A checklist of marine larval trematodes ( Digenea ) in molluscs from Argentina , Southwestern Atlantic coast

A checklist of cercariae parasitizing marine molluscs from Patagonian coast, Argentina, based on literature sources and new records here presented. In total, cercariae of 31 species of marine digeneans, including 11 new records here presented, are known to infect 20 species of molluscs (14 in nine gastropods species; 17 in 11 bivalve species). These records include two species of Aporocotylidae, four Bucephalidae, one Fellodistomidae, five Gymnophallidae, one Hemiuroidea, four Lepocreadiidae, two Microphallidae, five Monorchiidae, one Notocotylidae, one Philophthalmidae, two Renicolidae, one Schistosomatidae and two Zoogonidae. For each digenean species, their hosts, habitat type, localities, infection site, prevalence, life cycle advances, and access numbers in helminthological collections and GenBank, when available, are detailed. Regarding the life cycles, eight were completely elucidated and four were partially elucidated. Moreover, there are molecular data for 15 species. The data here presented constitutes an advance in the parasites biodiversity knowledge and their life cycles.


INTRODUCTION
The Patagonian Shelf extends for about 5,649 km along the Atlantic coast of South America from northern Uruguay (33°51ʹ S, 053°11ʹ W) to the southern tip of Argentina, bordering with Chile (54°55ʹ S, 064°52ʹ W).Its continental shelf is generally up to 100 m in depth, is the largest and one of the most productive ecosystems in the Southern Hemisphere (Miloslavich et al. 2011).In the Patagonian Shelf, two major marine currents coexist, the cold Malvinas and the warm Brazil currents.The Malvinas current originates in the Antarctic circumpolar current and carries a high nutrient load north along the Argentine coast.Biogeographically, the Patagonian Shelf is divided into two provinces, the Argentine and the Magellanic, that join around Peninsula Valdés.The Argentine Biogeographic Province extends from 36° to 43°S and the Magellanic Biogeographic Province extends from 43° to 56°S (Miloslavich et al. 2011).The estimated mollusc species number is about 200 in the Argentine province and about 720 species in the Magellanic province (Bigatti and Penchaszadeh 2008).The great diversity of molluscs is given by the physiographic and climatic features of the Magellanic province, which is highly homogeneous and it has a lot of endemic taxa (Balech and Erlich 2008).Argentina's coast has mostly sandy beaches and some rocky formations.These rocky shores are dominated by two mussel species and by a diverse macroalgal community with an obvious tidal zonation.
Molluscs generally act as first intermediate hosts of digenean life cycles (Rohde 2005;Cremonte 2011) and their larval stages develop in the gonad and the digestive gland of them.The taxonomic identification of larval digenean is an important step towards the elucidation of their life cycles and the posterior use as a predictive tool in ecological studies (Nolan and Cribb 2005).
Lothar Szidat was largely responsible for inaugurating the study of marine helminthes in Argentina (Lunaschi et al. 2007).He recorded two bucephalid cercariae parasitizing the mussel Brachidontes rodrigezi (d'Orbigny) (Szidat 1963).Many freshwater cercariae (e.g., Ostrowski de Núñez 1974; Suriano and Martorelli 1983;Etchegoin et al. 1996;Martorelli and Etchegoin 1996;Martorelli et al., 2013) were recorded; however the knowledge about marine cercariae is scarce.Five isolated records were published by Ageitos de Castellanos (1961), Morris (1984), Martorelli (1991) and Szidat (1963Szidat ( , 1965)).Since 2009 to date several advances in studies of cercariae from marine gastropods were done in Argentina by our research group.Despite the importance of digeneans in coastal marine areas, the data are scattered in literature and only 18 species of larval digenean in molluscs have been recorded (eight infecting six species of gastropods and 10 infecting six species of bivalves) in the Southwestern Atlantic coast.The aim of this study The records were based on naturally infected molluscs.For most of the species listed in this study, cercariae emerge from the molluscs in aquatic environment; in other cases, when the life cycle is abbreviated, the information is provided in the section of the life cycle advances.

RESULTS
A total of 31 cercariae were identified infecting 20 species of molluscs, one at superfamily level, 14 at family level, 10 at genera level and six at species level.From 31 marine cercariae, 14 are infecting nine gastropods species and 17 are infecting 11 bivalve species.Figure 1 shows the localities where the cercariae were recorded.Gilardoni et al. (2011b).

Phylum
is to present information and new data of occurrence of larval digenean found in the commonest gastropods and bivalves inhabiting coast from Argentina.

MATERIALS AND METHODS
A survey of the scientific data on larval digeneans found in marine molluscs from the Argentine coast was done through scientific papers between 1961 and 2014.Furthermore, we included new records from our research group.Specimens described by our research group were sampled during low tide on several occasions along different sites from the coast of Patagonia, Argentina.Molluscs were transported to the laboratory and placed in small flasks filled with seawater at room temperature (20-23°C).Emerged cercariae were studied alive, stained with neutral red or Nile blue under light microscope before being fixed.After 48 h, all molluscs were necropsied in order to detect prepatent infections and to study larval stages.The morphology was studied alive and photographed.Several naturally emerged cercariae were killed with heated seawater, immediately fixed with 10% formalin (Cribb and Bray 2010), stained with acetic carmine, dehydrated through ascendant ethanol series, cleared with methylsalicylate and mounted on glass slides with Canada balsam.Prevalences were calculated following Bush et al. (1997).Specimens of cercariae were deposited in the Parasitological Collection of Centro Nacional Patagónico (CONICET), Puerto Madryn, Chubut province, Argentina.A sample of cercariae specimens were preserved in ethanol 96% and frozen for molecular analyses.These analyses were performed on ribosomal DNA (mainly ITS1 e ITS2 regions) using standard techniques of DNA extraction, amplification by PCR and DNA sequencing (see protocols in Cremonte et al. 2013Cremonte et al. , 2015)).
The taxonomy of digenean species is in accordance with Cable (1954Cable ( , 1956Cable ( , 1963)), Holliman (1961), Yamaguti (1975) and Stunkard (1983).The taxonomic identity of the mollusc hosts were updated according to WoRMS (2015) and the species names were corroborated by malacological specialists.The marine cercaria species found in molluscs from Argentina are presented in alphabetic order for each family; each record contains information of the species name, authority and year, the name of mollusc host species, authority and family; the habitat where the respective life cycles of the digeneans occur (inter-or subtidal); the Argentine localities where of the molluscs were collected.Moreover, data related to prevalence of infection and life cycle were presented when it is available.If material deposited in helminthological collections or molecular sequences GenBank of other larval stages were available, the respective accession numbers were clarified between parentheses.References: Ageitos de Castellanos (1961), Szidat (1965), Morris (1984).

DISCUSSION
This bibliographic survey, with new record, presents the diversity of marine digenean cercariae in molluscs from Argentine coast over 54 years of parasitological studies.A total of 31 cercariae were known to parasitize 20 species of molluscs; 14 of which parasitized nine gastropods species and 17 were parasitized 11 bivalve species.
This study is the third checklist of marine larval trematodes from South America.The two previous checklists recorded marine and freshwater cercariae from Chile (Muñoz and Olmos 2008) and Brazil (Pinto and Melo 2013), and these includes records since 1980 and 1912, respectively.The Chilean checklist summarized 63 species of digeneans; nine of them are larval trematodes (sporocysts or rediae with cercariae) parasitizing seven molluscan species and five of them are from marine molluscs.About 500 digeneans are known in vertebrates from Brazil.From 46 larval trematodes species recorded in 25 molluscan species in Brazil, only five are marine.The records of larval trematodes from freshwater molluscs (at least 95 species) from Argentina are much higher (e.g., Ostrowski de Núñez 1992; Ostrowski de Núñez et al. 1997;Flores and Semenas 2008;Martorelli et al. 2013;Alda and Martorelli 2014), than Chile and Brazil.However, the number of records from marine molluscs is higher than Chile and Brazil (31 digenean species from 20 molluscs).We found 12 families, corresponding to Gymnophallidae, Monorchiidae, Bucephalidae, Lepocreadiidae, Aporocotylidae, Microphallidae, Renicolidae, Zoogonidae, Fellodistomidae, Hemiuridae, Philophtalmidae and Schistosomatidae.From Chilean marine cercariae, two species belong to Fellodistomidae, one each to Bucephalidae, Gymnophallidae and Plagiorchiidae.From Brazilian marine cercariae, three species belong to Bucephalidae, one to Cyathocotylidae and one (Cercaria maritima) to an unidentified family.
Studies of larval digeneans are necessary to elucidate life cycles and to link cercariae to their respective adult parasites.Moreover, morphological descriptions should be complemented with molecular data, because the majority of larvae can only be identified to family by their morphologically.On the other hand, the distribution of records is not uniform throughout the country mainly because most studies were made in a few Patagonian localities.We hope that this review can stimulate new studies on marine larval digeneans and contribute to the biodiversity knowledge from South American marine parasites.