Melanoides tuberculatus (Gastropoda: Thiaridae) como hospedeiro intermediário de Heterophyidae (Trematoda: Digenea) na região metropolitana do Rio de Janeiro, Brasil

rimtsp Revista do Instituto de Medicina Tropical de São Paulo Rev. Inst. Med. trop. S. Paulo 1678-9946 Instituto de Medicina Tropical São Paulo, SP, Brazil No final da década de 60, caramujos da espécie Melanoides tuberculatus, originários do nordeste africano e sudeste asiático, foram introduzidos no Brasil. Os primeiros registros de espécimes infectados com cercárias foram feitos no Estado do Rio de Janeiro em 2001. O presente estudo relata a ocorrência de M. tuberculatus infectados com larvas de trematódeos na cidade do Rio de Janeiro. O sedimento dos criadouros foi coletado e peneirado através de malhas de 0,25 polegadas. Os caramujos foram transportados em recipientes com água dos criadouros, sendo medidos e posteriormente individualizados. Eles foram expostos à luz e temperatura artificiais para induzir a emergência cercariana. As cercárias mais ativas foram processadas pelos métodos de coloração diferencial e de impregnação por nitrato de prata. Os caramujos negativos foram subsequentemente dissecados. Aproximadamente 700 caramujos foram coletados. Seu comprimento variou de 1,2 a 3,3 cm. A taxa de prevalência foi de 15,76% apesar de 53,76% dos caramujos estarem infectados em uma das áreas. Os caramujos estavam infectados com rédias e cercárias do tipo pleurolofocerca. A morfologia e a quetotaxia cercarianas foram compatíveis com as da família Heterophyidae principalmente devido à presença de nadadeiras medianas dorsais e ventrais e à ausência de receptores sensoriais CI dorsais. PARASITOLOGY Melanoides tuberculatus (Gastropoda: Thiaridae) as intermediate host of Heterophyidae (Trematoda: Digenea) in Rio de Janeiro metropolitan area, Brazil Melanoides tuberculatus (Gastropoda: Thiaridae) como hospedeiro intermediário de Heterophyidae (Trematoda: Digenea) na região metropolitana do Rio de Janeiro, Brasil Tami BogéaI; Fernanda Martins CordeiroII; Janaína Silva de GouveiaII IUniversidade Federal do Rio de Janeiro, Laboratório de Histologia Animal e Comparada, Centro de Ciências de Saúde, Dept. Histologia e Embriologia, Bloco B, Sala 017, Cidade Universitária, 21970-000 Rio de Janeiro, RJ, Brazil IIUniversidade Estácio de Sá (SESES), Laboratório de Parasitologia Animal, Campus CERA, Ciências Biológicas, Estrada da Boca do Mato 850, Vargem Pequena, 22783-320 Rio de Janeiro, RJ, Brazil Correspondence SUMMARY In the late 1960s, Melanoides tuberculatus snails were introduced in Brazil from North/East Africa and Southeast Asia. The first records of specimens infected with cercariae were registered in Rio de Janeiro State in 2001. The present study reports the occurrence of M. tuberculatus infected with larval trematodes in Rio de Janeiro City. Bottom sediment was collected with dip nets and sieved through 0.25 inch-mesh screening. Snails were transported to the laboratory in vials with stream water, then measured and individually isolated in glass vials with distilled water. They were exposed to artificial light and temperature to induce cercarial emergence. The most actively emerging cercariae were processed by differential staining and silver nitrate impregnation methods. Negative snails were subsequently dissected. Approximately 700 snails were collected. Snail total lengths ranged from 1.2 to 3.3 cm. The prevalence rate was 15.76% although 53.76% of the snails were found infected in one of the sites. Infected snails were infected with rediae and pleurolophocercous cercariae. Cercarial morphology and chaetotaxy were consistent with those of the family Heterophyidae mostly due to the presence of median dorsal and ventral fins on the tail and the absence of CI dorsal sensory receptors. Keywords: Melanoides tuberculatus; Cercaria; Heterophyidae; Rio de Janeiro City; Parasitism; Biodiversity. RESUMO No final da década de 60, caramujos da espécie Melanoides tuberculatus, originários do nordeste africano e sudeste asiático, foram introduzidos no Brasil. Os primeiros registros de espécimes infectados com cercárias foram feitos no Estado do Rio de Janeiro em 2001. O presente estudo relata a ocorrência de M. tuberculatus infectados com larvas de trematódeos na cidade do Rio de Janeiro. O sedimento dos criadouros foi coletado e peneirado através de malhas de 0,25 polegadas. Os caramujos foram transportados em recipientes com água dos criadouros, sendo medidos e posteriormente individualizados. Eles foram expostos à luz e temperatura artificiais para induzir a emergência cercariana. As cercárias mais ativas foram processadas pelos métodos de coloração diferencial e de impregnação por nitrato de prata. Os caramujos negativos foram subsequentemente dissecados. Aproximadamente 700 caramujos foram coletados. Seu comprimento variou de 1,2 a 3,3 cm. A taxa de prevalência foi de 15,76% apesar de 53,76% dos caramujos estarem infectados em uma das áreas. Os caramujos estavam infectados com rédias e cercárias do tipo pleurolofocerca. A morfologia e a quetotaxia cercarianas foram compatíveis com as da família Heterophyidae principalmente devido à presença de nadadeiras medianas dorsais e ventrais e à ausência de receptores sensoriais CI dorsais. INTRODUCTION Snails of the species Melanoides tuberculatus (Müller, 1774) (Gastropoda: Thiaridae) have been monitored worldwide because they may serve as intermediate hosts of parasitic trematodes. They originate from North/East Africa and Southeast Asia, where they participate in paragonimiasis and chonorchiasis transmission cycles11. M. tuberculatus was introduced in Brazil around 196718, when four specimens were collected in Santos, São Paulo State. Since then, its distribution has been expanding7,19. In 2001, THIENGO et al.16 reported the occurrence of this species infected with pleurolophocercous cercariae in two municipalities of Rio de Janeiro State. In the present study, we report the occurrence of M. tuberculatus infected with larval trematodes in the metropolitan area of Rio de Janeiro, Brazil. Cercarial morphology and chaetotaxy are also described in detail. MATERIAL AND METHODS Collection and identification of infected snails: Approximately 700 snails of M. tuberculatus, with shell lengths varying from 1.2 to 3.3 cm, were collected from June 2003 through October 2004. The collecting sites were as follows: (1) drainage system of a vegetable garden in a local farm in Vargem Pequena (Granja site), (2) public sewage system of Vargem Pequena (Boca do Mato site), (3) artificial ponds of a social club in Vargem Grande (Pacuí site), (4) streams near a rain forest State park in Vargem Grande (PEPB site), (5) drainage system of a condo in Vargem Pequena (Condo site), and (6) streams of a rain forest City park in Prainha (PNMP site). Bottom sediment was collected with dip nets and was sieved through 0.25-inch-mesh screening. The snails were transported to the laboratory in vials containing stream water. In the laboratory, they were measured and individually isolated in glass vials with 5 mL of distilled water. They were then exposed to artificial light (60 w) and temperature (approximately 30 0C) for two hours a day every two days for up to two weeks to induce cercarial emergence. The most actively emerging cercariae were collected with fine glass pipettes and processed accordingly. Forty days after the collection, the negative snails were dissected for detection of larval stages of digeneans. Specimen preparation and study: Twenty cercariae were processed as whole mounts4. They were fixed in hot 5% formalin, stained with Semichon's acetocarmine, dehydrated in graded ethanol series, cleared in methyl salicylate, and mounted in Entelan (Merck, Darmstadt, Germany). Specimens were then measured. For each measurement, the range is given followed in parentheses by mean, standard deviation, and number of specimens measured. All measurements are in micrometers. Taxonomic identifications followed descriptions by Cable4, Schell12, and Travassos et al.17. Voucher specimens have been deposited in the Instituto Oswaldo Cruz Helminthological Collection, Brazil. The standard procedure for silver nitrate impregnation as described by Combes et al.6 allowed the mapping of sensory receptors on the cercarial tegument. Seventy-seven cercariae were fixed in cold 3% silver nitrate solution, exposed to indirect sunlight for up to two min, rinsed in cold distilled water, and mounted in Aquatex (Merck, Darmstadt, Germany). Illustrations recording the distribution of sensory receptors on the cercarial tegument of each specimen were made. The chaetotaxic pattern was subsequently determined using procedures described elsewhere.2,3 RESULTS One hundred and thirteen snails were found infected with rediae and cercariae of a single type. Although the estimated prevalence rate for the area was 15.76%, the number of infected snails varied significantly among the six sites (Table 1). Fifteen out of 20 cercariae were investigated with differential staining. The general cercarial morphology (Fig. 1) is described as follows: body elongate-oval, 142.5 - 199.5 (172.4 ± 19.5; 14) long by 22.5 - 135.0 (99.4 ± 31.0; 14) wide, covered with minute conical spines. Slender tail, usually sigmoid, attached posteroventrally 160.5 - 255.0 (206.4 ± 27.5; 14) long by 22.5 - 75.0 (34.6 ± 12.3; 14) wide, with median dorsal and ventral fins, a terminal spine, and conspicuous annulations on the tegument. Protrusible subterminal oral sucker 33.0 - 52.5 (43.7 ± 6.4; 13) long by 30.0 - 45.0 (38.1 ± 5.1; 13) wide, with eight enlarged acicular spines 3.0 - 7.5 (6.2 ± 1.5; 12) long. Pair of eyespots 7.5 - 15.0 (11.6 ± 2.5; 11) long by 6.0 - 13.5 (8.0 ± 2.18; 11) wide. Prepharynx present. Globular pharynx 15.0 - 27.0 (16.7 ± 3.9; 10) in diameter; oesophagus well developed 3.0 - 22.5 (10.5 ± 8.1; 5) long by 10.5 - 13.5 (13.8 ± 2.0; 5) wide. Intestinal ceca extending posteriorly to excretory vesicle level. Ventral sucker not conspicuous. Seven pairs of penetration glands anterolateral to ventral sucker, with ducts openings concealed in a tegumental fold when oral sucker is retrated, and exposed when it is protruded. Large saccate excretory vesicle. A brownish pigment dispersed throughout the cercarial body except in oral sucker. A total of 64 out of 77 cercariae were investigated with silver nitrate impregnation. The cephalic, anterior, ventral sucker, posterior, and caudal nerve regions were schematically subdivided into 44 sites. Approximately 87 sensory receptors were found on the cercarial tegument. The chaetotaxic pattern (Fig. 2) is summarized as follows, showing receptors numbers on each side of the cercaria: Cephalic region: CI = 1 CI2, 1 CI4; CII = 2 CII2, 2CII4, 2 CII5, 3 H2, 3 HL2; CIII = 1 CIII2, 3 CIII4, 5 CIII5, 3 H3, 3 HL3; CIV = 2 CIV2, 2 CIV4, 5 CIV5, 1 H4, 2 HL4; CV = 2 CV2, 2 CV4, 3 CV5, 1 H5, 1 HL5; Anterior region: AI = 2 AI V, 4 AI L, 3AI D; AII = 2 AII V, 3 AII L, 3 AII D; AIII = 2 AIII V, 2 AIII L, 1 AIII D; Posterior region: PI = 1 PI L, 1 PI D; PII = 1 PII L, 2 PII D; PIII = 1 PIII L,1 PIII D; Ventral sucker region: M = 3 M V, 1 M L, 2 M D; Tail: U = 2 UD DISCUSSION This study reports that M. tuberculatus snails are been infected by larval trematodes in Rio de Janeiro City. The presence of median dorsal and ventral fins is characteristic of pleurolophocercous cercariae. This type has been described in families Acanthostomidae, Cryptogonimidae, Opisthorchiidae, and Heterophyidae12, which infects reptiles, mammals, and birds in Brazil13. Chaetotaxic investigations were useful to refine the taxonomical classifications at the family level since the distribution of sensory receptors is phylogenetically based2. Thus, the pattern was consistent with that of the family Heterophyidae. This was mostly due to the absence of CI dorsal receptors and similar caudal and dorsal body patterns1. The infection of M. tuberculatus has been maintained in the collecting sites. Throughout this study, infected snails were consistently collected in five of six foci, indicating that local vertebrates are been used as definitive hosts. Further investigations are currently been carried out to examine local vertebrates for heterophyids as well to evaluate the risks for human health5. The presence of M. tuberculatus snails in Prainha Natural City Park and near Pedra Branca State Park is of particular concern. Considering that such exotic snails have a high reproductive capacity and unknown local predators14, they may colonize both parks rapidly. This could affect and even endanger native species. Several investigators have demonstrated that M. tuberculatus may exclude populations of Biomphalaria snails, which are vectors of schistosomiasis in Brazil8,9,10. The present results indicate that a better assessment of the role of these thiarids as intermediate hosts of trematodes is required. This is particularly encouraged to evaluate the impact of using this species in the biological control of Biomphalaria in Brazil15. ACKNOWLEDGMENTS We thank Dr. Lycia de Brito-Gitirana (UFRJ), for granting access to laboratory facilities. We are also indebted to Drs. Silvana Thiengo (FIOCRUZ), for the snail identification, and Fernando Régis diMaio (SESES), for support. Renato Silva and André Bazzanella helped out with the snail collections. Dilma Pimentel and Rui Moreira kindly granted access to collecting sites in private areas. The technical support of Luciana Fávero and Luiza Coelho (SESES) was greatly appreciated. Collections at "Parque Natural Municipal da Prainha" were conducted according to conditions set forth in a collecting permit issued to T.B. by "Secretaria Municipal do Meio Ambiente." This work was supported by a Post-doctoral fellowship and a research grant of the "Conselho Nacional de Desenvolvimento Científico e Tecnológico", Brazil, (no. 150021/2003-8) awarded to T.B. Correspondence to Dr. T. Bogéa Universidade Estácio de Sá (SESES) Laboratório de Parasitologia Animal, Campus CERA, Ciências Biológicas Estrada da Boca do Mato 850, Vargem Pequena 22783-320 Rio de Janeiro, RJ, Brazil Received: 14 October 2004 Accepted: 04 January 2005 1. BAYSSADE-DUFOUR, C. - L'appareil sénsoriel des cercaries et la systématique des trématodes digenétiques. Mém. Mus. Natn. Hist. Nat. (Paris) Ser. A, 113: 1-81, 1979. L'appareil sénsoriel des cercaries et la systématique des trématodes digenétiques Mém. Mus. Natn. Hist. Nat. 1979 1 81 113 BAYSSADE-DUFOUR C. 2. 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