Freshwater gastropods of the Baixada Maranhense Microregion , an endemic area for schistosomiasis in the State of Maranhão , Brazil : I-qualitative study

introduction: The Baixada Maranhense Microregion currently has the highest prevalence of schistosomiasis in the State of Maranhão, likely because this parasitosis is characterized as an occupational disease, and increased contact with water increases the risk of infection by Schistosoma mansoni. This paper reports the results of the fi rst comprehensive freshwater malacological survey performed in the Baixada Maranhense Microregion. Methods: Freshwater mollusks were collected from the twenty-one municipalities of the Baixada Maranhense Microregion and from Bacurituba and Cajapió and were evaluated for infection by trematodes. Results: A total of 9,129 mollusks were collected (sixteen species), which included the fi rst records of six species in the State of Maranhão: Gundlachia radiata, G. ticaga, Hebetancylus moricandi, Plesiophysa guadeloupensis, Pomacea bridgesii diffusa and Omalonyx sp. Biomphalaria glabrata was found in fi ve municipalities, whereas B. straminea was found in nine. Biomphalaria glabrata and B. straminea were observed in syntopy in Pinheiro and São Bento. Of the 990 specimens of B. glabrata and the 2,109 specimens of B. straminea that were exposed to and/or analyzed for the presence of larval trematodes, only a single specimen of B. glabrata (0.1%) from São Bento shed S. mansoni . Other larval trematodes were fi rst observed in mollusks from the State of Maranhão. Conclusions: These results indicate that the study area is epidemiologically important due to the presence of two natural vectors of schistosomiasis and the active transmission of schistosomiasis, which was confi rmed in the infected specimen that was collected in this study.

The Baixada Maranhense Microregion, which is one of the seven ecological regions of Maranhão, represents the main group of lacustrine basins in the northeast and is a complex ecological system that consists of temporary, shallow, marginal and permanent lakes that are commonly known as fl ooded grasslands.Despite being an interesting ecological system, the Baixada Maranhense Microregion is not protected from environmental damage, such as extensive-breeding buffalo farming, irrigation projects and dam construction 6 .Currently, the Baixada Maranhense Microregion shows the highest prevalence of schistosomiasis in the state 4 .This parasitosis is characterized as an occupational disease because activities such as fi shing, hunting and farming, which result in increased contact with water, also increase the risk of infection by Schistosoma mansoni (Sambon, 1907) 7 .
Therefore, this paper reports the results of the first comprehensive freshwater malacological survey performed in the Baixada Maranhense Microregion.This study investigated the distribution of snail vectors of S. mansoni and contribute to a better understanding of schistosomiasis transmission within this Microregion. resULTs

MeTHODs
The Baixada Maranhense Microregion is 17,909km 2 and corresponds to approximately 5.4% of the total area of Maranhão, which includes 21 municipalities 8 .Freshwater mollusks were collected from each of the municipalities (Anajatuba, Arari, Bela Vista do Maranhão, Cajari, Conceição do Lago Açu, Igarapé do Meio, Matinha, Monção, Olinda Nova, Palmeirândia, Pedro do Rosário, Penalva, Peri-Mirim, Pinheiro, Presidente Sarney, Santa Helena, São Bento, São João Batista, São Vicente Férrer, Viana and Vitória do Mearim) and from Bacurituba and Cajapió, which are two neighboring municipalities with geographic characteristics that are similar to those of the Baixada Maranhense Microregion.The specimens were obtained from different biotopes (streams, rivers, marsh areas, drainage and sewage ditches, ponds, fl ooded areas and irrigation canals) in November 2011 and in March, July and October 2012.Four collectors performed 10-minute searches in each biotope, and all collection sites were georeferenced.
In the Laboratório de Malacologia of Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro-RJ, specimens were placed in aquaria containing dechlorinated water, substrate and food (fresh lettuce leaves).To search for larvae, the samples were processed following the protocol of Mattos et al. 9 .Except for Omalonyx sp., all of the snails were individually exposed to artifi cial light to determine if they were infected with S. mansoni larvae or any other trematode of medical or veterinary importance, and the trematode larvae were identifi ed using key specifi cs according to Schell 10 .The mollusks were examined using a stereomicroscope, and negative mollusks were crushed to confi rm the absence of larval trematodes 11 .Live cercarial stages were obtained by exposure under an incandescent light, and other larval stages were determined during crushing or anatomical dissections.Ten specimens of each sample were anesthetized in Hypnol 0.05% solution for fi ve hours, killed by hot water (70°C) and fi xed in Railliet-Henry solution for morphological analysis 11 .Mollusk samples were deposited at the Coleção de Moluscos of Instituto Oswaldo Cruz (CMIOC).
Mollusks were confi rmed in 176 of the 230 biotopes that were surveyed (table 1), including S. mansoni vectors (Figure 1).Some species were recorded for the fi rst time, as shown in Figure 2.
Five gastropod families were obtained: Ampullariidae, Ancylidae, Planorbidae, Physidae and Succineidae.A total of 9,129 individuals were collected and identifi ed, including 990 specimens of Biomphalaria glabrata (Say, 1818); 122 specimens of Biomphalaria schrammi (Crosse, 1864); The highest diversity of mollusks species was observed in the municipality of São Bento, where 12 species were reported.In contrast, the municipality of Cajari had the lowest species diversity (only P. maculata).
Of the species of medical and veterinary importance, B. glabrata was identifi ed in fi ve municipalities, whereas B. straminea was found in nine.These species were observed in syntopy in two biotopes of Pinheiro and in four biotopes of São Bento.Of the 990 specimens of B. glabrata and 2,109 specimens of B. straminea that were exposed and/or analyzed for the presence of larval trematodes, only a single specimen of B. glabrata (0.1%) from the municipality of São Bento shed S. mansoni cercariae.
Echinostome cercaria were the most frequent cercariae found in the mollusks, and the highest percentage of infected snails was recorded in São João Batista (14.2%), followed by Conceição do Lago Açu (8.8%).Drepanotrema lucidum was infected with the highest diversity of cercariae (fi ve different types).
The Baixada Maranhense Microregion is epidemiologically important because of the presence of two natural vectors of schistosomiasis and the occurrence of active transmission of schistosomiasis, which was confi rmed based on the infected specimen that was collected.According to the information available at the Schistosomiasis Information System (SISPCE) 2 , the Baixada Maranhense Microregion shows a higher prevalence of schistosomiasis than Peri Mirim (7.2% in 2010; 4.07% in 2011) and São Bento (6.12% in 2010; 6.48% in 2011; and 6.13% in 2012).
This study revealed an extended distribution of two species of medical importance: B. glabrata in Bacurituba and The present study confi rmed the occurrence of the nonvector planorbid species B. schrammi and D. depressissimum in São Bento and D. lucidum in Arari, which coincides with the results of a previous study that was performed in the Amazon Region by Paraense 12 .The most common species of Drepanotrema that were observed in this study included   D. anatinum and D. lucidum.Similarly, in the freshwater malacological survey studies that were performed in the state of Rio de Janeiro, D. anatinum was the most frequently observed in the four Mesoregions (Metropolitana, Centro Fluminense, Norte and Noroeste Fluminense), whereas D. lucidum was the most frequently observed in the Sul Fluminense Mesoregion [13][14][15][16] .The diversity of cercarial types shed from D. lucidum was also observed in other studies; Echinostome cercaria was the most frequent type 17 .Drepanotrema lucidum from the state of Mato Grosso presented the largest variety of larval trematode forms, and Echinostome cercaria were present in most of the gastropod families that were studied in all of the Brazilian regions 17 .Echinostomatidae is the most common trematode family that parasitizes freshwater gastropods in Brazil 9 .The biological interactions between snails and trematodes found in Maranhão state extends the list of cercariae in mollusks from Brazil that was recently related by Pinto and Melo 18 .
Brevifurcate-apharingeate-cercaria can cause cercarial dermatitis 19,20 ; these types of cercaria were found in specimens of B. glabrata from São Bento and P. maculata from Anajatuba.Some trematode larvae of Schistosomatidae can cause cercarial dermatitis, which is an effective skin immune response caused by cercarial penetration.The apparent emergence of cercarial dermatitis should be understood as the spread of disease to a new area (e.g., Chile and the United Kingdom) or the increased contact between people and larval trematodes 21 .Although not recorded in Brazil, the intermediate mollusk hosts are widely distributed in the country.These trematodes have also been found in birds 22 .
This study provides the fi rst record of the ampullariid P. bridgesii diffusa in the state of Maranhão.Currently, this species, which is native to the Amazon Basin, is found in Brazil, Bolivia and Peru 23 .The Baixada Maranhense Microregion is situated in the transition zone between the Amazon and the Northeast 24 , which is more precisely located in the Amazonia Maranhense, where P. bridgesii diffusa was recorded in a contiguous area that covers three municipalities.The wide distribution of the congeneric species P. maculata within the region and the large number of specimens that were collected confi rm that the species has a wide range of morphological, behavioral and physiological adaptations that have possibly contributed to the high diversity and distribution of those mollusks in a large population density 25 .Pornacea maculata is distributed from the western region of Brazil, the border of Paraguay, to the south and north of the Amazon Basin, and it is an important component of the biodiversity of freshwater mollusks of the Pantanal 26 .
Ancylidae that were found in this study are widely distributed in South America 27 .Furthermore, our results are similar to those of previous studies that reported G. radiata and G. ticaga as the most common species.Indeed, in the state of Rio de Janeiro, G. ticaga was the most common species 13-16;28,29 , whereas, in the state of Goiás 30 , G. radiata and G. ticaga were among the species that were found.
Similarly, this paper reports the fi rst record of P. guadeloupensis in Maranhão.In Brazil, P. guadeloupensis was previously reported in the States of Bahia, Espírito Santo, Minas Gerais, Paraíba, Pernambuco, Rio de Janeiro, Rio Grande do Norte and Sergipe 34 .In the present study, the specimens were found in two neighboring municipalities, Pedro do Rosário (locality of Três Palmeiras) and Santa Helena (locality of Mangabeira).
This paper is the fi rst extensive malacological survey of freshwater mollusks in the state of Maranhão.Additional malacological surveys with the intention of obtaining quantitative data to contribute to future studies, such as the monitoring of snail vectors of schistosomiasis and the gain of a comprehensive knowledge of freshwater mollusks from that region, are underway.Additionally, further molecular studies on the larval forms of trematodes that infect mollusks will be performed to aid in their identifi cation.

TABLE 2 -
Types of cercariae and the municipalities where they were found in the Baixada Maranhense Microregion, Maranhão, Brazil.