On the diversity of mollusc intermediate hosts of Angiostrongylus costaricensis Morera & Cespedes, 1971 in southern Brazil

Veronicellid slugs are considered the most important intermediate hosts of Angiostrongylus costaricensis, an intra-arterial nematode of rodents. Studies undertaken in three localities in southern Brazil led to identification of molluscs other than veronicellid slugs as hosts of A. costaricensis: Limax maximus, Limax flavus and Bradybaena similaris. These data indicate a low host specificity of larval stages of A. costaricensis, as it has been reported to other congeneric species.

Effect of Niclosamide (Bayluscide WP 70 (R)), Anacardium occidentale hexane extract and Euphorbia splendens latex on behavior of Biomphalaria glabrata (Say, 1818), under laboratory conditions

The repellent effect of the molluscicides Niclosamide (Bayluscide WP 70 (R)), Anacardium occidentale and the latex of Euphorbia splendens on Biomphalaria glabrata was observed through the investigation of the occurrence of escape behavior among molluscs that were exposed to dosages lower than the LD 50. The total number of individuals out of water among the surviving snails in the control group provided a "Natural Escape Index". The comparison between this total and the total number of surviving snails in each group exposed to the different dosages of the molluscicides after 24 hr provided the "Molluscicide Escape Index" and the detection of a "Repellency Range" to these snails. The escape indexes for Niclosamide, A. occidentale and E. splendens were 10, 6.22 and 6.44 respectively. Repellency occurred at the following concentration ranges: 0.01, 0.02 and 0.03 ppm Bayluscide, 0.1, 0.2 and 0.3 ppm A. occidentale and 0.05, 0.10, 0.15 and 0.20 ppm E. splendens. The Natural Escape Index obtained in the control group was zero.

Eurytrema coelomaticum: influence of the infection on the reproduction and nucleic acids contents in the albumen gland and ovotestis of Bradybaena similaris

The number of eggs laid per snail in Bradybaena similaris and the nucleic acids (DNA and RNA) in the albumen gland and ovotestis were quantified in snails infected with sporocysts of the digenetic trematode Eurytrema coelomaticum. The total number of eggs laid per mollusc was reduced by 96.32% at the end of the larval development. The DNA concentration increased by 700% and the RNA concentration was reduced by 8,38% by the time when the daughter sporocysts of E. coelomaticum were released from B. similaris. The relation between these values and the inhibition of the reproduction observed in infected molluscs is discussed.

Abdominal Angiostrongylosis in Southern Brazil - Prevalence and Parasitic Burden in Mollusc Intermediate Hosts from Eighteen Endemic Foci

Angiostrongylus costaricensis is a parasitic nematode of rodents and molluscs are the intermediate hosts. Nocturnal collection of molluscs and search for infective third stage larvae of A. costaricensis was carried out in 18 endemic foci identified by the notification of a confirmed diagnosis in human biopsies or surgical specimens. Molluscs were digested in acidic solution and isolation of larvae eventually present was done in a Baermann funnel. Larvae identified by the presence of a delicate groove in the tail were counted to assess the individual parasitic burden. Four species were found infected, with ranges of prevalence in parenthesis: Phyllocaulis variegatus (7% to 33.3%); Bradybaena similaris (11.7% to 24.1%); Belocaulus angustipes (8.3% ) and Phyllocaulis soleiformis (3.3% to 14.2%). Parasitic burden varied from 1 to 75 with P. variegatus, 1 to 98 with B. similaris, 1 to 13 with B. angustipes and 1 larvae in each of two specimens of P. soleiformis. P. variegatus was present in all sites and was found infected with the highest prevalence figures and the highest individual parasitic burdens. These data stress the importance of veronicellid slugs as intermediate hosts for A. costaricensis in the endemic areas in Rio Grande do Sul, Brazil

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mnl I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Some Aspects of the Development of Lobatostoma jungwirthi Kritscher, 1974 (Aspidogastrea) in Snails and Cichlid Fishes from Buenos Aires, Argentina

The molluscs Heleobia castellanosae and Ampullaria canaliculata and cichlid fishes were collected from an artificial pond at Tres de Febrero Park (Buenos Aires city), between June 1994 and May 1995. One hundred and eighty of 2,467 H. castellanosae examined were infected with pre-adults of Lobatostoma jungwirthi, 112 of them in concurrent infections with digeneans. L. jungwirthi was significantly more prevalent in larger snails, especially those infected with larval digeneans, but the prevalence of infection did not fluctuate significantly throughout the year. A. canaliculata and cichlids smaller than 10 cm were not infected, but larger Cichlasoma facetum harboured adults of L. jungwirthi in their intestines. Experimental infections of C. facetum and Gymnogeophagus meridionalis with pre-adult aspidogastreans obtained from H. castellanosae were successful. The development of L. jungwirthi in the snail host involves five arbitrary growth phases. Its life-cycle is heteroxenous, similar to that described for L. manteri, with H. castellanosae as the intermediate host and cichlid fishes as the definitive host in this pond.

Penetration Sites and Migratory Routes of Angiostrongylus costaricensis in the Experimental Intermediate Host (Sarasinula marginata)

The intermediate hosts of Angiostrongylus costaricensis are terrestrian molluscs, mostly of the family Veronicellidae. The present work aimed at clarifying more accurately the sites of penetration and the migratory routes of A. costaricensis in the tissue slugs and at verifying the pattern of the perilarval reaction at different times of infection. Slugs were individually infected with 5,000 L1, and killed from 30 min to 30 days after infection. From 30 min up to 2 hr after infection, L1 were found within the lumen of different segments of the digestive tube having their number diminished in more advanced times after exposition until complete disappearance. After 30 min of exposition, percutaneous infection occurred, simultaneously to oral infection. Perilarval reaction was observed from 2 hr of infection around larvae in fibromuscular layer, appearing later (after 6 hr) around larvae located in the viscera. A pre-granulomatous reaction was characterized by gradative concentration of amebocytes around larvae, evolving two well-organized granulomas. In this work we confirmed the simultaneous occurrence of oral and percutaneous infections. Perilarval reaction, when very well developed, defined typical granulomatous structure, including epithelioid cell transformation. The infection also caused a systemic mobilization of amebocytes and provoked amebocyte-endothelium interactions.

Anesthesia of Biomphalaria spp. (Mollusca, Gastropoda): sodium pentobarbital is the drug of choice

The anesthetic effect of some water-soluble anesthesic or narcotic drugs currently used in mice was tested in molluscs of the Biomphalaria genus. Sodium thiopental was very toxic to the snails resulting in high rates of mortality in all the treatment schedules tested. Cetamine base, at concentration of 0.25 mg/ml of water, resulted in partial snail anesthesia (40% of snails were anesthetized) only after 20 h of exposition. The association of Cetamine base with Tiazine chloridrate did not improve the anesthesic effect, and higher concentrations of these drugs were toxic to the snails. Sodium pentobarbital at 0.4 mg/ml in water for 8 h was the best treatment schedule to anesthetize Biomphalaria snails. In this schedule, the snails were anesthetized without any toxic effect. The procedure provides a powerful tool for in vivo studies that demande a complete state of snail anesthesia.

Infection by Schistosoma mansoni sambon 1907 in the first four months of life of Biomphalaria straminea (Dunker, 1848) in Brazil

Compatibility between Schistosoma mansoni and Biomphalaria straminea when exposed to the parasite on the first four months of age was assessed for five parasitological aspects: indices of infection and mortality, duration of precercarial and cercarial periods, and rate of cercarial emission. Infections were made on molluscs from laboratory colonies, at the following ages: 8, 13, 18, 21, 53, 83 and 114 days. Two B. straminea colonies were used (Camorim, PE and Picos, PI), and one B. glabrata colony (Ressaca, MG) was used as control. The main results are as follows: (I) infection was significantly associated with mollusc age, being proportionally higher in sexually immature than in mature molluscs for the three colonies; (II) for B. straminea from Camorim, mortality did not differ significantly between infected and non-infected snails; for B. straminea from Picos significantly more deaths occurred among infected than among non-infected snails, while the opposite was observed for B. glabrata from Ressaca; (III) for the three colonies, the precercarial period was significantly shorter for immature molluscs than for mature ones; (IV) the duration of the cercarial period was extremely variable for the three colonies; (V) sexual maturity did not influence cercarial emission for the three colonies.

The Histopathology of the Infection of Tilapia rendalli and Hypostomus regani (Osteichthyes) by Lasidium Larvae of Anodontites trapesialis (Mollusca, Bivalvia)

It is described the histopathology of the infection of Tilapia rendalli (Osteichthyes, Perciformes, Cichlidae) and Hypostomus regani (Osteichthyes, Siluriformes, Loricariidae) by lasidium larvae of Anodontites trapesialis (Mollusca, Bivalvia, Mycetopodidae). The larvae were encysted within the epidermis of the host, being surrounded by a thin hyaline membrane, 3-6 µm thick, of parasite origin. A proliferative host cell reaction did not occur. The histopathology of the infection shows that the lesions induced by the parasites are minimal. However, the numerous small lesions produced by the release of the larvae may provide optimal conditions for the infection by opportunistic pathogens, namely fungus, which may eventually cause the death of the host.

The molluscicidal activity of niclosamide (Bayluscide WP70®) on Melanoides tuberculata (Thiaridae), a snail associated with habitats of Biomphalaria glabrata (Planorbidae)

The aim of this study was to determine the toxicity of niclosamide (Bayluscide ®) on Melanoides tuberculata and Biomphalaria glabrata under laboratory conditions. The latter species is the intermediate host of Schistosoma mansoni (Sambon 1917). M. tuberculata was successfully used as competitor of B. glabrata in biological control programs in French West Indies. Both molluscicide and biological control using M. tuberculata have proved to be successful in reducing the population density of B. glabrata. The associated use of molluscicide in this area would be an effective measure if M. tuberculata were less susceptibility to the molluscicide than B. glabrata. Three hundreds individuals each of B. glabrata and of M. tuberculata, collected in Sumidouro, State of Rio de Janeiro, were used in the experiment. The molluscs were exposed to 14 different concentrations of niclosamide as recommended by the World Health Organization. Probit analysis was used to determine the LC 50 and LC 90. The LC 50 and LC 90 values for B. glabrata were 0.077 mg/l and 0.175 mg/l, respectively and the LC 50 and LC 90 values for M. tuberculata were 0.082 mg/l and 0.221 mg/l respectively. As the lethal concentrations of niclosamide were approximately the same to both species, this could be a disadvantage when controlling B. glabrata with niclosamide in an area of M. tuberculata occurrence. It migth therefore be preferable to utilize the latex extracted from the Euphorbia splendens, which presented a much higher efficiency for B. glabrata than to M. tuberculata.

Response of the Pacific oyster Crassostrea gigas, Thunberg 1793, to pesticide exposure under experimental conditions.

Pesticide run-off into the ocean represents a potential threat to marine organisms, especially bivalves living in coastal environments. However, little is known about the effects of environmentally relevant concentrations of pesticides at the individual level. In this study, the suppression subtractive hybridisation technique was used to discover the main physiological function affected by a cocktail of three pesticides (lindane, metolachlor and carbofuran) in the Pacific oyster Crassostrea gigas. Two oyster populations exposed to different pollution levels in the wild were investigated. The pesticide concentrations used to induce stress were close to those found in the wild. In a time course experiment, the expression of three genes implicated in iron metabolism and oxidative stress as well as that of two ubiquitous stress proteins was examined. No clear regulation of gene or protein expression was found, potentially due to a low-dose effect. However, we detected a strong site- and organ-specific response to the pesticides. This study thus (1) provides insight into bivalve responses to pesticide pollution at the level of the transcriptome, which is the first level of response for organisms facing pollution, and (2) raises interesting questions concerning the importance of the sites and organs studied in the toxicogenomic field.

Planorbidae, Lymnaeidae and Physidae of Ecuador (Mollusca: Basommatophora)

In the course of a trip to Ecuador I had the opportunity of collecting topotypic specimens of the following nominal species of pulmonate molluscs: Biomphalaria cousini Paraense, 1966; Planorbis equatorius Cousin, 1887; P. canonicus Cousin, 1887; Lymnaea cousini Jousseaume, 1887 and P. boetzkesi Miller, 1879. Additional findings were: Helisoma trivolvis (Say, 1817), Biomphalaria peregrina (Orbigny 1835), Drepanotrema anatinum (Orbigny, 1835), D. kermatoides (Orbigny, 1835), D. lucidum (Pfeiffer, 1839), D. surinamense (Clessin, 1884), Lymnaea columella Say, 1817 and Physa acuta Draparnaud, 1805. P. boetzkesi and P. canonicus are considered junior synonyms of Gyraulus hindsianus (Dunker, 1848) and Biomphalaria peregrina (Orbigny, 1835), respectively.

Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS) region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.

Diagnostic of Biomphalaria snails and Schistosoma mansoni: DNA obtained from traces of shell organic materials

Freshwater snails belonging to the genus Biomphalaria act as intermediate hosts for the parasite trematode Schistosoma mansoni in Africa and in the neotropical region. Identification of such molluscs is carried out based on morphological characters and the presence of cercariae is verified through squeezing snails between two glass slides or by exposing them to artificial light. However, sometimes, the material collected includes molluscs with decomposed bodies or, yet, only empty shells, which precludes their identification and S. mansoni detection. Due to these difficulties, we have developed a methodology in which DNA may be extracted from traces of organic material from inside shells in order to identify molluscs through polymerase chain reaction and restriction fragment length polymorphism and to detect S. mansoni into these snails, by using low stringency polymerase chain reaction. Species-specific profiles obtained from B. glabrata, B. straminea, and B. tenagophila snails and their shells, maintained in laboratory for ten years, showed the same profiles. S. mansoni profiles showed to be present in shell specimens as far as the eighth week after being removed from aquarium.