STUDY AREAS

The mollusks were collected from October to December 2016 and from April to July 2017, in peridomicile irrigation ditches of three areas of Peruíbe, on the southern coast of São Paulo (Figure 1). The selected areas were: A - Nova Peruíbe: 24°17'50"S, 47°1'24"W (peripheral area) with 14 ditches; B - Jardim Caraguava: 24°17'42"S, 47°0'56"W (peripheral area) with 28 ditches; and C - Ruínas: 24°16'8"S, 46°56'12"W (urban area) with 11 ditches. All areas were georeferenced using the GPS (global position system) eTrex Summit^® (Garmin).

Source: Tabwin version 4.1.5 - Datasus; Google Earth. Image produced by Nayla Zanella Carramão.

Area A: Nova Peruíbe; Area B: Jardim Caraguava; Area C: Ruínas.

Figure 1 - Location of Peruíbe in Baixada Santista, São Paulo State, Brazil, and satellite image of malacological collection areas 

Area C (Ruínas) was selected based on the environmental similarities with areas A (Nova Peruíbe) and B (Jardim Caraguava), such as the presence of irrigation ditches with water and vegetation in the peridomicile, as well as insect larvae, tadpoles, mollusks, and some fishes. Around the ditches, some animals were noticed, such as dogs, cats, birds (black vultures, ducks, chickens), and horses. All areas had basic sanitation; however, sanitary effluents were discharged into the ditches, contaminating the soil due to the lack of paving.

COLLECTING MOLLUSKS

It was not necessary to request a license from the Brazilian Institute for the Environment and Renewable Natural Resources to carry out the collections, as the Endemics Control Superintendence (Superintendência de Controle de Endemias - SUCEN) is responsible for the surveillance and control actions of vectors and intermediate hosts¹.

The methodology used in the collections followed the SUCEN's guide: Procedimento Operacional Padrão para o Programa de Vigilância e Controle da Esquistossomose Mansônica¹⁶. Mollusks were collected along the ditch, three samples at each 100 m stretch.

A resistant plastic cup (200 mL) was considered a sample, with approximately 20 to 30 mollusks, depending on the animal's size. Some aquatic vegetation was used to maintain the humidity inside the cup and, it was sealed with gauze. The samples were identified as to the date of collection, sample number, locality name (neighborhood), ditch name (same as the name of the street), and the ditch stretch number.

The collections were performed with the aid of a collecting shovel, consisting of a wire mesh screen (2 mm) at right angles, with a long wooden handle and a 30 cm stainless steel straight-tipped forceps. All samples were packed in plastic boxes to be transported safely at the end of the collections.

TREATMENT OF MOLLUSKS

The initial procedures took place in the afternoon, as soon as the field samples arrived at the laboratory. Mollusks were quantified and separated by genus, according to the conchological criteria¹. After screening, the animals were grouped according to their genus. Each container received up to 15 mollusks, chlorine-free water, and a small amount of fresh lettuce.

PARASITOLOGICAL EXAMINATION

The examinations for assessing cercariae infection in mollusks occurred individually in wells of the culture plate with water, except animals of larger diameter, that were placed in small transparent glass containers (7 cm). The mollusks were exposed to artificial light (photostimulation) for 2 h, always during the day.

The emerged larvae forms from the mollusks were observed in the same cell culture plates they were, which were placed on the base of the stereoscopic microscope in 10-15x magnification. For a better detail of the larval forms, it was necessary to place the larvae between a microscope slide and cover glass to be analyzed under the optical microscope.

Parasitological research was completed with the crushing of mollusks, aiming to find possible cercariae in their internal organs (viscera). For this purpose, an 8.0/12.0 cm glass plate was used, which received up to five mollusks, depending on the animal's size; and, between two plates, the shells were crushed with light pressure^16,17. The global and specific infection rates of the mollusks were determined after exams, according to the method described by Ruiz¹⁸.

PREPARATION OF THE MICROSCOPE SLIDES

The larvae found in the water and viscera of the crushed mollusks were captured with a pipette and transferred to microscope slides with a drop of water to be examined fresh and, subsequently, analyzed under an optical microscope with the lugol (0.1%) and neutral red (0.05%) stains. The larvae were photographed in 40x magnification, except for Apharyngostrigea sp., a sturdier larva, which was photographed in 10x magnification.

The morphological identification of the larvae was based on the following criteria: body and tail shape; size and position of their suckers; the presence or absence of specialized structures, such as oral stylets and ocelli; the presence of pharynx; among other structures, as shown by the taxonomic keys of Pinto and Melo⁹, Martorelli et al.¹⁵, Naruto¹⁹, Frandsen and Christensen²⁰, Shell²¹, and Boaventura et al.²².

IDENTIFICATION OF BIOMPHALARIA SPP.

After the initial parasitological examinations, two or three specimens of Biomphalaria spp. that did not release cercaria were separated for species identification, according to the protocols of the Brazilian Ministry of Health¹, Deslandes²³, Pan American Health Organization²⁴, and Paraense²⁵.

The larvae identification was made, and the pictures were taken at the Malacology Laboratory of the Regional Center of São Vicente/SUCEN.

MOLLUSKS COLLECTED AND IDENTIFIED

A total of 5,969 mollusks from 53 ditches distributed in the three areas were collected and examined: 1,163 mollusks in area A; 3,841 in area B; and 965 in area C. The identification of mollusks revealed B. tenagophila, B. straminea, Drepanotrema sp. (Planorbidae), Lymnaea sp. (Lymnaeidae), Physa sp. (Physidae), Pomacea sp. (Ampullariidae), and Melanoides tuberculata (Müller, 1774) (Thiaridae) (Table 1).

IDENTIFICATION OF TREMATODES AND INFECTION RATES

Nine larval forms were recorded and classified into seven morphological types, i.e.: xiphidio cercaria - Cercaria lutzi (Ruiz, 1943); echinostome cercaria - Cercaria granulifera (Lutz, 1924) and Echinostoma sp.; strigea cercaria - Cercaria caratinguensis (Ruiz, 1953) and Apharyngostrigea sp.; brevifurcate pharyngeate distome cercaria - Cercaria ocellifera (Lutz, 1917); brevifurcate apharyngeate distome cercaria - Cercaria blanchardi (Pirajá da Silva, 1912) or larva of S. mansoni; pleurolophocercous cercaria - Centrocestus formosanus (Nishigori, 1924); and amphistome cercaria - Amphistoma sp.. Larvae C. lutzi, C. granulifera, C. caratinguensis, and C. ocellifera appeared more frequently in this study (Table 2).

Among the specimens of B. tenagophila collected in area B (Jardim Caraguava), six specimens carried S. mansoni larvae, representing an infection rate of 0.16%. However, the highest rates of B. tenagophila infection were with C. lutzi (9.02%) in area B, and C. granulifera (3.96%) in area C (Ruínas). When compared by the area surveyed, mollusk infection rates resulted in 11.40%, 4.76%, and 1.63% for Jardim Caraguava, Ruínas, and Nova Peruíbe, respectively (Table 2).

MORPHOLOGICAL DESCRIPTION OF LARVAE FOUND IN PARASITOLOGICAL EXAMS

Two different larvae of echinostome cercaria (Figure 2: A and D) were observed. One of them, called Echinostoma sp., presented: natatory membrane in the single tail (Figure 2: B); oral and ventral suckers of similar sizes; several cystogenic cells scattered throughout the body; the presence of the esophagus and two long intestinal caeca, which reach the posterior region of the body (Figure 2: A). Larvae cultivated in brown redia (Figure 2: C).

Photos: Nayla Zanella Carramão.

A: Body and tail of Echinostoma sp.; B: Natatory membrane of Echinostoma sp.; C: Brownish redia of Echinostoma sp.; D: Body and tail of Cercaria granulifera; E: Structures of the body and tail of the Cercaria granulifera; F: Brownish redia of Cercaria granulifera. Dye used - Diluted Lugol 0.1% (A, B, E); Without dye - fresh examination (D, F). Scale: 50 μm. vo: Oral sucker; fa: Pharynx; gr: Granules; c.cef: Cephalic collar; cc: Cystogenic cells; co: Concretions; es: Esophagus; vv: Ventral sucker; ce: Caeca; ve: Excretory bladder; mn: Natatory membrane; clc: Caudal cells; esp: Spines.

Figure 2 - Larvae of echinostome cercaria 

The second echinostome cercaria (Figure 2: D and E) presented: muscular pharynx, esophagus long, and bifurcated in two intestinal caeca, reaching the posterior region of the body; excretory bladder on each side of the body, with collecting ducts that have, in the most dilated parts, 35-45 spherical refringent granulations or easily visible concretions; oral and ventral suckers; the presence of two refringent granules in front of the pharynx and near the base of the oral sucker (Figure 2: E); simple, long, and narrow tail covered by spines. These characteristics are from C. granulifera, the larval form of Paryphostomum segregatum (Dietz, 1909). Development in redias⁸ (Figure 2: F).

A larva of xiphidio cercaria, C. lutzi (Figure 3: A and B), presented: oral sucker well-developed and stylet, and ventral sucker in the median region of the body; easily observed penetration glands, five on each side of the body; excretory bladder Y-shaped and simple tail. Development in sporocysts (Figure 3: C).

Photos: Nayla Zanella Carramão.

A, B: Body and tail of Cercaria lutzi; C: Sporocysts of Cercaria lutzi; D: Body and tail of Cercaria caratinguensis; E: Body and tail of Apharyngostrigea sp. Fresh examination. Scale: 50 μm. ve: Excretory bladder (Y); eo: Oral stylet; vo: Oral sucker; gp: Penetration glands; vv: Ventral sucker; oc: Ocelli; esp: Spines; clc: Caudal cells; f: Furca; pg: Genital primordium.

Figure 3 - Larvae of xiphidio cercaria (A, B, C) and strigeo cercaria (D, E) 

Two strigea cercaria larvae (Figure 3: D and E) were found, one of them similar to C. caratinguensis, with: large body; oral and ventral suckers, the latter was located in the middle part of the body; ocelli without pigmentation; long bifurcated tail, with furcations flattened laterally and longer than the stem, and five pairs of cells were observed in its interior. Development in sporocysts^20,21.

The second strigea cercaria (Figure 3: E) presented features similar to the larva Apharyngostrigea: large and oval body; oral and ventral suckers (spherical); pharynx; two bag-shaped penetration glands on each side of the body, just below the oral sucker; genital primordium near the tail; bifurcated tail, with medium and wide furcations, flattened dorsum ventrally, and a markedly globular caudal trunk; and brown-colored granules inside the body and tail. Development in sporocysts^20,21.

The larvae of the brevifurcate pharyngeate distome type (Figure 4: A and B) resemble C. ocellifera, of the family Clinostomidae, with: dorsal membrane in the elongated body; pigmented ocelli; large and oval oral sucker, ventral sucker not visualized; long tail and bifurcated in short furcations. Development in rediae⁸.

Photos: Nayla Zanella Carramão.

A: Body and tail of Cercaria ocellifera; B: Body of Cercaria ocellifera; C: Body and tail of Centrocestus formosanus; D: Body and tail of Cercaria blanchardi (Schistosoma mansoni); E: Body and tail of Anfistoma sp.. Dyes: neutral red 0.05% (C) and lugol 0.1% (D, E). Scale: 50 μm. vo: Oral sucker; oc: Ocelli; md: Dorsal membrane; f: Furca; vv: Ventral sucker; fa: Pharynx; ce: Caeca; ve: Excretory bladder; gp: Penetration glands.

Figure 4 - Larval types of brevifurcate pharyngeate distome cercaria, pleurolophocercous cercaria and amphistome cercaria - with pigmented ocelli (A, B, C, E); and brevifurcate apharyngeate distome - without pigmented ocelli (D) 

The amphistome cercaria type larva (Figure 4: E) presented: elongated body, with scattered cystogenic cells and two pigmented ocelli; large oral and ventral suckers, with the ventral sucker located in the posterior region of the body; and simple tail. Development in redias^8,26.

The pleurolophocercous cercaria larva (Figure 4: C) presented characteristics of C. formosanus, with: oval body and numerous cystogenic cells; large oral sucker, and ventral sucker difficult to visualize; muscular pharynx; two long intestinal cecae, below the ocelli and excretory bladder in the back part of the tail; simple tail with small natatory membrane. Development in rediae^27,28.

The larva C. blanchardi, a brevifurcate apharyngeate distome cercaria type (Figure 4: D), presented: elongated body; developed oral sucker and small ventral sucker, located in the subequatorial area of the body; adhesion and penetration glands; absent pharynx; cylindrical tail, bifurcated in short furcations. Development in sporocysts¹⁹.