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Introduction
Naididae Ehrenberg, 1828, is a family of small aquatic oligochaetes that play an important role in many marine and freshwater ecosystems worldwide (Brinkhurst & Jamieson, 1971). The group includes detritivorous, grazers and carnivorous species (Pinder & Ohtaka, 2014). One member of the family is the genus Chaetogaster von Baer, 1827 which, in addition to free-living worms, includes Chaetogaster limnaei von Baer, 1927, an ectosymbiont species for which a close commensal association with several mollusc taxa, including gastropods and bivalves, has been broadly reported (Barbour, 1977; Buse, 1974; Conn et al., 1995; Gelder, 1980; Gruffydd, 1965a, b; Ibrahim, 2007; Streit, 1974).
Chaetogaster limnaei has no dorsal chaetae and the ventral chaetae, up to 14-20 per bundle, are absent in segments 3 to 5 (Gruffydd, 1965a; Brinkhurst, 1986). The species is also characterized by having a vestigial prostomium and chaetae with strongly curved teeth (Gruffydd, 1965a; Gelder, 1980; Brinkhurst, 1971, 1986). These features, as well as its habitat, separate C. limnaei from the other species of the genus. Two subspecies have been proposed for C. limnaei, based on habitat and feeding differentiation, C. limnaei limnaei Von Baer, 1927 and C. limnaei vaghini Gruffydd, 1965. Chaetogaster limnaei limnaei inhabits the mantle cavity of snails, where it reduces parasite infection by feeding on trematode larvae in addition to other organisms, so it has been considered a commensal (Gruffydd, 1965a, b; Khalil, 1961; Michelson, 1964; Rodgers et al., 2005; Zimmermann et al., 2011). However, Gamble and Fried (1976) reported C. limnaei limnaei consuming host snail epithelial tissue, indicating a parasitic relationship. Chaetogaster limnaei vaghini, on the other hand, lives in the renal system of the host, where it feeds on kidney cells, so it has been considered as endoparasitic (Gruffydd, 1965a, b). This subspecies separation is not supported by recent molecular phylogenetic analysis based on COI sequences, which provides evidence that these forms belong to the same taxonomic entity (Smythe et al., 2015). In South America, C. limnaei has been found associated with snails Aplexa rivalis (Maton & Rackett, 1807) (Physidae), Biomphalaria straminea (Dunker, 1848) (Planorbidae) and Pseudosuccinea columella (Say, 1817) (Lymnaeidae) in Brazil (Callisto et al., 2005; Martins & Alves, 2008, 2010). Di Persia (1980) found C. limnaei in Argentina. In Chile, Gluzman (1990) and Valdovinos (2008) included Chaetogaster sp. in their checklists without specifying habitat or host, while Fuentealba-Jara (2011) recorded this worm associated with the freshwater snails of the genus Uncancylus Pilsbry, 1913. Only Chaetogaster diastrophus (Gruithuisen, 1828) has been recorded in freshwater ecosystems at the Chilean Altiplano (SINAB, 2017).
In the present study, we report the first finding of C. limnaei in Chile based on samples retrieved from the gastropod Physa acuta Draparnaud, 1805, an invasive snail recently reported in the country (Bousset et al., 2014; Collado, 2017).
Materials and methods
Eleven adult physid snails were sampled from the Illapel River (Fig. 1), Coquimbo Region, Northern Chile (31°37’55.30” S, 71°09’20.30” W) on March 2016 and preserved in ethanol. The snails were assigned to Physa acuta following Collado (2017). The worms were isolated by dissection of the mantle cavity of snails and observed using stereo and light microscopes (Motic). Photographs of the chaetae were obtained using a scanning electron microscope (SEM) Hitachi SU3500. The specimens were identified as C. limnaei according to taxonomic keys (Brinkhurst, 1971, 1986; Kathman & Brinkhurst, 1998) and additional literature regarding the morphology of the species (Cichy et al., 2016; Gelder, 1989; Gruffydd, 1965a, b; Khalil, 1961). Genomic DNA was extracted using the CTAB method (cetyltrimethyl ammonium bromide) (Winnepennickx et al., 1993) of one specimen assigned to this species. The mitochondrial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the primers 16Sar-L and 16Sbr-H (Palumbi, 1996). PCR profile was described by Collado and Méndez (2012). Nucleotide sequences were obtained by automatic sequencing in forward and reverse directions at Macrogen Company (South Korea). DNA sequences were edited in the software BioEdit (Hall, 1999) and aligned with Clustal X (Thompson et al., 1997). Phylogenetic analyses were performed using maximum parsimony (MP) and Bayesian inference (BI) methods. The MP analysis was based on heuristic search, the TBR algorithm and the random addition of sequences using the program PAUP* 4.0 (Swofford, 2003). The statistical confidence of the nodes was evaluated performing 100 bootstrap pseudoreplicates (Felsenstein, 1985). The BI analysis was performed in the software MrBayes v. 3.1.2 (Ronquist & Huelsenbeck, 2003), previously selecting the best model of sequence evolution with jModelTest (Posada, 2008). The analysis was run for 3 million generations sampling every 1,000 generations; posterior probabilities were obtained using a burn-in period of 10%. The analyses were performed together with 16S rRNA sequences from other congeners and naidid taxa (Bely & Sikes, 2010; Envall et al., 2006, 2012; Sjölin et al., 2005).
Figure 1. Illapel River in the Choapa River Basin, locality of occurrence of Chaetogaster limnaei in northern Chile.
Genomic DNA of C. limnaei is housed in the Laboratorio de Malacología y Sistemática Molecular, Universidad del Bío-Bío, Chillán, Chile. The 16S sequence obtained in the present study was deposited in GenBank (accession number: KY436156).
Results
Specimens of C. limnaei (Fig. 2A-C) were found living in the mantle cavity of the invasive freshwater snail P. acuta sampled in Illapel River. The worms were characterized by the absence of dorsal chaetae, the absence of ventral chaetae in anterior segments and the presence of an inconspicuous prostomium. Other worm species were not present in the host snails. The incidence of C. limnaei on P. acuta was estimated to be 27.3%.
Figure 2. Chaetogaster limnaei, Illapel River, Chile. A, B, Lateral view of living specimens using stereo and light microscope, respectively; C, 2 bundle of chaetae imaged using SEM. Scale bar: A, B = 0.5 mm; C = 30 μm.
Amplification of the 16S gene in the specimen of C. limnaei from Illapel River produced a 503 base pair fragment. BLASTn analysis indicated 100% identity with a sequence of C. limnaei from Sacramento, USA (accession number: GQ355405) (Bely & Sikes, 2010). The MP analysis recovered a single tree (not shown) similar to the topology recovered by the BI analysis (Fig. 3). In both trees our sequence of C. limnaei formed a monophyletic group (1.00 posterior probability under BI; 100% bootstrap under MP) together with C. limnaei from Sacramento, USA, within a well-supported clade (1.00 posterior probability under BI; 87% bootstrap under MP) composed by other species of the genus Chaetogaster for which relationships are poorly supported.
Discussion
The present study allowed the identification of C. limnaei in the invasive P. acuta in northern Chile. The molecular approach was useful to confirm the accuracy of the species identification based on morphological characters. Although the 16S rRNA sequence obtained in the present study is identical to that of C. limnaei from Sacramento, USA (Bely & Sikes, 2010), further molecular investigation is required to establish the route of introduction of C. limnaei to Chile. Besides, it is important to note that the route of introduction of P. acuta to this country is also unknown, although more than one introduction event of the species has been suggested (Collado, 2017).
As a commensal, C. limnaei feeds in a variety of prey including diatoms, filamentous algae, ciliates, flagellates, foraminiferans, rotifers and trematode cercariae, although its omnivory is limited by particle size (Buse, 1974; Conn et al., 1996; Gelder, 1989; Gruffydd, 1965a; Khalil, 1961; Michelson, 1964; Stoll et al., 2013; Streit, 1974). In the present study, we speculate the existence of a commensal relationship between C. limnaei and P. acuta in Illapel River since the examination of the snails showed no damage to the paleal tissues, a hypothesis that needs to be further investigated. Stoll et al. (2013), on the other hand, hypothesized an epizoic antibiosis relationship between C. limnaei and P. acuta at high infestation rates as suggested by the lower growth rates of the infected snail hosts. A strong positive correlation between the size of P. acuta and the infection intensity of C. limnaei has also been observed (Mitchell & Leung, 2016).
Chaetogaster limnaei has been considered a cosmopolitan species (Brinkhurst & Jamieson, 1971; Callisto et al., 2005). It has been found associated to more than 40 species of freshwater snails from at least 10 families, mainly members of Lymnaeidae, Physidae, and Planorbidae (Smythe et al., 2015). In the present study we found C. limnaei within a population of the invasive P. acuta in Chile. Potential snail hosts in this country include native species of the genus Biomphalaria Preston, 1910, Uncancylus, Chilina Gray, 1828, Lymnaea Lamarck, 1799, Heleobia Stimpson, 1865, Physa Draparnaud, 1801 and Potamolithus Pilsbry, 1896. A more extensive sampling is necessary to test the occurrence of C. limnaei in species of these genera, as well as other freshwater ecosystems in Chile.
