Central Annals of Aquaculture and Research

Cite this article: de Freitas Tallarico L, Miyasato PA, Nakano E (2016) Rearing and Maintenance of Biomphalaria glabrata (Say, 1818): Adults and Embryos under Laboratory Conditions. Ann Aquac Res 3(1): 1013.

*Corresponding author

Lenita de Freitas Tallarico, Laboratório de Parasitologia/Malacologia, Instituto Butantan, Avenida Vital Brasil, 1500, CEP 05503-900, São Paulo, SP, Brazil, Tel: 55-11-2627-9770; Fax: 55-11-2627-9581; Email:

Submitted: 20 May 2016

Accepted: 08 June 2016

Published: 10 June 2016

ISSN: 2379-0881

Copyright© 2016 de Freitas Tallarico et al.

OPEN ACCESS

Keywords•Ecotoxicology•Schistosomamansoni•Embryos•Breeding; Moluscicides

Short Communication

Rearing and Maintenance of Biomphalaria glabrata (Say, 1818): Adults and Embryos under Laboratory ConditionsLenita de Freitas Tallarico1,2*, Patrícia Aoki Miyasato1, and Eliana Nakano1

1Laboratório de Parasitologia/Malacologia, Instituto Butantan, Brazil2Faculdade Nossa Cidade – Estácio, Brazil

Abstract

The freshwater snail Biomphalaria glabrata has been studied by many features. The life cycle and reproduction are well described, besides research in the development of molluscicides, maintenance of schistosomiasis cycle and most currently as environmental pollution bioindicators. The knowledge of the best conditions of snails breeding and cultivation are the most relevant information and contribute to the success of the studies with those organisms. The parameters for analyses of snails are described in tables to simplify the employment during the research and comply with the requirements for the preparation of laboratory testing standards. In this paper a review of the recommendations for B. glabrata rearing is described.

INTRODUCTIONFreshwater snails of the genus Biomphalaria (Preston,

1910) are widely studied due their importance in public health, as intermediate hosts of Schistosoma mansoni Sambon 1907 trematode parasite and for being good bioindicator for environmental studies, as described by Tallarico [1]. According to World Health Organization the control of schistosomiasis is of international concern, on their own or jointly with other measures, is to reduce morbidity within to decrease general parasite transmission in the snail and associated aquatic stages by use of moluscicides and environmental changes [2]. Another important statement by WHO explains that invertebrates are recommended for toxicity tests in evaluating of any chemical agent in the potential toxic effects, either for humans, animals and plants [3]. Furthermore, toxicological assays employing Biomphalaria species have revealed its importance as a biomarker providing endpoints for the aquatic life risk assessment and protection [1].

Adaptation to adverse environmental conditions is an important aspect of Biomphalaria [4]. This genus shows wide geographical distribution, low dispersion and is easily collected in the freshwater. For the maintenance of snails, little space is enough and the exposure systems require relatively small quantities of test samples for analysis [5].

Among the genus Biomphalaria, the species B.Glabrata (Say, 1818) is the most used in laboratory conditions [6] (Figures 1

and 2). Snails are simultaneous hermaphrodite and different reactions in both sexes do not occur, they can reproduce throughout the year under controlled conditions and have a short life span, with an egg-to-egg monitoring during two months [5].

The B. glabrata breeding is important for several approaches in scientific researches. It is necessary to keep an abundant population of snails, both for the study of schistosomiasis, such as the development of new molluscicides or parasite cycle maintenance, as well as for toxicity of chemical compounds and environmental analyses. In addition to maintaining the intermediate host to Schistosoma mansoni cycle, our laboratory

Images courtesy: Márcio M. Yamamoto

A) B)

Figure 1 Adults of Biomphalaria glabrata (Say, 1818). (A) Wild-type snail. (B) Non-pigmented, albino snail.

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has used B. glabrata to evaluate toxic and mutagenic effects of chemical and physical agents using mortality index, alterations in embryonic development and chromosome aberrations as biomarkers [5,7].

The knowledge of the best conditions of snails breeding and maintenance are relevant information to the success of the culture. In this paper a review of the recommendations for B. glabrata rearing is described.

MATERIALS AND METHODSIn this work, a literature review was conducted on the

importance of the rearing of Biomphalaria glabrata and its application in scientific research. All available printed literature and world database indexed on the internet were consulted for this preparation. Molluscs, schistosomiasis, toxicology, Biomphalaria, rearing and cultivation were the descriptors used. In addition, information collected for more than thirty years of breeding snails in the Parasitology Laboratory of the Instituto Butantan - São Paulo, Brazil, were included. All the information

has been summarized in the tables.

RESULTS AND DISCUSSIONSome particular requirements are necessary to optimize

the conditions for the growth and maintenance of B. glabrata, both snails as embryos. Fertility, growth, mortality rates and shell size are essential indicators of the physiological state of the snails and it is important to have all the information about them [8]. Furthermore, many aspects for a proper rearing of snails are necessary, such as food, temperature, oxygen, pH, conductivity, and mate selection [1]. Another relevant topic is the characterization of the reproductive cycle under defined conditions. The normal embryonic development of B. glabrata is well defined by Camey and Verdonk [9], the stages are described in detail and easily recognizable. The observation of embryos may be visible due the transparency of capsules spawning and eggs are placed one by one in a single layer. The complete embryonic development and hatching of young snails can be observed after seven to nine days at around 25ºC, and after about 30 days they can reach sexual maturity and begin to spawn [5,7,10].

In this work, the best conditions about reproduction, water quality and ambient requirements for the maintenance and rearing of B. glabrata adults and embryos described by several authors are presented in Table (1) and the requirements for achievement toxicological studies are listed in Table (2), [6-8,11-18]. Some complementary or divergent considerations are addressed in articles and will be described below.

The container material for culturing can be diversified, the most commonly used are plastic and glass. For rearing of infected animals, the use of glass aquaria is advisable, since the decontamination process with this type of material is more safe

A) B)

Figure 2 Maintenance of Biomphalaria glabrata (Say, 1818) in laboratory conditions. (A) Containers for rearing. (B) Individual containers.

Table 1: Detailed summary of the existing literature data concerning recommended maintenance and culturing conditions of the freshwater snail Biomphalaria glabrata (Say, 1818) [6-8, 11-15].Parameters Culturing conditions

Test species: Biomphalaria glabrata

Age of organisms: Known - adult snails with at least two months old and with a minimal shell diameter of 10mm

Amount of liquid per stock vessel: 20 L

Stock vessels: Container of 50 x 23 x 17 cm

Number of stock vessels: 50

Maintenance water: Filtered and dechlorinated water

Water exchange: ± 15 days (if necessary) Aeration:pH:

Constant aeration during the maintenance7.0 ( ± 0.2)

Photoperiod: 12 h light and 12 h darkness

Room temperature 25°C ( ± 2)

Light quality: Ambient light

Feeding: Three times a week; test snails were not fed 24 h before test start

Food: Two large fresh lettuce leaves without the talus, and fish food flakes

Duration of culture: Over 6 months

Culture rotation: Weekly, remove the egg masses deposited in container

Daily control: Ambient temperature, amount of food, quality of food

Sensitivity control: Quarterly assays with a reference substance

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and efficient. The cover is recommended to prevent the snails escape and to the presence of insects in containers. The screen cover is preferably indicated by allowing better aeration [13,14].

According to Balaban and Fried [15], B. glabrata maintained in de ionized water and artificial spring water showed similar percentages in snail’s survival, with percentage of 90by day 21, but there were a significantly higher number of eggs per spawning produced in the artificial spring water cultures.

Sifted clay, ground sterile or sand and oyster flour can be placed on the tank bottom and added with10% calcium carbonate. The snails are scrapers and the substrate can be used as a dietary supplement, marine mollusk shells can thus serve as food.

Fresh lettuce are widely used in the snails feeding, further portion of sieved rodent chow added with 10% calcium carbonate can be made. The ration should be wet to form a paste and be deposited on the aquarium bottom. Another possibility is to mix alfalfa and rodent chow, wheat germ, calcium carbonate, vitamin E and powder milk [6,13]. In our rearing, the fish food flakes has been a convenient and nutritional supplement to the organisms. Snails with 1-3 mm diameter can eat dehydrated lettuce and can be supplemented with the Cyanobacteria nostoc or fish feed [17]. Embryos nursery can also receive the same feed one week prior to hatching, to form a layer of algae or fungi on the bottom and walls of the tanks. The dead snails and the excess food must be removed to prevent the formation of mold and bacterial growth, as evidenced by cloudy and foul-smelling water leading to decreased water oxygenation [6,7,13,14,18].

The animal growth and fecundity can be influenced by agglomeration of B. glabrata. The wide range of population density is 2 to 50 snails per liter but the infected snails are more fragile and should be kept between 15 and 20 snails per liter [8].

Rotifers, ostracods and oligochaetes are the most common invertebrates that can interfere with the growth of B.glabrata and the release of cercariae from the infected snails. Among them, rotifers often contaminate the aquaria, many adhere to the shells and around the egg masses, they can be removed from the snails by mechanical means, directing a stream of tap water onto the surface of the shell and capsule of spawning, additionally a paintbrush or swab can be used [18].

CONCLUSIONIn the Laboratory of Parasitology - Instituto Butantan,

breeding snails has been kept successfully for more than 30 years and they are used in several studies [7]. The lifecycle of Schistosoma mansoni (BH strain) is being maintained for over 14 years and all animals used were treated according to the rules of the Ethics Committee (CEUAIB). The laboratory took part in the sequencing project on S. mansoni transcriptome [19] and currently collaborates in the works involved with the identification of new proteins to be investigated as vaccine candidates and potential drugs. A good development and performance of the culture in the laboratory are due to the care and daily observation of biological, physical and chemical parameters, which have ensured the longevity of creation. The most relevant parameters for breeding snails are described in tables to simplify the employment in the

Table 2: Review of the existing literature data concerning the requirements for the toxicity assays with Biomphalaria glabrata (Say, 1818) adults and embryos [7,12,16,17].Parameters Requirement

Adults EmbryosMortality in seven days before test start:Assay duration:

10%7 days

10%7 days

Exposition duration: 24 h 24 h

Observation time: Daily Daily

Solution renewal: Static assay Static assay

Temperature: 25°C ( ± 2) 25°C ( ± 2)

Photoperiod: 12 h light and 12 h darkness 12 h light and 12 h darkness

Dilution water: Reconstituted soft water Reconstituted soft water

Feeding: None None

Aeration: No No

Test vessels: 180 mL 20 mL

Test solution volume: 178 mL 10 mL

Number of replicate test vessels: 3 3

Number of replicate test vessels in the control: 3 3

Number of organism per replicate: 10 100

Organism stage at the start of bioassay: 2 monthsBlastulae (0-15 h after the first egg cleavage), Gastrulae (24-39 h), Trocophore (48-87 h) and Veliger (96-111 h) stages

Endpoints: Mortality Mortality and Malformation

Assay acceptability: Control Mortality ≤ 10% Control Mortality ≤ 10%

Results expressions: Median lethal concentration (LC50); 7 days with 95% confidence intervals

Median effect concentration (EC50); 7 days with 95% confidence intervals

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de Freitas Tallarico L, Miyasato PA, Nakano E (2016) Rearing and Maintenance of Biomphalaria glabrata (Say, 1818): Adults and Embryos under Laboratory Conditions. Ann Aquac Res 3(1): 1013.

Cite this article

research and comply with the requirements for the preparation of laboratory testing standards.

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