercariae was morphologically compared with other opisthorchiid cercariae known. The possible occurrence of human amphimerosis in Brazil is discussed.

Effectiveness of health education in improving knowledge, attitude and practice related to foodborne zoonotic trematodes in Vietnam, with a particular focus on Clonorchis sinensis.

OBJECTIVE: Foodborne zoonotic trematodes (FZT), which infect the liver, lungs and intestines of humans, are an emerging public health concern in tropical countries including Vietnam. In northern Vietnam, Clonorchis sinensis is recognised as the most important species of the FZT. Up to now, small-scale studies conducted in the country have indicated that health education could improve participants' knowledge and practices related to clonorchiasis, however strong evidence is still lacking. We conducted an intervention trial between 2020 and 2021 in four communes in Yen Bai and Thanh Hoa province, aiming to evaluate the impact of an extended educational package on knowledge, attitude and practices related to clonorchiasis, as well as on knowledge on the large liver fluke and minute intestinal flukes. METHODS: To this end, baseline and post-intervention questionnaires were conducted. Generalised estimating equations models were run to analyse the impact of the intervention on knowledge, attitudes and practices over time in the intervention versus control group. Finally, 172 people in the intervention group and 162 in the control group were included for analysis. RESULTS: Results demonstrated that the intervention significantly improved clonorchiasis knowledge, attitudes and practices, with higher odds for a correct knowledge response (odds ratio (OR) = 2.80, 95% confidence interval (CI) = 1.84-4.27, p-value < 0.001), increased average mean attitude score (mean = 0.363, 95%CI = 0.182-0.544, p-value < 0.001), and a reduced odds of consuming raw fish (OR = 0.15, 95%CI = 0.06-0.40, p = 0.002) in the intervention group compared to the control group post-intervention versus baseline. Additionally, participants in the intervention group exhibited enhanced basic knowledge of the large liver fluke and minute intestinal flukes post-intervention. CONCLUSIONS: These findings demonstrate the potential of health education programs in preventing clonorchiasis in endemic areas, emphasising the importance of continued health education as a critical component of integrated control programs for clonorchiasis.

Phylogenetic relationship of Prohemistomum vivax to other trematodes based on the internal transcribed spacer region and mitochondrial genes.

Prohemistomum vivax is a zoonotic small cyathocotylid trematode that inhabits the intestines of fish-eating birds and mammals. Here, we amplified the internal transcribed spacer (ITS) sequence and six mitochondrial protein-coding genes (PCGs) from P. vivax. The ITS region was 1389 base pairs long and had a partial 18S ribosomal RNA gene, a full ITS1, 5.8S rRNA, and ITS2 sequence, and a partial 28S rRNA gene. The ITS region of P. vivax showed a minimum pairwise distance (0.3-0.6%) from the ITS sequences of Cyathocotylidae sp. 1 and 2 metacercariae from Clarias gariepinus. This result suggests that these metacercariae belong to P. vivax metacercariae. We first amplified mitochondrial genes from P. vivax, including cytochrome c oxidase subunit III (cox3) partial sequence; tRNA-His, cytochrome b (cytb), and NADH dehydrogenase subunit 4L (nad4L) complete sequences; and NADH dehydrogenase subunit 4 (nad4), cytochrome c oxidase I (cox1), and NADH dehydrogenase subunit 5 (nad5) partial sequences. P. vivax was most closely related to Cyathocotyle prussica (NC_039780) and Holostephanus sp. (OP082179), with cox1, cox3, and cytb genes conserved among the three trematodes. The ML phylogenetic tree of ITS sequences supports the order Diplostomida, divided into two main clades (the superfamily Diplostomoidea and Schistosomatoidea). The phylogeny of concatenated amino acid sequences of P. vivax six PCGs revealed that diplostomoids and Clinostomum sp. evolved in a clade with Plagiorchiida members, away from Schistosoma species. These results may yield ribosomal and mitochondrial genetic markers for molecular epidemiological investigations of cyathocotylid intestinal flukes.

Detection of echinostomatid trematode eggs at the forest-oil palm interface in Sabah, Malaysia.

In this study, we report the occurrence of echinostomatid eggs in feces of wildlife, domestic animals and humans frequenting the forest­oil palm plantation interface in the Kinabatangan (Sabah, Malaysia), and discuss potential implications for public health. Using microscopy, we detected echinostomatid eggs in six host species, including Asian palm civets (Paradoxurus hermaphroditus [13/18]), leopard cats (Prionailurus bengalensis [3/4]), long-tailed macaques (Macaca fascicularis [1/10]), domestic dogs [3/5] and cats [1/1], and humans [7/9]. Molecular analysis revealed a close genetic proximity of civet echinostomatids to Artyfechinostomum malayanum, a zoonotic parasite of public health relevance. The intermediate hosts for A. malayanum have been reported in at least 3 districts in Sabah, suggesting that all the necessary elements required for the completion of the parasite's life cycle are present. Our findings point at the presence of zoonotic trematodes in an area with high human­wildlife interaction and highlight the potential public and animal health concern of zoonotic trematode infection in the context of Southeast Asia's rapidly changing ecosystems.

Disturbance and disease: host-parasite interactions in freshwater streams remain stable following wildfire.

Increases in the intensity and frequency of wildfires highlight the need to understand how fire disturbance affects ecological interactions. Though the effects of wildfire on free-living aquatic communities are relatively well-studied, how host-parasite interactions respond to fire disturbance is largely unexplored. Using a Before-After-Control-Impact design, we surveyed 10 stream sites (5 burned and 5 unburned) in the Willamette River Basin, Oregon and quantified snail host infection status and trematode parasite community structure 1 year before and two years after historic wildfires. Despite the severity of the wildfires, snail host populations did not show significant shifts in density or size distributions. We detected nine taxa of trematode parasites and overall probability of infection remained consistent over the three-year study period. However, at the taxon-specific level, we found evidence that infection probability by one trematode decreased and another increased after fire. In a larger dataset focusing on the first year after fire (9 burned, 8 unburned sites), we found evidence for subtle differences in trematode community structure, including higher Shannon diversity and evenness at the burned sites. Taken together, host-parasite interactions were remarkably stable for most taxa; for trematodes that did show responses, changes in abundance or behavior of definitive hosts may underlie observed patterns. These results have implications for using parasites as bioindicators of environmental change and suggest that aquatic snail-trematode interactions may be relatively resistant to wildfire disturbance in some ecosystems.

Trace elements assessment in Cerastoderma glaucum from port areas in the Tunisian Mediterranean coast: The influence of parasites on bioaccumulation.

In the present study, the seasonal concentration of seven trace elements was investigated in sediment and the cockle Cerastoderma glaucum tissues from two port areas in the North-East and South of Tunisia comparing cockles non-parasitized (NP) and parasitized (P) with digenean parasites. Elements concentration in sediments analyzed in both sites revealed that Zinc (Zn), Chromium (Cr), and Lead (Pb) were the most abundant ones, while Cadmium (Cd) and Mercury (Hg) were less abundant. The bioaccumulation of trace metals and Arsenic (As) in the tissues of cockles seems to be modulated by both the infection state and the parasite species. The relationship between bioaccumulation of metals and As, trematode species and abiotic parameters showed that the availability of certain metals for uptake by P cockles of both sites was influenced by the salinity and temperature of the water. Our results corroborate the possibility of using digenean infecting bivalves in biomonitoring aquatic ecosystems.

Prevalence of Foodborne Trematodes in Small Indigenous Fish Species in Local Markets of Phnom Penh, Cambodia.

Background: This study aimed to determine the prevalence and mean infection intensity of zoonotic foodborne trematodes (FBT) in small indigenous species (SIS) fish hosts. Materials and Methods: A total of 8630 specimens of unknown origin were collected from the markets in Phnom Penh City. Fish were identified, weighed, and separated into 20 g subsamples for each fish species, and were examined using the artificial digestion method. A total of 10 species of FBT were detected in 11 species of fish out of the 23 species of fish in the total sample. Results: All infected FBT species were intestinal flukes. No liver flukes were found. The overall prevalence of FBT infection was 29.3% ± 5.3%, and the mean intensity was 0.85 ± 1.89 metacercariae (Mc)/g. The highest FBT prevalence was observed for Isthmiophora hortensis (10.7%) and Centrocestus formosanus (7.9%). Other species identified were Diplostomum spp., Echinochasmus japonicus, Echinostoma revolutum, Echinostoma sudanense, Haplorchis taichui, Haplorchis pumilio Heterophyes spp., and Procerovum varium. The highest mean intensity was found for Diplostomum spp. (1.75 ± 3.03 Mc/g). Trichopsis vittata was one of the main species constituting the SIS in urban markets (31.72%) and showed the highest prevalence of FBT (75.0%). In addition, T. vittata had the highest diversity of intestinal flukes compared with other fish species. Amblypharyngodon chulabhornae showed the highest mean intensity. but low diversity (only two species) and low prevalence. Conclusions: T. vittata could be considered a potential indicator for targeted monitoring of FBT intestinal flukes, but A. chulabhornae could also be considered for quantitative studies considering its high mean intensity.

Trematode parasites as indicators of an animal component in the diet of the Water Vole Arvicola amphibius.

The intestinal trematode fauna of the Water Vole Arvicola amphibius, (previously A. terrestris), was investigated to determine whether it might provide evidence of an animal component in the diet of this aquatic herbivorous small mammal. Interrogation of the electronic Host-Parasite Database of the Natural History Museum London revealed the presence of fourteen species of intestinal trematode in water voles, infection with each of which would require the ingestion of tissue from an animal intermediate host. The results obtained using these parasite indicators provide convincing evidence of animal components in the diet of A. amphibius and support anecdotal reports of water voles feeding on animal material in the field.

Glycan Complexity and Heterogeneity of Glycoproteins in Somatic Extracts and Secretome of the Infective Stage of the Helminth Fasciola hepatica.

Fasciola hepatica is a global helminth parasite of humans and their livestock. The invasive stage of the parasite, the newly excysted juvenile (NEJs), relies on glycosylated excreted-secreted (ES) products and surface/somatic molecules to interact with host cells and tissues and to evade the host's immune responses, such as disarming complement and shedding bound antibody. While -omics technologies have generated extensive databases of NEJs' proteins and their expression, detailed knowledge of the glycosylation of proteins is still lacking. Here, we employed glycan, glycopeptide, and proteomic analyses to determine the glycan profile of proteins within the NEJs' somatic (Som) and ES extracts. These analyses characterized 123 NEJ glycoproteins, 71 of which are secreted proteins, and allowed us to map 356 glycopeptides and their associated 1690 N-glycan and 37 O-glycan forms to their respective proteins. We discovered abundant micro-heterogeneity in the glycosylation of individual glycosites and between different sites of multi-glycosylated proteins. The global heterogeneity across NEJs' glycoproteome was refined to 53 N-glycan and 16 O-glycan structures, ranging from highly truncated paucimannosidic structures to complex glycans carrying multiple phosphorylcholine (PC) residues, and included various unassigned structures due to unique linkages, particularly in pentosylated O-glycans. Such exclusive glycans decorate some well-known secreted molecules involved in host invasion, including cathepsin B and L peptidases, and a variety of membrane-bound glycoproteins, suggesting that they participate in host interactions. Our findings show that F. hepatica NEJs generate exceptional protein variability via glycosylation, suggesting that their molecular portfolio that communicates with the host is far more complex than previously anticipated by transcriptomic and proteomic analyses. This study opens many avenues to understand the glycan biology of F. hepatica throughout its life-stages, as well as other helminth parasites, and allows us to probe the glycosylation of individual NEJs proteins in the search for innovative diagnostics and vaccines against fascioliasis.

A Molecular Phylogenetic Study of the Eye Fluke Philophthalmus lacrymosus (Trematoda: Philophthalmidae) Found in Larus dominicanus (Aves: Laridae) from Brazil.

PURPOSE: Species of the genus Philophthalmus are eye flukes with a complex taxonomy, which began to be improved with the help of molecular data only recently. However, most described species have never been placed into a phylogenetic context. In this study, eye flukes previously found on kelp gulls, Larus dominicanus, from Brazil and identified as Philophthalmus lacrymosus were subjected to molecular analysis. METHODS: For the molecular analyses, we analyzed parasites found in six infected gulls (one worm per bird) collected from different municipalities of the state of Santa Catarina, Brazil. We carried out the amplification and sequencing of the partial region of the 28S and cox1 genes and the data obtained were compared with sequences available to philophthalmid species and subjected to phylogenetic analysis. RESULTS: The isolates of P. lacrymosus from Brazil grouped in well-supported clades with five other species of Philophthalmus with sequences available for comparison. Interspecific divergences of 0.1-1.6% in 28S and 8.2-14.9% in cox1 were found in relation to other isolates of Philophthalmus spp. Two cox1 haplotypes differing in one nucleotide (0.1%) were found between the six eye flukes isolates in gulls from different localities. The Brazilian isolates grouped in a subclade with parasites identified as P. lacrymosus in Portugal; however, the molecular divergences found in cox1 (8.2-8.5%) strongly suggest that these isolates belong to different species. The phylogenetic trees obtained and the intergeneric divergences to species of the genera Cloacitrema and Parorchis did not support the validity of the genus Natterophthalmus, for which P. lacrymosus was proposed as the type species in the past. CONCLUSION: As P. lacrymosus was described from Brazil, we recommend that this name be applied to the South American isolates and that the Portuguese isolates be provisionally considered as Philophthalmus sp., a probable cryptic species. Moreover, data obtained supports the previous morphology-based synonymizing between Natterophthalmus and Philophthalmus. Considering our results and most of previous reports of P. lacrymosus in South America, we suggest this species presents a marine life cycle.

Infection with Trematodes in Littorina obtusata Snails (Gastropoda: Littorinidae) with Different Shell Color Genotypes.

The prevalence of infection with trematode parthenitae was studied in Littorina obtusata littoral periwinkles with different shell color genotypes. Activities of genes responsible for a purple or orange single-pigment background shell coloration was not found to affect the prevalence of trematode infection in periwinkles. In L. obtusata with a yellow-purple background shell color and a pattern of white pigment spots on the shell, the prevalence of infection with Microphallus piriformes and M. pygmaeus was lower than the theoretical expectation. The prevalence of infection in periwinkles with purple, orange, or white stripes on the shell did not differ from that of unstripped periwinkles. The differences found were presumably associated with genetically determined susceptibility to infection. Possible consequences of differential infection with trematodes in periwinkles with different shell color genotypes are discussed in terms of the stability of parasitic systems and possible changes in the phenotypic structure of the host population.

Finding of an Unusually Colored Sporocyst of the Genus Leucochloridium in a Succinea putris Snail.

A Leucochloridium sp. Carus, 1835 sporocyst with a mature colored broodsac was found in a Succinea putris L., 1758 snail in the Boksitogorsk district of Leningrad Region (Russia). The pigmentation of the sporocyst's broodsac was different from those of other Leucochloridium Carus, 1835 species previously described for Europe. The obtained sporocyst is most similar by the coloration of its broodsac to the trematodes of the same genus from Japan. The genotyping of the investigated sporocyst by the rDNA gene fragments (complete ITS1, ITS2, 5.8S and partial 18S and 28S sequences) was conducted. Genetic distances between the obtained sporocyst and the previously described species of the genus Leucochloridium were higher than intraspecific ones in the most cases. These data and the data of morphological analysis imply that the investigated sporocyst belongs to a separate species of the Leucochloridium genus, previously not described in the European region.