Phenoloxidase is involved in the immune reaction of Helix lucorum to parasitic infestation by dicrocoeliid trematode.

INTRODUCTION: Phenoloxidases are known to play a role in the immune defences of arthropods and molluscs. In the invertebrates, phenoloxidases mediate three major physiologically important processes: sclerotization, wound healing, and defence reactions. Helix lucorum serve as the first intermediate host for the larval stages of dicrocoeliid trematodes which infects animals as well as human beings. OBJECTIVE: The aim of the study is to investigate the effect of larval forms of dicrocoeliid trematodes to phenoloxidase acitivity in H. lucorum, Linneaus, 1758, in Bitlis, Turkey. The effect of the snail's shell colour to phenoloxidase activity was also investigated. MATERIAL AND METHODS: Land snails (n=200) were collected by hand from their natural habitats during the period May - June 2019 in Bitlis, Turkey. Evaluation of the process was performed by measuring immune reaction of the snails against larval forms of dicrocoeliid trematodes. Phenoloxidase activity assay was carried out using a spectrophotometer device based on 3,4-Dihydroxy-L-phenylalanine (L-dopa) hydrolysis. RESULTS: The natural infection rate of the land snails with the developmental stages of dicrocoeliid trematodes was 20%. Phenoloxidase activity was found to be significantly higher (*p<0.05) in larval forms of dicrocoeliid trematodes infected snails when compared with non-infected snails. No effect of shell colours to phenoloxidase activity was observed. CONCLUSIONS: To the best of the authors' knowledge, this study is the first to report that the phenoloxidase system is involved in the immune reaction of Helix lucorum to parasitic infestation by larval forms of dicrocoeliid trematodes.

Importance of Land Snails in Dicrocoeliosis Epidemiology.

Dicrocoeliosis is a helminthosis caused by the small liver fluke Dicrocoelium spp. (Trematoda, Dicrocoeliidae) parasitizing in the bile ducts and gall bladder of ruminants as well as many other animal species including humans. In the biological life cycle of Dicrocoelium, land snails are first intermediate hosts and ants are second intermediate hosts. Sporocysts and cercaria, which are larval stages, live in the hepatopancreas of land snails and metacercaria, which is also the larval stage, lives in the abdomen and brain of ants. Land snails, which are the first intermediate host of this parasite in Turkey, include Helicopsis derbentina, Helicopsis protea, Helicopsis krynickii, Cernuella virgata, Trochoidea pyramidata, Cochicella acuta, Monacha carthusiana, Helicella candicans, Helix aspersa, Helix lucorum, and Chondrus tournefortianus. Dicrocoeliosis is widespread in ruminants and affects their liver, which can lead to weight loss and reduced milk production. The number of reports on dicrocoeliosis is increasing due to the expansion of dry habitats and parasites becoming resistant to antihelminthic drugs. This study provides information on the epidemiology and control methods of Dicrocoelium.

Biological control of snail hosts transmitting schistosomiasis by the water bug, Sphaerodema urinator.

The water bug, Sphaerodema urinator (Hemiptera : Belostomatidae), shares the same habitat of the freshwater snails in ponds, lakes, and streams. Studies conducted in lakes show that fish and crayfish predators play an important role in determining the abundance of freshwater snails. In contrast, shallow ponds and marches often lack fish and crayfish but have abundant insect predators. This study has been carried out to evaluate the predatory potential of S. urinator adult on two freshwater snails that serves as intermediate hosts of Schistosoma. Laboratory evaluation of predation by S. urinator on these intermediate hosts revealed that the adult bug could kill and consume the two intermediate hosts: Bulinus truncatus and Biomphalaria alexandrina. The number of snails consumed differed according to the snail type, size, and density. The times taken for searching and handling times were depending on the snail size, type, and vulnerability of the predator. The predation rate varied also with respect to snail type and density. Prey size is a major factor influencing predator preferences. This study indicated that the predator, S. urinator, may be a suitable bio-control agent in connection with Schistosoma intermediate hosts in the aquatic area.

Prevalence of Larval-Stage Dicrocoeliidae (Digenea) Trematodes in Helix lucorum (Mollusca: Pulmonata) in Van Province.

OBJECTIVE: The aim of this study was to investigate the prevalence of larval-stage Dicrocoeliidae trematodes in Helix lucorum, a land snail found in Van Province. METHODS: Helix lucorum snails were collected in April, May, and June 2017 from Edremit and Gevas, the central districts of Van Province, especially from natural areas where ruminants predominate. The snails were anesthetized with magnesium chloride, were removed from their shells, and their digestive glands were disrupted. The disrupted parts were examined under a microscope. RESULTS: In Van Province, H. lucorum snails were found to be intermediate hosts for Dicrocoelium trematodes with a prevalence of 22%. The larval stages detected in the microscope are photographed and shown in detail. The number of infection with larval stages of the parasite was found to be highest in May. CONCLUSION: Helix lucorum the land snail, serves as an intermediate host for some developmental stages of the Dicrocoeliid trematodes, is also consumed as nutrients by humans in some countries. Based on the obtained results in this study, it can be concluded that this snail would have important effects on animal health in the Van region which has a hard climate and a border with Iran.

Evaluation of different methods for the experimental infection of the land snail Helix aspersa with Aelurostrongylus abstrusus lungworm.

The laboratory maintenance of parasitic life cycles is crucial to support research in many fields of parasitology. The land snail Helix aspersa (syn. Cornu aspersum), an intermediate host of feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior, is adopted to produce infective stages of those nematodes in laboratory condition. The aim of this study was to compare the most common methods of experimental infection of H. aspersa with first stage larvae (L1) of A. abstrusus (i.e., contact of the snail foot with the L1) with the injection of these larvae in the foot of the snail, instrumental to reduce the infection time and to maximize the output of third-stage larvae (L3). Three groups (i.e., A, B, C) of 15 H. aspersa snails were infected with L1 of A. abstrusus (n=250 for each snail), whereas a fourth group (group D) was not infected (control). Snails were individually placed for 48h on a microfilm containing L1 (group A), on a potato slice previously irrigated with a suspension of L1 (group B), or they were inoculated by injection of L1 in the posterior-ventral portion of the foot (group C). Eighteen days after the infection all snails were analyzed and tissues were digested to recover L3. No difference in mortality rate was recorded among snail groups and the mean number of retrieved L3 was significantly larger in group C (71.5±52.9) compared to group B (38.2±44.9; p=0.0161) and group A (19±23.3; p<0.0001). The injection of A. abstrusus L1 in the foot of H. aspersa proved to be a fast, easy to apply and effective method, resulting in the largest number of infective larvae retrieved.

Encystment/excystment response and serotypic variation in the gastropod parasite Tetrahymena rostrata (Ciliophora, Tetrahymenidae).

Tetrahymena rostrata, which is characterized by a particular encystment-excystment cycle involving autogamy, has been recently found infecting the kidney of edible Helix aspersa snails under farming conditions. In this work, the effects of several factors on its encystment/excystment behaviour and the occurrence of different serotypes were investigated. The encystment/excystment response under starvation conditions was seriously affected by temperature. While a peak of encystment at 48 h followed by a progressive spontaneous excystment was observed at 18 and 25 °C, the encystment response was practically inhibited at 5 °C and clearly slowed down at 10 °C. At 30 °C, most of surviving ciliates remained encysted throughout the experiment, with spontaneous excystment being detected only after switching the temperature to 18 °C. Soil components also affected the encystment/excystment behaviour at 18 °C, with spontaneous excystment occurring in the presence of a sterile-filtered soil extract or mineral water but being strongly minimized with a non-filtered soil extract. Resting cysts formed in the latter extract exhibited a 3­4 times thicker and ultrastructurally more complex wall than that formed in mineral water and retained the excystment ability for about 4 weeks. Incomplete desiccation did not affect significantly the encystment response, while the mucus and kidney extracts from snails as well as a ciliate extract strongly stimulated a rapid excystment. Finally, two different serotypes infecting H. aspersa in heliciculture farms of Galicia (NW Spain) were identified, but no differences were observed between the encystment/excystment responses exhibited by two isolates belonging to each serotype.

Praziquantel efficacy against Brachylaima sp. metacercariae (Trematoda: Brachylaimidae) parasitizing the edible landsnail Cornu aspersum and its HPLC-MS/MS residue determination.

Cornu aspersum is a terrestrial edible snail, often parasitized by Brachylaima (Trematoda) metacercariae. Ingestion of undercooked snails by humans allows metacercariae to develop to adult in the intestine causing brachylaimiasis (expected mortality rate 5-10%). The cosmopolitan character of the trematode and of its vector, enhanced in a future climate change scenario, and the absence of adequate sanitary controls of the snails in marketplaces clearly increase the risk of human brachylaimiasis. The treatment of farmed snails with praziquatel (PZQ) would be a tool to control this food-borne disease. The objectives of this study were: to report the prevalence of Brachylaima metacercariae parasitizing C. aspersum in marketplaces, to assess the efficacy and tolerance of PZQ in C. aspersum and to develop an HPLC-MS/MS analytical method to quantify PZQ residue in the edible snail (the acceptable daily intake of PZQ is 0.17 mg/kg bw in humans). Prevalence of parasitization of C. aspersum by Brachylaima metacercariae in public marketplaces reached the 80%. PZQ was orally administered to snails, mixed with the usual snail food. In dose determination assays three doses were individually tested (10 days): 1.2 mg PZQ/snail, 1.8 mg PZQ/snail (efficacy 97.3%, p < 0.001) and 2.4 mg PZQ/snail (efficacy 98.7%, p < 0.001). In dose confirmation tests (n = 200) the 2.4 mg PZQ/snail dose was group tested (10 snails/box, 7 days): 2 g of PZQ supplemented snail food were disposed daily in the treatment group boxes and 2 g of snail food (placebo) in the control boxes (efficacy 94.6%, p < 0.001; prevalence dropped from 68.7% in control group to 10.1% in treatment group, p < 0.001). Received dose was 220.2 mg PZQ/kg snail with shell. In the analytical method, linearity, lower limit of quantification (0.05 µg/ml), selectivity, carry over, accuracy, precision, dilution integrity, matrix effect and stability were tested. Sixty snails were treated (11 mg PZQ/g snail food) and analyzed. PZQ was detected and quantified (0.093 mg PZQ/g snail). PZQ treatment of C. aspersum is effective, well tolerated by the snail, affordable and easy to reproduce in snail farms.

[The prevalence of Dicrocoeliid (Trematoda: Digenea) larval stages in Helix lucorum Linneus 1758 (Mollusca: Pulmonata) in the vicinity of Kastamonu]. / Kastamonu civarinda dagilis gösteren Helix lucorum Linnaeus, 1758 (Mollusca: Pulmonata)'da Dicrocoeliid (Trematoda: Digenea) larval safhalarinin yayginligi.

OBJECTIVE: The aim of this study was to investigate the prevalence of Dicrocoeliid larval stages in Helix lucorum Linnaeus, 1758 (Mollusca: Pulmonata) living in the vicinity of Kastamonu. METHODS: Snails were collected in the vicinity of Tasköprü, Kastamonu in March, April, May 2013. They were dissected while alive and their hepatopankreas were removed and placed on clean glass slide with a drop of 0.6% NaCl solution. The hepatopankreas were incised with a mounted needle and a thin film of the hepatopankreas fluid was drown out on a slide for examination of live parasites. After examination, larval stages fixed in %70 alcohol, formaldehyde-ethyl alcohol, 10% formalin and bouin. RESULTS: H. lucorum which is a land snail species has been reported for the first time being an intermediate host in the life cycle of Dicrocoeliid (Trematoda: Digenea) species with the prevalence of 27.6% from our country. Morphological and histological features of larval stages were determined. CONCLUSION: H. lucorum is an intermediate host in the life cycle of Dicrocoeliid species. The prevalence of infection was highest in April. This land snail species is important for the health of animal and human.

Larval development of the feline lungworm Aelurostrongylus abstrusus in Helix aspersa.

The cat lungworm Aelurostrongylus abstrusus affects the domestic cat and other felids all over the world. Feline aelurostrongylosis is of importance in clinical feline medicine and is gaining more and more attention for the present expansion in the geographical range of A. abstrusus. Global warming and changes in phenology of snail intermediate hosts have been incriminated in this spreading. Helix aspersa, a potential vector of A. abstrusus, is endemic in most regions of the world, where it has become recently a pest outside its native Mediterranean range. The present work evaluated key features of larval development of A. abstrusus in H. aspersa at two different temperature conditions. The results showed that A. abstrusus may reach the infective stage in muscular foot and viscera of H. aspersa and that environmental temperatures may influence the biological cycle of the cat lungworm. In particular, the higher the average temperature, the higher the rate of larval development, i.e., about 50% of larvae of A. abstrusus reached the infective stage between temperatures of 18.8-29.5 °C, while only 17.8% of larvae completed their development at temperatures of 6.7-22 °C. Biological and epidemiological implications are discussed.

Parasitic infections in mixed system-based heliciculture farms: dynamics and key epidemiological factors.

Heliciculture is an excellent alternative to obtain edible snails but its viability is seriously threatened by pathogens. A parasitological survey was conducted in 3 mixed system-based heliciculture farms in Galicia (NW Spain), with the species Tetrahymena rostrata, Tetrahymena limacis, Tetratrichomonas limacis, Cryptobia helicogenae, Brachylaima aspersae (metacercariae and sporocysts), Alloionema appendiculatum, Nemhelix bakeri, and Riccardoella limacum being commonly found infecting Helix aspersa aspersa (petit-gris) snails. With the exception of C. helicogenae, N. bakeri, and B. aspersae sporocysts, all species were also detected in Helix aspersa maxima (gros-gris) snails, although generally with lower parameters. Most monoxenous infections, and consequently multiple parasitism, exhibited a rising trend during the first 2 months of intensive mating, with tendencies being slowed down or even reversed during the third month as a result of accumulated mortality and a sampling-derived reduction in host density. No parasites were vertically transmitted and infections were initially acquired from invading gastropod and micromammal reservoirs during fattening. Finally, artificial hibernation reduced significantly the prevalence of most species. These results confirm the importance of parasites in heliciculture and emphasize the need to prevent the entry of wild reservoirs into the farms and to rapidly remove the carcasses of dead snails from the reproduction units and fattening pens.

Intensity of parasitic mite infection decreases with hibernation duration of the host snail.

Temperature can be a limiting factor on parasite development. Riccardoella limacum, a haematophagous mite, lives in the mantle cavity of helicid land snails. The prevalence of infection by R. limacum in populations of the land snail Arianta arbustorum is highly variable (0-78%) in Switzerland. However, parasitic mites do not occur in host populations at altitudes of 1290 m or higher. It has been hypothesized that the host's hibernation period might be too long at high elevations for mites and their eggs to survive. To test this hypothesis, we experimentally infected snails and allowed them to hibernate at 4°C for periods of 4-7 months. Winter survival of host snails was negatively affected by R. limacum. The intensity of mite infection decreased with increasing hibernation duration. Another experiment with shorter recording intervals revealed that mites do not leave the host when it buries in the soil at the beginning of hibernation. The number of mites decreased after 24 days of hibernation, whereas the number of eggs attached to the lung tissue remained constant throughout hibernation. Thus, R. limacum survives the winter in the egg stage in the host. Low temperature at high altitudes may limit the occurrence of R. limacum.

Morphological and molecular characterization of renal ciliates infecting farmed snails in Spain.

Renal infections by parasitic ciliates were studied in adult snails of Helix aspersa aspersa and Helix aspersa maxima collected from 2 mixed rearing system-based heliciculture farms located in Galicia (NW Spain). The occurrence of ciliates was also examined in slugs (Deroceras reticulatum) invading the greenhouses where first growing and fattening of snails is carried out. Histological examinations revealed a severe destruction of the renal epithelium in heavily infected hosts. Three ciliate isolates, one from each host species, were obtained and grown in axenic cultures. Cultured and parasitic ciliates were characterized morphologically and morphometrically. In addition, the encystment behaviour, the occurrence of autogamy, and the sequences of the mitochondrial cytochrome-c oxidase subunit 1 (cox1) and the small subunit ribosomal RNA (SSU rRNA) genes were also studied in the 3 isolates. A polymorphic life cycle involving resting and reproductive cysts, together with the morphological and morphometrical characteristics and the confirmation that autogamy occurs within cysts, demonstrate that our ciliates belong to the species Tetrahymena rostrata (Kahl, 1926) Corliss, 1952. The 3 isolates formed a well-supported clade using both genetic markers, and were clearly separate from the strain ATCC(R) 30770, which has been identified as Tetrahymena rostrata. We argue that our Spanish isolates should be regarded as Tetrahymena rostrata, and that the ATCC isolate should be regarded as a misidentification as neither cytological nor cytogenetical support for its identity has been presented.

[Occurrence of the flagellated protozoan, Cryptobia helicis Leidy, 1846 (Kinetoplasta: Bodonea: Cryptobiidae) in the garden snail, Helix aspersa]. / Bahçe Salyangozu Helix aspersa'da Kamçili Protozoon Cryptobia helicis Leidy, 1846 (Kinetoplasta: Bodonea: Cryptobiidae)'in Bulunusu.

In this survey, the prevalence and cytological features of the flagellated protozoan, Cryptobia helicis living in the bursa copulatrix of the garden snail, Helix aspersa Müller 1774 found in the vicinity of Izmir, Turkey was investigated. The prevalence of Cryptobia helicis in garden snails collected in the spring of 2005 was found to be 68.65%. This study is the first record of the occurrence of Cryptobia helicis in the garden snail Helix aspersa found in Turkey.

Natural infection of Helix aspersa (Mollusca: Pulmonata) by Dicrocoeliidae (Digenea) larval stages in Izmir, Turkey.

In this study, the prevalence of larval stages of Dicrocoeliidae in the garden snail Helix aspersa Müller, 1774 commonly found in the vicinity of Izmir, Turkey was investigated and some of its histological and morphological features were determined. The molluscs were collected during the spring of 2005. As the result of the investigation, a land snail species, H. aspersa, was found to be an intermediate host in the life cycle of Dicrocoeliidae and it had a prevalence of 0.97 % in the study area. In the present study, H. aspersa has been reported for the first time as an intermediate host of Dicrocoeliid species. The prevalence of infection was highest in March.

Increased response to cadmium and Bacillus thuringiensis maize toxicity in the snail Helix aspersa infected by the nematode Phasmarhabditis hermaphrodita.

To determine the effect of nematode infection on the response of snails to selected toxins, we infected Helix aspersa with 0-, 0.25-, 1-, or 4-fold the recommended field dose of a commercial nematode application for agricultural use. In the first experiment, the snails also were exposed to cadmium via food and soil at concentrations of 0, 30, 60, 120, or 240 mg/kg in a full-factorial design. In the second experiment, snails were infected with nematodes and also fed either Bt (expressing Bacillus thuringiensis toxin) maize or non-Bt maize. The snails were weighed at the beginning and end (after four weeks) of the experiments, and mortality was checked daily. Neither exposure of snails to nematodes nor exposure of snails to cadmium or Bt toxin affected the survival rates of snails. The number of dead snails was highest for combinations of nematode treatments with cadmium concentrations of 120 and 240 mg/kg. In both experiments (Bt and cadmium), the growth rate decreased with increasing nematode dose. The Bt maize was not harmful to the snails in the absence of nematodes, but infected snails grew faster when fed non-Bt maize. The growth rate of snails exposed to cadmium decreased with exposure to increasing Cd concentrations and differed significantly between the no-nematode treatment and the treatments with nematode doses of one- and fourfold the recommended field dose. Snails treated with the highest dose of nematodes accumulated the highest cadmium concentrations.

Hemozoin formation in Echinostoma trivolvis rediae.

Rediae of the trematode Echinostoma trivolvis, from naturally infected Helisoma trivolvis snails, form a black pigment while inside the snail host. Here we examine the black pigment to show that the insolubility characteristics in detergent and weak base solution are identical to Plasmodium falciparum hemozoin. Laser desorption mass spectrometry of the purified pigment demonstrates the presence of heme. Examination of purified pigment under polarized light microscopy illuminates ordered birefringent crystals. Field emission in lens scanning electron microscopy reveals irregular ovoid crystals of 200-300 nm in diameter. The purified pigment crystals seeded extension of monomeric heme onto the crystal which by Fourier Transform Infrared analysis is beta-hematin. Rediae of a second echinostome parasite, Echinostoma caproni, from experimentally infected Biomphalaria glabrata, do not produce measurable or recoverable heme crystals. These observations are consistent with heme crystal formation by a hematophagous parasite within a non-vertebrate intermediate host.

Larval stages of Brachylaima fuscatum in the terrestrial snail Limicolaria aurora from southern Nigeria.

Of 150 specimens of the gastropod snail Limicolaria aurora examined from the Edo and Delta states of Nigeria, 63.4% were infected with larval digeneans comprising mother sporocysts (12.1%) daughter sporocysts (20.4%) cercariae (43.1%) and metacercariae (24.5%). Attempts to experimentally infect three 14-day-old chicks (Gallus domesticus) and two laboratory-bred 4-month-old mice (Mus musculus) by oral feeding and peritoneal injection with cercariae were negative, although experimental infections of chicks via a cloacal drop yielded 62 immature and 37 mature worms from the intestinal caeca and ileum. The worms were identified as Brachylaima fuscatum (Trematoda: Brachylaimidae). The study also revealed that L. aurora acts as an intermediate host for B. fuscatum, in addition to Eulota sp., Helix sp., Helicella sp., Oxychilus sp. and Agrolimax sp.

Differential display analysis of hemocytes from schistosome-resistant and schistosome-susceptible intermediate hosts.

Hemocytes from schistosome-resistant and schistosome-susceptible Biomphalaria glabrata differ fundamentally in their behavior towards an invading parasite. When the schistosome infects a resistant snail host it is quickly surrounded by hemocytes, encapsulated and destroyed. Hemocytes from susceptible hosts fail to kill the parasite. To detect the differences between these two host phenotypes, we used differential-display reverse-transcription PCR (DDRT-PCR), based on RNA extracted from isolated hemocytes. A number of differentially expressed fragments from resistant and susceptible snails were detected by DDRT-PCR and confirmed using single-strand conformation polymorphism. These methods proved to be sensitive enough to allow comparison and verification of differential gene expression in our system, where only small numbers of cells are available. The most interesting phenotype-specific fragments detected so far show sequence homologies to an adhesion molecule, defensin, serine/ threonine kinases, peroxidases and glycosidases.

Study on the life cycle of a sexually transmitted nematode parasite of a terrestrial snail.

A technique for the artificial infection of the snail Helix aspersa by its parasite the nematode Nemhelix bakeri is described. The snail is relaxed by injection of an anesthetic, and 1 gravid female worm is introduced into the genitalia through the genital pore. Half of the injected snails were successfully infected. Following the course of infection over time indicated a 1:1 sex ratio, that the maximum number of progeny produced by injected female worms was 7, and that the development time of female worms was 56 days. The first generation of gravid females was found 100 days after infection. A survey of naturally parasitized snails was also conducted. The sex ratio of worms was in equilibrium, with a mean number of 2.4 larvae per female. The development time (56 days) and the body size (2.47-4.00 mm) of female N. bakeri are similar to those of a related species Cosmocercoides dukae (52-57 days and 1.66-4.34 mm), although the life cycle and biogeographic distribution for each of them are distinct.

A time-lapse study of interactions between Echinostoma paraensei intramolluscan larval stages and adherent hemocytes from Biomphalaria glabrata and Helix aspersa.

In vitro interactions between intramolluscan stages (sporocyst, daughter rediae, and metacercariae) of the trematode parasite Echinostoma paraensei and adherent hemocytes from the gastropods Biomphalaria glabrata (intermediate host) and Helix aspersa (non-host) were visualized by time-lapse videomicroscopy. Hemocytes of either species not exposed to E. paraensei displayed extensive mobility and activity of cellular extensions. Image analysis disclosed no significant change in the total surface area occupied by hemocytes in a selected field of view over 2 hr. Echinostoma paraensei exerted life stage-specific effects on the behavior of B. glabrata hemocytes; the cells moved away from sporocysts and daughter rediae but not encysted metacercariae. In the presence of sporocysts, hemocytes rounded up, whereas hemocytes adjacent to rediae assumed a stringy, beady appearance. Hemocytes close to the parasite were affected more rapidly than more distant cells. In 2 hr, a hemocyte-free "halo" formed around the parasite larvae, significantly reducing the hemocyte-occupied surface area (to 43% by sporocysts and to 70% by rediae). The changes induced by sporocysts and rediae are similar to those noted in both in vivo and in vitro studies of the B. glabrata-E. paraensei model system and are interpreted as manifestations of parasite-mediated interference with host hemocyte function. Helix aspersa (non-host) hemocytes were not affected, suggesting that E. paraensei-mediated effects on hemocytes exhibit a degree of specificity.