Cercariae ( Digenea : Strigeidae , Diplostomidae ) in Biomphalaria straminea ( Planorbidae ) from a rice field in Northeastern Argentina

The rice fields can provide habitats for many species of aquatic invertebrates, as insects, molluscs, crustaceans; and vertebrates, as fish, amphibians, reptiles and birds, which may act as hosts in the life cycles of digenean parasites. In this context, the goal of the present study was to describe the cercariae found in Biomphalaria straminea from a rice field in Northeastern Argentina. This freshwater snail inhabits a wide variety of environments, favoring shallow, temporary and standing or slow-flowing freshwater bodies. For this study, snails were collected from the cultivated parcels and irrigated channels during the flooding periods (from the time of sowing to soon after harvesting of the rice) between December 2010 and May 2011 and December 2011 and April 2012, in a rice field from Corrientes province, one of the main rice producers of Argentina. A total of 5 510 snails were examined of which 26 were infected with three different larval trematodes belonging to Strigeidae, Furcocercaria V (0.40 %) and Furcocercaria XX (0.04 %), and Diplostomidae, Furcocercaria XIX (0.04 %). Furcocercaria XX and Furcocercaria XIX were present in one rice cultivation cycle, while Furcocercaria V was present in both rice cultivation cycles. The prevalence of the different furcocercariae was somewhat higher in the second rice cultivation cycle. The cercariae described are new records for Argentina that added to 53 cercariae previously reported for Biomphalaria spp. in different aquatic environments of country. The study of the digenean larval in rice fields is important because contribute to the knowledge of the biodiversity of these environments. Rev. Biol. Trop. 65 (2): 551-563. Epub 2017 June 01.

The species of Biomphalaria Preston, 1910 (Gastropoda: Planorbidae) are widely distributed in Argentina, mainly in the Northeast (Rumi & Núñez, 2013).In particular, Biomphalaria straminea (Dunker, 1848) is more restricted to the Northeast and pampean regions, being very frequent in the Río de La Plata basin, namely in the Paraná and Uruguay rivers (Núñez, Gutierrez Gregoric, & Rumi, 2010;Rumi & Núñez, 2013).This species, natural vector of Schistosoma mansoni Sambon, 1907 in Brazil, inhabits a wide variety of environments, favoring shallow, temporary and standing or slow-flowing freshwater bodies.Thus, rice fields provide favorable conditions for the development of dense populations of this planorbid snail.
In Argentina, the larval trematodes diversity of Biomphalaria species, have been studied mainly in natural environments.Recently, Fernández et al. (2013Fernández et al. ( , 2014) ) described larval trematodes in B. straminea from a rice field in Corrientes province, Argentina, one of the main rice producers of Argentina (ACPA, 2014).In this context, the goal of the present work was to complete the description of the cercariae found in the sampling of molluscs of this environment.We provide descriptions of new larval trematodes and assign them to taxonomic families and genera.In some cases, we add data about the period of emergence of cercariae and potential second intermediate hosts, and information about their prevalence.

MATERIALS AND METHODS
The study site was an agricultural area with cultivated rice parcels connected or associated to the Paraná river basin, located approximately 30 km South from Corrientes city, in Corrientes province, Argentina (27°40' N -58°48'21.6"W).
Snails were collected during the flooding periods (n= 5 510), from the time of sowing to soon after harvesting of the rice, between December 2010 and May 2011 (n= 3 494), and December 2011 and April 2012 (n= 2 016).Samples were taken manually from the border of cultivated parcels and irrigated channels.Two persons sampled in the morning during 1.5 hours, using simple mesh nets, locally named "copos" (25 cm frame diameter).The width of the studied channels (n= 2) was approximately 2 m, and 260 m in length.In the laboratory, the snails were kept individually in vials with 20 mL of tap water, and were observed for the emergence of cercariae.Seemingly uninfected snails were dissected to check for other larval intramolluscan stages (e.g.immature infections and metacercariae).
Cercariae were studied alive, with and without vital dyes (0.01 % aqueous solutions of neutral red and Nile blue sulphat).Drawings were made using a camera lucida attached to a Carl Zeiss Jena microscope.Measurements given in the description of each cercaria are based on heat-killed and formalin-fixed specimens and expressed in micrometers (μm), with range followed by the mean ± SD in parentheses.We adopted the "open nomenclature" recommended by Odening (1971) for new species of cercariae.Cercariae fixed in hot 4 % formalin were preserved in vials with 70 % ethanol, and deposited in the Helminthological Collection of the Centro de Ecología Aplicada del Litoral (CECOAL), Corrientes, Argentina.
Photographs were taken with a Leica DFC 295 camera mounted on a Leica DM 2500 microscope.Specimens to be studied by scanning electron microscopy (SEM) were dehydrated in an ethanol series, dried using the critical point technique, coated with goldpalladium and examined under a Jeol 5800 LV Scanning Electron Microscope.
To determine the second intermediate host, laboratory-reared Cnesterodon sp. were exposed to the emerged cercariae.The fish were maintained in small aquaria under controlled conditions until dissection, which was carried out five hours post-exposure (PE).Overall prevalence of each trematode taxon and prevalence in each season was calculated following Bush, Lafferty, Lotz and Shostak (1997).

RESULTS
A total of 26 snails were infected with the cercariae here described.The prevalence of individual larval trematodes was lower than 1 %; Furcocercaria XX and Furcocercaria XIX were present in one rice cultivation cycle, while Furcocercaria V was present in both rice cultivation cycles, with higher prevalence in the second rice cultivation cycle (Fig. 1).The cercariae described are added to 53 previously reported for Biomphalaria spp. in different aquatic environments of Argentina (Table 1).Cercariae emerge from sporocysts.At resting position, the cercariae maintained themselves suspended in water with their bodies bent at a characteristic angle, tail stem straight, and furcae spread at an angle of more than 90° (Fig. 4).

Superfamily Diplostomoidea
Emerged cercariae penetrated in exposed Cnesterodon sp.Five hours post-exposure (PE) the fish were dissected, showing many free young metacercariae, not yet encysted in the body musculature.
Remarks: Cercariae with four penetration gland-cells (two anterior and two lateral to the ventral sucker), 20 flame cells and absent eyespots have been described in the genus Hysteromorpha Lutz, 1931.These cercariae penetrate primarily into fishes and their definitive hosts are mainly aquatic birds of the families Phalacrocoracidae and Ardeidae and genus Burhinus (Niewiadomska, 2005).Hugghins (1954) described the life cycle of Hysteromorpha triloba (Rudolphi, 1819) whose cercaria is similar to Furcocercaria XIX in the morphology of the penetration gland cells, presence of three circles of spines surrounding the opening of the ventral sucker, number and position of sensory hairs on the body (one pair) and several pairs in the tail stem, resting position, size of the body (168-221/41-50 H. triloba vs. 166-205/44-62 Furcocercaria XIX) and anterior organ (42-53/20-28 vs. 41-51/23-30), by penetrating into fishes.However, Furcocercaria XIX differs in the arrangement of the penetration gland-cells (anterior to ventral sucker), the presence of caudal bodies, shorter tail stem  and furcae (165-195 vs. 196-227), and larger ventral sucker (30-34 vs. 17-20).
In Argentina, two cercariae have been reported that could be comparable to Furcocercaria XIX in number and arrangement of the penetration gland-cells: Furcocercaria sp.A (cf. Tylodelphys sp.) Ostrowski de Núñez   with spines smaller than body spines, and 10-14 pairs of sensory hairs, without caudal bodies.Furcae 154-230 (204±26) long and 201-34 (27±5) wide, with spines smaller than tail stem spines.
At resting position, the cercariae maintained themselves suspended in the water with their bodies directed downward, body and tail stem in a straight line and furcae spread at an angle of approximately 90 ° (Fig. 7).
Emergence of cercariae was monitored during two days in two infected snails.At 14.1-27.1 °C cercariae had emergence peaks between 11 am and 3 pm.
Remarks: Furcocercaria V shows the typical characteristics of the cercariae of Cotylurus Szidat, 1928 regarding the presence of four penetration gland-cells, colourless eye-spots, 20 flame cells and transverse commissure anterior to ventral sucker, only differ in the arrangement of penetration gland-cells (anterior to ventral sucker) (Niewiadomska, 2002).Three species of this genus have cercariae with features similar to Furcocercaria V, besides those mentioned above.The cercaria of C. flabelliformis (Faust, 1917) is similar in having 4-5 circles of spines surrounding the opening of its ventral sucker, as well as in size of the suckers, tail stem and furcae, the resting position and the absence of caudal bodies; but differs from Furcocercaria V by having larger body (139-231 vs. 120-195), using snails of genus Lymnaea Lamarck, 1799 and Physa Draparnaud, 1801 as first intermediate hosts, and encysting in snails Lymnaea Lamarck, 1799.Cotylurus flabelliformis may also penetrate snails of Planorbidae and Physidae, but only if they are infected by other species of trematodes, where its encysts within sporocysts and/ or rediae (Cort, Olivier, & Brackett, 1941).The cercaria of C. brevis Dubois & Rausch, 1950 is similar to Furcocercaria V in the number and position of sensory hairs in the body (one pair posterior to ventral sucker); but differs in the number of circles of spines (three) surrounding the opening of ventral sucker, the presence of caudal bodies, a larger body (120-360/30-110 vs. 120-195/46-96) and furcae (240-270 vs. 154-230), and by using snails of genus Lymnaea as first intermediate hosts.The cercaria of Cotylurus (Cotylurus) lutzi Basch, 1969 from Biomphalaria glabrata in Bahía, Brazil is similar to Furcocercaria V in the number of sensory hairs in the tail stem (10-12 pairs) and the hours of emergency (near noon); but differs in having a shorter body (130-174/31-49 vs. 120-195/46-96), tail stem (115-166/29-39 vs. 140-235/32-55) and furcae (143-174/13-16 vs. 154-230/21-34) and more sensory hairs in the body (five pairs).The cercaria of Cotylurus (Cotylurus) lutzi encysts in ovotestis of molluscs or hyperparasitizes the redia or sporocysts of other trematodes, developing encysted metacercariae of the "tetracotyle" form (Yamaguti, 1975).
Cercariae emerge from sporocysts.At resting position, cercarial body and tail stem remain aligned in a straight line and furcae spread at an angle of approximately 90° (Fig. 11).
Remarks: Furcocercaria sp.XX shows similarities to those of genera Strigea Abildgaard, 1790, Apatemon Szidat, 1928, Pharyngostomum Ciurea, 1922and Parastrigea Szidat, 1928 due to presence of ten flame cells and non-pigmented eyespots; however, the first three genera have fewer penetration gland-cells (4 or 2 pairs).Only Parastrigea has seven pairs of penetration gland-cells lateral and posterior to ventral sucker and anterior excretory commissure, which may not be closed (Niewiadomska, 2002).The adult stages of this genus use birds and exceptionally mammals as definitive hosts (Yamaguti, 1971).
In Corrientes Province, Argentina, Ostrowski de Núñez et al. (1997) described two cercariae similar to Furcocercaria XX in the number of flame cells (10) and the presence of non-pigmented eyespots: Furcocercaria sp.VIII from B. orbignyi and B. peregrina and Furcocercaria sp.XIII from B. tenagophila and B. orbignyi.However, both cercariae differ in having fewer penetration glands (4 pairs) and lacking an incomplete transverse commissure.

DISCUSSION
Cercariae of two species of the family Strigeidae (Furcocercaria V, Furcocercaria XX) and one of the family Diplostomidae (Furcocercaria XIX) were found in a rice field in Corrientes, Argentina.This species are added to the eight cercariae previously reported parasitizing B. straminea in the same environment (Fernández et al. 2013(Fernández et al. , 2014)), and are new records of cercariae from Argentina.
The furcocercariae identity to genus level cannot be elucidated because the arrangement (Furcocercaria XIX and Furcocercaria V) and the number of penetration gland-cells (Furcocercaria XX) did not match with any species known.However, according to the characteristics of the families to which the cercariae described are tentatively assigned, theirs potential definitive hosts would be mainly aquatic birds.In this sense, the rice fields in Northeastern Argentina harbor numerous species of aquatic birds that use these environments as refuge, food sources and breeding sites (Blanco et al., 2006;Blanco & de la Balze, 2011).Thus, many of these bird species might act as definitive hosts in the life cycles of theses trematodes that are found as larvae parasitizing snails.Furthermore, the positive results of experimental infections of Furcocercaria XIX in fish, suggest that its second intermediate host should be found among these aquatic vertebrates, which are part of the diet of several bird species present in this agricultural environment.
In general terms, the prevalence of the different furcocercariae was low, and regarding the rice cultivation cycles, it was somewhat higher in the second rice cultivation cycle.Similar results were previously observed in the same rice field (Fernández et al., 2014), and this could be related to diverse factors, such as the presence of greater numbers of definitive hosts in the environment during the second rice cultivation cycle, or fewer snails in the environment during the same period.
The study of the freshwater species in rice fields is important because it can contribute to the conservation of biodiversity in regions where wetlands have been turned into rice fields (Maltchik et al. 2011).In this sense, given the importance of this agroecosystem type in Northeast Argentina, further research efforts should be encouraged to obtain more knowledge about the larval trematode species that parasitize snails in rice fields (using morphological, molecular and life-cycle data) as well as of their hosts.

ACKNOWLEDGMENTS
We would like to thank the authorities of the Agricultural Experimental Station El Sombrero, Corrientes of Instituto Nacional de Tecnología Agropecuaria (INTA) for allowing access to the rice field and for providing accommodation during the sampling period.We thank Margarita Ostrowski de Núñez for reviewing a draft version of the manuscript and making useful suggestions and to two anonymous referees, who helped to improve the manuscript.This study was funded by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

TABLE 1
Previous records of larval trematodes infecting Biomphalaria species in different environments of Argentina