Molecular detection of Schistosoma species in unusual snail hosts: a note of caution.

The interaction between snails and species of Schistosoma results from an evolutionary process with an intrinsic host-parasite specificity to the snail genus. Faced with this fact, the recent molecular-based report on the potential infection of the thiarid Melanoides tuberculata with human schistosome should be cautiously interpreted. The high sensibility of molecular tools can result in false positives, perhaps by amplifying DNA from an external (contaminant) or invasive stage of schistosome found in this non-permissive snail host. Thus, parasitological data are mandatory to extrapolate the importance of the finding for the epidemiology and control of schistosomiasis.

The complete mitochondrial genome of Melanoides tuberculata (Müller, 1774) in Guangdong, China.

The Melanoides tuberculata is an invasive species, which is natively distributed in Africa and Southeast Asia. This study describes the first mitochondrial genome of the M. tuberculata based on the whole genome sequencing data. The complete sequence length of the mitogenome is 15,821 bp, including 37 genes (2 rRNA genes, 22 tRNA genes and 13 protein-coding genes). Phylogenetic analysis using the 13 species of Cerithioidea species showed that the M. tuberculata is closely related to P. dartevellei, forming the sister group to C. sinensis and C. obtuse.

Detection of schistosome infection in the invasive snail Melanoides tuberculata (Gastropoda: Thiaridae) by polymerase chain reaction from Plateau State, Nigeria: a novel turning point in disease epidemiology and control?

BACKGROUND: Melanoides tuberculata is a freshwater snail that serves as an intermediate host for 11 parasitic flukes. This study was conducted with the aim of identifying and screening the snail intermediate hosts in the study site for schistosomiasis using the molecular technique. METHODS: DNA was extracted from the snails by the hexadecyl trimethyl ammonium bromide method and the Dra1 primer was used to amplify the Dra1 repeated sequence of Schistosoma haematobium. RESULTS: The presence of schistosome DNA in M. tuberculata by polymerase chain reaction was confirmed. CONCLUSIONS: Our findings show that M. tuberculata is a potential intermediate host of schistosomes.

Philophthalmus gralli in domestic waterfowl: An environmental study in an urban area from Brazil.

Philophthalmosis is a zoonotic ocular disease that affects mainly domestic and wild avian species in different parts of the world. However, aspects related to its epizoology in domestic animals are not completely known. In the present study, 47 Muscovy duck (Cairina moschata), 8 mallards (Anas platyrhynchos platyrhynchos) and 6 African geese (Anser cygnoides), free-living in an urban lake in Belo Horizonte, Minas Gerais, Brazil, were evaluated for eye fluke infection. The animals were manually contained, and the conjunctival sac and nictitating membrane were evaluated by direct ophthalmic examination (DOE), in which the fluke burden was estimated by a semiquantitative scale. Moreover, the conjunctival sac lavage (CSL) technique was applied for the search of parasite eggs. When possible, the detected trematodes were carefully removed for fixed for taxonomic identification. In order to characterize the parasite-host-environmental system, malacological collections were performed at the locality, and the collected intermediate host mollusks (Melanoides tuberculata) were evaluated for infection with larval philophthalmids. In addition, wild vertebrates found dead in the locality, including one common gallinule (Gallinula galeata) and two capybaras (Hydrochoerus hydrochaeris), were necropsied for the search of eye flukes. Samples from the developmental stages obtained from the hosts were subjected to morphological and molecular studies (amplification and sequencing of a partial region of the Nad-1 gene). In the DOE, the presence of eye flukes was verified in 67% (4/6), 31% (15/47) and 25% (2/8) in geese, Muscovy ducks and mallards, respectively. In most cases, the birds showed mild parasitism (1-5 trematodes), with no apparent eye damage, characterizing a subclinical infection. In only one goose, which had a high fluke burden (20 trematodes), it was possible to obtain eggs by CSL. Regarding the malacological survey, 73/4545 (1.6%) specimens of M. tuberculata evaluated were found infected with megalurous cercariae, compatible with representatives of the family Philophthalmidae. Additionally, 147 flukes were recovered from a specimen of G. galeata found dead at the lake. The morphological and molecular study of parasites obtained from different hosts evaluated in this study enabled the identification of Philophthalmus gralli, a species native from Asia and introduced in the Americas. Factors such as the wide distribution of the intermediate host and the participation of domestic and wild reservoirs in the parasite transmission chain may favor the occurrence of parasite spillover from wild to domestic birds. Furthermore, the occurrence of the life cycle of P. gralli in an urban waterbody serves as an alert to the possibility of human cases of this zoonotic eye parasite.

Are Freshwater Snails, Melanoides sp. and Invasive Tarebia granifera (Gastropoda: Thiaridae) Suitable Intermediate Hosts for Calicophoron microbothrium (Trematoda: Paramphistomoidea)? An Experimental Study.

Prosobranch snails and adult Paramphistomoidea flukes were collected from water bodies and cattle abattoir located in Mpumalanga province of South Africa, respectively. The snails were identified based on morphological characters as well as the ITS-2 and 16S markers as Melanoides sp. and Tarebia granifera, respectively, and the Paramphistomoidea flukes were identified as Calicophoron microbothrium using the ITS-1/5.8S/ITS-2 marker. After confirming identification, the snails were bred to first filial generation (F1) under laboratory conditions. Ninety snails were randomly selected from the laboratory-bred F1 snails and 25 Melanoides sp. and 20 T. granifera were exposed to C. microbothrium miracidia, and the same numbers were maintained as non-exposed controls. Results showed that C. microbothrium successfully established in Melanoides sp. and produced cercariae, and the prepatent period recorded was 21 days. Three snails shed cercariae at day 21 postexposure (PE), and rediae and free cercariae were detected in the soft tissues of one snail on dissection at day 44 PE. The same fluke did not establish in T. granifera. Melanoides sp. started producing offspring at day 7 PE, and T. granifera at day 14 PE. In conclusion, our results showed that Melanoides sp. used in this study is a suitable intermediate host for C. microbothrium under experimental conditions, and given the wide distribution of this snail species, it is important to determine its role in the natural transmission of other Calicophoron species that have been reported in South Africa.

First report of Melanoides tuberculata (Mollusca: Thiaridae) harboring a xiphidiocercaria in Brazil: A new parasite introduced in the Americas?

Xiphidiocercariae were found in the invasive snail Melanoides tuberculata collected during a malacological survey in Ceará-Mirim, State of Rio Grande do Norte, Northeastern Brazil in November 2018 and submitted to morphological and molecular analyses. The morphology revealed similarities between the larvae here reported for the first time in M. tuberculata from Brazil and other xiphidiocercariae described in thiarid snails from Asia and Africa. Phylogenetic analyses based on 28S and ITS-2 sequences revealed that the larvae correspond to an unidentified species of the family Lecithodendriidae. Aspects related to the morphology and taxonomy of xiphidiocercariae found in M. tuberculata are briefly discussed. It is possible that the parasite here reported is a newly introduced species transmitted by M. tuberculata in the American continent.

Haplorchis pumilio (Trematoda: Heterophyidae) as a new fish-borne zoonotic agent transmitted by Melanoides tuberculata (Mollusca: Thiaridae) in Brazil: A morphological and molecular study.

The fishborne zoonotic trematode Haplorchis pumilio (Looss, 1896) is here reported for the first time in Brazil based on morphological, experimental and molecular studies. Pleurolophocercous cercariae emerged from the invasive snail Melanoides tuberculata collected in the municipality of Ceará-Mirim, state of Rio Grande do Norte, Northeast region of Brazil, in November 2018, were used for experimental infection of guppies, Poecilia reticulata. Metacercariae were extracted from the caudal musculature of these fishes and were used to infect mice. Adult parasites recovered in the small intestine of the mice were morphologically identified as H. pumilio. Molecular sequences were obtained for the Brazilian and Peruvian isolates of H. pumilio, and were compared with data available in GenBank. Analyses of fragments of the nuclear genes 28S (1219 bp) and ITS-2 (290 bp) revealed 98.48-100% similarity between the South American and Asian isolates of H. pumilio. Moreover, new sequences of the mitochondrial gene cox-1 obtained for the Brazilian (797 bp) and Peruvian (646 pb) isolates were 100% similar to a Mexican isolate of this species and 97.54% similar to an isolate from Thailand. This finding reveals the potential for occurrence of human haplorchiasis in Brazil, especially because of the increasing popularity of raw fish dishes in the country.

Biota sediment accumulation and bioconcentration factors of trace metals in the snail Melanoides tuberculata form the agricultural drains of the Manzala Lagoon, Egypt.

The freshwater mollusk, Melanoides tuberculata, contains bioconcentration trace metals in its tissues from the surrounding area. M. tuberculata was used as a bioindicator for pollution with seven trace metals (Mn, Fe, Ni, Cu, Zn, Cd, and Pb) accumulated in snail soft tissues near Bahr El-Baqar and El-Serw agricultural drains of industrial activities in Port Said and Damietta, respectively. The biota sediment accumulation factors (BSAF) in this study reveals that all the soft tissues of the M. tuberculata snails were macro-concentrators with the exception of Ni; it was deconcentrated, and thus provides that the selective tissues as good biomonitors. The bioconcentration factor (BCF) showed the highest concentrations of Fe and Cu in the soft tissues of the M. tuberculata snail, which has the potential to be used as a biomonitoring agent for Fe and Cu contamination of the water.

Non-marine invasive gastropods on Ilha Grande (Angra dos Reis, Rio de Janeiro, Brazil): distribution and implications for conservation / Moluscos continentais invasores na Ilha Grande (Angra dos Reis, Rio de Janeiro, Brasil): distribuição e implicação para a conservação

Abstract Invasive species are one of the main causes of biodiversity loss and the knowledge of their distribution is essential for conservation efforts. The present study inventoried the land and freshwater invasive molluscs of 25 villages of Ilha Grande (Rio de Janeiro, Brazil), an island with Atlantic Rainforest remnants. Three invasive species are found: Melanoides tuberculata (Müller, 1774), Physa acuta Draparnaud, 1805, both on two localities; and Achatina fulica Bowdich, 1822, on 16 localities. Most records were done on the island continental side, in anthropic areas, probably due to ease of access. One record was inside a conservation area, that calls concern about the spread of those species on the island and possible effects.

Resumo As espécies invasoras são uma das maiores causas de perda de biodiversidade e o conhecimento acerca da sua distribuição é essencial para os esforços de conservação. O presente estudo inventariou os moluscos continentais invasores em 25 vilas da Ilha Grande (Rio de Janeiro, Brasil), uma ilha com remanescentes de Mata Atlântica. Três espécies invasoras foram encontradas: Melanoides tuberculata (Müller, 1774), Physa acuta Draparnaud, 1805, ambas em duas localidades; e Achatina fulica Bowdich, 1822, em 16 localidades. A maior parte dos registros foi no lado continental da ilha, provavelmente devido a facilidade de acesso. Um dos registros foi dentro de uma área de conservação, o que chama a atenção para a dispersão destas espécies na ilha, e seus possíveis efeitos.

Mitochondrial Genetic Diversity of the Freshwater Snail Melanoides tuberculata.

According to geological history, Peninsular Malaysia and Borneo formed at different times and were once connected during Quaternary glaciations. To determine how this history has influenced phylogeography, our study examined the population genetic structure of the tropical freshwater gastropod Melanoides tuberculata across Peninsular Malaysia and Borneo using the sequences from mitochondrial DNA 16S rRNA and cytochrome oxidase subunit I genes (1168 bp). In total, 104 specimens were collected from seventeen populations. All mtDNA haplotypes were identified as belonging to two highly divergent lineages, and these lineages were almost allopatric in their distributions. Our study found that the freshwater fauna in Malaysia might be divided into four regions: northeast Peninsular Malaysia, northwest Peninsular Malaysia, south Peninsular Malaysia, and Borneo. The phylogeography of M. tuberculata in Malaysia was shaped by the landforms of Peninsular Malaysia and by the paleo-river systems in the Sunda continental shelf. In addition, our study found that these two lineages in Malaysia have invaded the globe. These results suggest that Malaysia is located in important shipping lanes throughout the world, and the populations of M. tuberculate might be widely distributed throughout the world by shipping.

Chronological analysis of the damages caused by the metacercariae of Centrocestus formosanus in the gills of Cyprinus carpio and lesions caused by the adult flukes in Ardeola ralloides: An experimental study.

Centrocestus formosanus is a digenean that requires three host species to complete its life cycle. This study was conducted to observe the damage caused by two life stages of the C. formosanus on its host species. The snail Melanoides tuberculata was induced to shed cercariae by exposing to sunlight and specimens of koi carps were experimentally infected with cercariae. Gills of two infected fish were killed and fixed in Bouin's solution daily for 21 days. Infected fish were continuously fed to a pond heron (Ardeola ralloides) for three weeks and therafter the bird was killed. Small intestine was resected as duodenum, jejunum, and ileum and fixed in formol saline. Gills and small intestine were prepared to study the histopathological damages. Flared opercula with protruding gills and increased respiratory rate were identified as the primary clinical signs of the fish. Encysted metacercariae were observed in the basal, middle and in the apical portion of the gills' filaments and gradual distortions and extensive proliferation of the cartilage of the gills resulted in loss of the respiratory epithelium. A progression of fibroblast to chondroblast encapsulation of the parasite was observed in the gill of fish as a host response. The duodenum of the heron was severely infected with adult parasites than jejunum and ileum. Flukes were observed in the villi, mucosae, submucosae, and also in the tunica muscularis of the duodenum. In conclusion, this study revealed that the heavy infection of C. formosanus could cause severe pathological lesions in both koi carps and pond heron.

Phenotypic plasticity of the gastropod Melanoides tuberculata in the Nile Delta: A pollution-induced stabilizing selection.

To understand the effect of metal pollution on the speciation process, we conducted comparative analyses of six populations of the gastropod Melanoides tuberculata, which dominated the Manzala lagoon (Nile Delta, Egypt). Geometric morphometric analysis was implemented to quantify the phenotypic plasticity of the species. The results from both Canonical Variate Analysis and Relative Warp indicated an overall decrease in the morphological breadth of M. tuberculata in the polluted sites. The favored phenotypes in the polluted sites have moderate whorl section, moderate ovate aperture, less-prominent radial ornament, and overall moderate-spired shells. Lack of morphological variations and dominance of intermediate phenotypes in the polluted sites indicate that stabilizing selection is driving the morphological pattern of this species. Moreover, analysis by using the partial least square model confirmed that metal pollution is the major predictor of the observed shape variations, whereas other biotic/abiotic traits are a minor predictor.

Fishborne Zoonotic Trematodes Transmitted by Melanoides tuberculata Snails, Peru.

We investigated the transmission of the fishborne trematodes Centrocestus formosanus and Haplorchis pumilio by Melanoides tuberculata snails in Peru. We report on results of experimental, morphological, and molecular approaches and discuss the potential risk for future human cases, given the existence of food habits in the country involving the ingestion of raw fish.

Cost of resistance to trematodes in freshwater snail populations with low clonal diversity.

BACKGROUND: The persistence of high genetic variability in natural populations garners considerable interest among ecologists and evolutionary biologists. One proposed hypothesis for the maintenance of high levels of genetic diversity relies on frequency-dependent selection imposed by parasites on host populations (Red Queen hypothesis). A complementary hypothesis suggests that a trade-off between fitness costs associated with tolerance to stress factors and fitness costs associated with resistance to parasites is responsible for the maintenance of host genetic diversity. RESULTS: The present study investigated whether host resistance to parasites is traded off with tolerance to environmental stress factors (high/low temperatures, high salinity), by comparing populations of the freshwater snail Melanoides tuberculata with low vs. high clonal diversity. Since polyclonal populations were found to be more parasitized than populations with low clonal diversity, we expected them to be tolerant to environmental stress factors. We found that clonal diversity explained most of the variation in snail survival under high temperature, thereby suggesting that tolerance to high temperatures of clonally diverse populations is higher than that of populations with low clonal diversity. CONCLUSIONS: Our results suggest that resistance to parasites may come at a cost of reduced tolerance to certain environmental stress factors.

The first record of Centrocestus formosanus (Nishigori, 1924) (Digenea: Heterophyidae) in Egypt.

The life cycle of Centrocestus formosanus (Digenea: Heterophyidae) was to be successfully completed in the laboratory in the present study. Hundreds of the thiarid snail, Melanoides tuberculata, were collected from the main water course Mansouriya Canal, Giza Governorate, Egypt. The snails were individually exposed to artificial light to determine possible infection with trematode larvae. Fifteen snails were found infected with opthalmopleurolophocercous cercariae (infection index of 1.97). These opthalmopleurolophocercous cercariae shedded from snails were collected and placed in an aquarium with fish intermediate host, Gambusia affinis, to obtain metacercariae encysted in the gills. The gills with metacercariae were fed to albino rats, Rattus norvegicus, to obtain the adult worms. Adult worms were recovered in the small intestine of rats at 7 days after infection and they were identified as Centrocestus formosanus based on the morphological characteristics and the comparison with the previous descriptions in the literature. They were small, 518 × 324 µm in average size and had characteristic 32 circumoral spines around the oral sucker. The morphological characteristics of the developmental stages, from cercariae to adults, of this heterophyid fluke were given here. Therefore, the presence of this fluke is to be confirmed for the first time in Egypt by the present study.

Effect of pond water depth on snail populations and fish-borne zoonotic trematode transmission in juvenile giant gourami (Osphronemus goramy) aquaculture nurseries.

Infection with fish-borne zoonotic trematodes (FZT) is an important public health problem in many parts of Southeast Asia. People become infected with FZT when eating raw or undercooked fish that contain the infective stage (metacercariae) of FZT. The parasites require specific freshwater snails as first intermediate host and a variety of fish species, both wild caught and cultured, as second intermediate host. Aquaculture production has grown almost exponentially in SE Asia and in order to produce fish free from FZT metacercariae, it is important to mitigate factors promoting transmission to fish. Here we report results from a cross-sectional study to look at the association between pond depth and infection with FZT in giant gourami nursery ponds. Density of intermediate host snails was positively associated with pond depth (count ratio associated with a 1m increase in pond depth was 10.4 (95% C.L.: 1.61-67.1, p<0.5)) and this may partly explain the higher prevalence and intensity of FZT infection in juvenile fish. High fry stocking density (>200 fry m(-3)) was associated with lower host snail density (count ratio=0.15) than low stocking density (<100 fry m(3)). Ponds stocked with 100-200 fry m(-3) had snail counts 0.76 (95% C.L.: 0.33-1.75, p n.s.) of those in ponds stocked with fry density of <100 fry m(-3). Since density of intermediate snail hosts was associated with FZT transmission to fish, effort should be taken to reduce snail density prior to stocking the fry, but focus should also be on habitats surrounding ponds as transmission may occur through cercariae produced outside ponds and carried into ponds with water pumped into ponds.

Focal Philophthalmus gralli infection possibly persists in Melanoides tuberculata over two years following the definitive hosts' removal.

Philophthalmosis is a zoonotic disease associated largely with the spread of the invasive freshwater snail Melanoides tuberculata, serving as an intermediate host. Here we examined Philophthalmus gralli focal fenced infection site reported recently as being associated with Tinamus major and M. tuberculata in Alajuela, Costa Rica. Removal of the definitive hosts allowed us to address also the long-term survival strategy of the parasite. Initially, the snail intermediate hosts displayed high prevalence of P. gralli infection across all its age cohorts. Two years following the removal of definitive hosts, the infection rate decreased by one order of magnitude, while the snails aging less than one year displayed zero infection prevalence. Additionally, phylogenetic analysis of mitochondrial (ND1) and nuclear (ITS1, ITS2) DNA loci revealed negligible intrasite DNA variability of the specimens obtained at the study site in Costa Rica (but not of those obtained earlier in Peru or New Zealand), supporting strongly the hypothesis on focal origin of the infection. The observed dynamics of infection suggests the explanation for the high variability in P. gralli prevalence in intermediate hosts experienced worldwide. We noticed that the reports claiming >20% prevalence of M. tuberculata infection by P. gralli originated exclusively from foci with known eye infection of the definitive hosts, while the P. gralli infection penetrance <2% is typically associated with sites, where the infection of definitive hosts was not observed, suggesting that the infected definitive hosts were present onsite only in the past, or were present only at a site upstream or downstream of the respective sampling site. Thus, this is the first evidence on the possible persistence of eye-trematode infection site for over two years following the last confirmed outbreak in its adult hosts.

Clonal diversity driven by parasitism in a freshwater snail.

One explanation for the widespread abundance of sexual reproduction is the advantage that genetically diverse sexual lineages have under strong pressure from virulent coevolving parasites. Such parasites are believed to track common asexual host genotypes, resulting in negative frequency-dependent selection that counterbalances the population growth-rate advantage of asexuals in comparison with sexuals. In the face of genetically diverse asexual lineages, this advantage of sexual reproduction might be eroded, and instead sexual populations would be replaced by diverse assemblages of clonal lineages. We investigated whether parasite-mediated selection promotes clonal diversity in 22 natural populations of the freshwater snail Melanoides tuberculata. We found that infection prevalence explains the observed variation in the clonal diversity of M. tuberculata populations, whereas no such relationship was found between infection prevalence and male frequency. Clonal diversity and male frequency were independent of snail population density. Incorporating ecological factors such as presence/absence of fish, habitat geography and habitat type did not improve the predictive power of regression models. Approximately 11% of the clonal snail genotypes were shared among 2-4 populations, creating a web of 17 interconnected populations. Taken together, our study suggests that parasite-mediated selection coupled with host dispersal ecology promotes clonal diversity. This, in return, may erode the advantage of sexual reproduction in M. tuberculata populations.

Predation effects of Melanoides tuberculatus Müller 1774) on periphytic biofilm colonization: an experimental approach

The present study evaluated the predation effects of the gastropod Melanoides tuberculatus on the taxa richness and density of the periphyton community. A laboratory experiment was conducted with two treatments: control (without M. tuberculatus) and with M. tuberculatus, each treatment being replicated four times. For periphyton colonization, glass slides were placed in 3.5 L of water from the environment of the gastropods. The analysis of species richness and density of the periphyton community on these artificial substrates showed significant differences (p <0.05) between treatments. In the treatment with M. tuberculatus, taxa richness and density were lower, with a predominance of Chantrasia macrospora in the last days of the experiment. It follows that predation by M. tuberculatus destabilizes the course of periphyton colonization, suggesting the possibility of disruption of natural food chains, however, more studies are needed in this perspectives.

O presente estudo teve como objetivo avaliar os efeitos da predação de Melanoides tuberculatus sobre a riqueza de táxons e densidade da comunidade perifítica. Foi realizado experimento em laboratório, com dois tratamentos: controle (ausência de M. tuberculatus) e com M. tuberculatus, sendo cada tratamento replicado quatro vezes. Para a colonização da comunidade perifítica foram colocados 3,5 L de água do ambiente de origem dos gastrópodes, em cada tratamento e 49 cm2 de substrato previamente colonizado e 20 lâminas como substratos artificiais, durante 24 dias. A análise da riqueza de táxons e densidades da comunidade perifítica nos substratos artificiais mostrou diferença (p<0,05) entre os tratamentos. Os tratamentos com M. tuberculatus apresentaram menor riqueza e densidade, com predominância da Chantrasia macrospora nos últimos dias do experimento. Conclui-se que a predação do M. tuberculatus desestabiliza o curso da colonização do perifiton e consequentemente pode causar a ruptura das cadeias naturais, contudo mais estudos nessa perspectiva são necessários.

Evolution of molluscan hemocyanin structures.

Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin molecules are oligomers composed of many protein subunits that in turn encompass subsets of distinct functional units. The structure and evolution of molluscan hemocyanin have been studied for decades, but it required the recent progress in DNA sequencing, X-ray crystallography and 3D electron microscopy to produce a detailed view of their structure and evolution. The basic quaternary structure is a cylindrical decamer 35nm in diameter, consisting of wall and collar (typically at one end of the cylinder). Depending on the animal species, decamers, didecamers and multidecamers occur in the hemolymph. Whereas the wall architecture of the decamer seems to be invariant, four different types of collar have been identified in different molluscan taxa. Correspondingly, there exist four subunit types that differ in their collar functional units and range from 350 to 550kDa. Thus, molluscan hemocyanin subunits are among the largest polypeptides in nature. In this report, recent 3D reconstructions are used to explain and visualize the different functional units, subunits and quaternary structures of molluscan hemocyanins. Moreover, on the basis of DNA analyses and structural considerations, their possible evolution is traced. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.