Journal of Parasitology METAZOAN SYMBIONTS OF THE YELLOW CLAM, Mesodesma donacium (BIVALVIA) IN SOUTHERN CHILE: GEOGRAPHICAL VARIATIONS. --Manuscript Draft-Manuscript Number:
13-301R2
Full Title:
METAZOAN SYMBIONTS OF THE YELLOW CLAM, Mesodesma donacium (BIVALVIA) IN SOUTHERN CHILE: GEOGRAPHICAL VARIATIONS.
Short Title:
Lopez et al.-Symbionts of M. donacium from southern Chile
Article Type:
Regular Article
Corresponding Author:
Zambra Lopez Universidad de Antofagasta Antofagasta, Antofagasta CHILE
Corresponding Author Secondary Information: Corresponding Author's Institution:
Universidad de Antofagasta
Corresponding Author's Secondary Institution: First Author:
Zambra Lopez
First Author Secondary Information: Order of Authors:
Zambra Lopez Leyla Cárdenas, PhD M. Teresa González, PhD
Order of Authors Secondary Information: Abstract:
Mesodesma donacium is a dominant species on sandy beaches along the Chilean coast. However, the only previous parasite records for this species were obtained for the northern Chilean coast (20ºS - 33°S), which dealt with cestodes, polychaetes and copepods. In this study, the symbiotic fauna of M. donacium in its southern distributional range is reported, and the geographical variations in the occurrence of this fauna are evaluated. A total of 565 individuals of M. donacium were captured by local fishermen from 5 localities: Mehuín (MEH) (39° 26'S), Carelmapu (CAR) (41° 44'S), Ancud (ANC) (41° 53'S), Cucao (CUC) (42° 35'S) and Quellón (QUE) (43° 24'S), covering a total distance of 450 km. To collect metazoan symbionts, the valves, mantle, gills, gonad and digestive gland of each specimen of yellow clam were examined, and symbiont identifications were made via morphological and genetic analyses. The prevalence and mean intensity of infestation were calculated for each symbiotic species. Univariate and multivariate analyses were performed to evaluate the differences in symbiotic load between localities. Seven metazoan symbiotic species were recorded. The most abundant species were Paranthessius mesodesmatis, Monorchiidae gen. sp. and Paravortex sp. The copepod P. mesodesmatis and metacercaria Monorchiidae gen. sp. showed a high prevalence in all localities, but their intensity of infection varied among localities. The turbellarian Paravortex sp. was most frequently associated with ANC and CUC. The digenean Sanguinicolidae gen. sp. was recorded only at CAR, and the polychaete Spionidae gen. sp. was recorded only at MEH. In its southern distributional range, M. donacium was characterized by an absence of cestodes. This absence can be explained by the absence of the definitive host. The local environmental conditions in the southern range of the host could explain the differences in symbiotic composition among localities.
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RH: LOPEZ ET AL.-SYMBIONTS OF M. DONACIUM FROM SOUTHERN CHILE METAZOAN SYMBIONTS OF THE YELLOW CLAM, MESODESMA DONACIUM (BIVALVIA) IN SOUTHERN CHILE: GEOGRAPHICAL VARIATIONS Z. López, L. Cárdenas*, and M. T. González Instituto de Ciencias Naturales “Alexander von Humboldt”, Facultad de Ciencias del Mar y de Recursos Biológicos, Universidad de Antofagasta, Av. Angamos 601, P.O. Box 170, Antofagasta, Chile. Correspondence should be sent to:
[email protected] ABSTRACT: Mesodesma donacium is a dominant species on sandy beaches along the Chilean coast. However, the only previous parasite records for this species were obtained for the northern Chilean coast (20ºS - 33°S), which dealt with cestodes, polychaetes and copepods. In this study, the symbiotic fauna of M. donacium in its southern distributional range is reported, and the geographical variations in the occurrence of this fauna are evaluated. A total of 565 individuals of M. donacium were captured by local fishermen from 5 localities: Mehuín (MEH) (39°26’S), Carelmapu (CAR) (41°44’S), Ancud (ANC) (41°53’S), Cucao (CUC) (42°35’S) and Quellón (QUE) (43°24’S), covering a total distance of 450 km. To collect metazoan symbionts, the valves, mantle, gills, gonad and digestive gland of each specimen of yellow clam were examined, and symbiont identifications were made via morphological and genetic analyses. The prevalence and mean intensity of infestation were calculated for each symbiotic species. Univariate and multivariate analyses were performed to evaluate the differences in symbiotic load between localities. Seven metazoan symbiotic species were recorded. The most 1
abundant species were Paranthessius mesodesmatis, Monorchiidae gen. sp. and Paravortex sp. The copepod P. mesodesmatis and metacercaria Monorchiidae gen. sp. showed a high prevalence in all localities, but their intensity of infection varied among localities. The turbellarian Paravortex sp. was most frequently associated with ANC and CUC. The digenean Sanguinicolidae gen. sp. was recorded only at CAR, and the polychaete Spionidae gen. sp. was recorded only at MEH. In its southern distributional range, M. donacium was characterized by an absence of cestodes. This absence can be explained by the absence of the definitive host. The local environmental conditions in the southern range of the host could explain the differences in symbiotic composition among localities. The parasites of marine invertebrates are still generally unknown. Studies of bivalve parasites are important and necessary for understanding the life cycles of parasites, especially for digeneans (Gibson et al., 2002; Jones et al., 2005; Bray et al., 2008) and cestode species (Cake, 1977; Carvajal and Mellado, 2007). In Chile, records of parasites in mollusks are limited (Carvajal, 1977; Campbell and Carvajal, 1979; Osorio and Castillo, 1984; Lasiak, 1991; Gallardo et al., 1992; Valderrama et al., 2004; Oliva and Sánchez, 2005; Muñoz et al., 2013a, 2013b). The trematode Proctoeces lintoni is far the most frequently studied species (see cites in Oliva and Huaquín, 2000; Valdivia et al., 2010). The yellow clam Mesodesma donacium (Lamarck, 1818) is an endemic species of the southern Pacific coast of South America. This species is distributed from Bay Sechura, Perú (5°S) to southern Chiloé (approximately 43°S), Chile (Osorio and Piwonka, 2002). Mesodesma donacium inhabits exposed sandy 2
beaches that are characterized by strong waves and highly active sediment dynamics (Guzmán et al., 1998). The species is buried in the substrate up to 10 cm depth (Arntz et al., 1987) in patches or identifiable banks along the coast (Jaramillo et al., 1994). Mesodesma donacium is heavily exploited by benthic fisheries along the Chilean coast; however, the only previous information on its parasites has been obtained for the northern Chilean coast (20°S – 33°S). These parasites include the plerocercoid Caulobothrium myliobatidis Carvajal 1976, the merocercoid Rhodobothrium mesodesmatum Carvajal 1975, the copepod Paranthessius mesodesmatis Humes, 1967 and the polychaete Polydora bioccipitalis Blake & Woodwick, 1972 (Bahamonde and López, 1962; Tómicic, 1975; Carvajal, 1977; Campbell and Carvajal 1979; Carvajal and Mellado, 2007; Riascos et al., 2007). Thus, there are no parasite records for M. donacium from its southern distributional range. We could expect that yellow clam from northern latitudes harbor different parasite fauna than those from southern latitudes based on the principle that the intermediate hosts of many parasites are highly specific (Huyse et al., 2005). In addition, several intermediate hosts and/or definitive hosts are not present in the southern distributional range of M. donacium. Similarly, the spatial distribution of ectoparasites (with direct life cycles) is often determined by the dispersal ability of the host species and the connectivity between the host species populations (MacKenzie and Abaunza, 1998), which could limit the latitudinal distributions of the parasites in the southern range of the host.
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In this study, the metazoan symbionts of M. donacium in its southern distributional range are reported for the first time; the identifications at higher taxonomic resolution level is developed using molecular analysis, and the geographical variations, at the local scale, in the occurrence of these symbionts are evaluated. Local scale refers to geographic space whose extension is rather small (distance of 450 km), it comprises localities within the same biogeographical areas, but these localities can present particular environmental/oceanographic/chemical characteristics. MATERIALS AND METHODS A total of 565 individuals of M. donacium were obtained between September 2009 and September 2010 from 5 locations in southern Chile: Mehuín (MEH) (39°26’S), Carelmapu (CAR) (41°44’S), Ancud (ANC) (41°53’S), Cucao (CUC) (42°35’S) and Quellón (QUE) (43°24’S) (Table I), spanning a distance of 450 km (Fig. 1). The yellow clams were collected by local fishermen. The collected specimens were frozen (-18 C) prior to examination in the laboratory. Each specimen was sexed and was measured at the maximum valvar length using a vernier device with an accuracy of 0.5 mm. The shell length, sex ratio, and number of examined M. donacium by locality are shown in Table I. The valves, mantle, gills, gonad, and digestive gland of each specimen were examined under a stereoscopic microscope after careful dissection. All recorded symbiotic species were fixed in 70% alcohol for taxonomic identification. The identification was based on the specialized literature (Humes, 1967; Gibson et al., 2002; Read, 2004; Jones et al., 2005; Brusa et al., 2006; Bray et al., 2008; Riascos et al., 2008). 4
The collected parasites were isolated, and each morphotype was transferred to a 1.5-ml microcentrifuge tube. DNA extraction was performed by adding 500 ml of 5% Chelex and 2.5 ml of 20 mg/ml proteinase K to each tube. The samples were incubated at 60 C for 4 hr and then boiled for 8 min (Leung et al., 2009). For digeneans, the V4 region of the 18S rRNA gene and mitochondrial Cytochrome Oxidase subunit I (COI) gene were amplified. For turbellarians, only the V4 region was amplified. For copepods, the D1-D2 region of the large subunit of the 28S rDNA and 18S gene was amplified with a polymerase chain reaction (PCR). The primers described by Hall et al. (1999) were used for the V4 region, Leung et al. (2009) for COI and Song et al. (2008) for 28S and 18S, with the protocols and optimal cycling parameters described for each gene. The PCR products for each specimen were sequenced using an automated capillary electrophoresis sequencer (ABI 3730XL, Macrogen Inc., Seoul, Korea) (http://www.macrogen.com). The sequences were edited using ProSeq v3.0 beta (Filatov, 2002) and aligned with Clustal 2 (Larkin et al., 2007). The phylogenetic trees were generated using Mega v6 software (Tamura et al., 2013) with the Neighbor-Joining (NJ) algorithm (Nei and Kumar, 2000) and the Maximum Composite Likelihood (ML) evolution model (Tamura and Nei, 1993). NJ and ML utilized 1,000 bootstrap analysis runs as nodal support. The best models for each phylogenetic tree were chosen according to the Akaike information criterion values supplied in Mega v6. The sequences obtained for the parasites analyzed in this study were contrasted with sequences obtained from the public database available in the website (www.ncbi.nlm.nih.gov) (Benson et al., 2013). 5
The prevalence and mean intensity were calculated according to Bush et al. (1997) for each morph obtained. However, the mean intensity for cercariae or rediae were not recorded due to the high number of parasite individuals found in each individual hosts. Statistical analyses were performed for those symbionts having a prevalence greater than 5% in at least 1 locality. A Mann-Whitney U test was used to assess differences in the intensity of infection between male and female hosts (Zar, 1999). To evaluate differences in the prevalence of species between the sexes, a 2x2 contingency table with a Yates correction was used (Zar, 1999). Correlations between maximum shell length and the intensity and prevalence were evaluated with Spearman correlation tests (Zar, 1999). For the analysis of prevalence, the host sizes were classified in 1-mm shell length classes. The statistical significance of differences in intensity for each parasite species among localities was assessed with a Kruskal-Wallis test. The possible differences in the prevalence of parasite species between localities were evaluated with independent 2x4 contingency tables (Zar, 1999). Then, we performed multivariate analyses (Legendre and Legendre, 1998): a discriminant analyses (DA) to evaluate differences in intensities (=individual hosts) of the parasites among localities. For this analysis, only parasite species were included to avoid a possible effect of host sizes on discrimination and classifications. Finally, correspondence analyses was used to evaluate possible associations between parasites and localities based on prevalence data and mean intensity data, respectively, for each one of these analyses. Only the parasites with prevalence 6
greater than 5% in at least 1 locality were considered. All analyses were performed in Statistica 8.0 (StatSoft, 2008). RESULTS Yellow clams from MEH and QUE were significantly larger than those from the other sampled localities (H = 314.02; P < 0.001; n = 565). Based on morphology, 8 morphs of metazoan symbionts were recorded. These included the copepod Paranthessius mesodesmatis, the turbellarian Paravortex sp., the spionid polychaete Spionidae gen. sp. and 5 morphs of digeneans (Fig. S1). The molecular analyses using the 28S and 18S genes (Fig. 2) supported the hypothesis that P. mesodesmatis collected in the southern range of the host is the same species found in the northern range of the host (bootstrap 99% and 100%), with a divergence between 0 and 0.3% (0 and 2 bp) in the 28S gene and 0% divergence in the 18S gene. Similarly, the identification of Paravortex sp. (Fig. 3) was supported by the molecular analyses, which showed that this species is associated with Graffila buccinicola (bootstrap 100%) and other members of the family Graffillidae. A divergence of 13% was found between Paravortex sp. and Graffila buccinicola, indicating that both species belong to the same family but not to the same genus. The molecular analyses of the 5 morphs of digeneans using the V4 region showed that morphs 1 (1a corresponds to cercaria and 1b corresponds to sporocyst) and morph 2 (Fig. 4) are associated with members of the family Monorchiidae with a bootstrap support of 99% and 98%, respectively, and a genetic divergence of 7.4% with Diplomonorchis leiostomi and 9.7% with 7
Provitellus turrum. Morph 3 (Fig. 4) was associated with members of the family Sanguinicolidae, with a divergence of 17.2% with Chimaerohemecus trondheimensis. Additionally, the analyses for the COI sequences of these morphs were concordant with the V4 region analyses, showing that the sequences of morphs 1 and 2 had 0% divergence in both genes and that these morphs should, therefore be considered the same species. Therefore, 4 species of digeneans, each belonging to a different family, were recorded: Monorchiidae gen. sp. in the sporocyst and cercaria stages (morphs 1a and ab, respectively) and metacercaria stages (morph 2), Sanguinicolidae gen. sp. at the cercaria stage (morph 3), Gymnophallidae gen. sp. at the metacercaria stage (morph 4) and Plagiorchiida gen. sp. at the metacercaria stage (morph 5). The prevalence and mean intensity of the symbionts by locality and site of infection of the host are shown in Table II. The metacercaria Monorchiidae gen. sp. showed a prevalence of 100% in most localities (MEH, CAR, ANC, and QUE) except CUC; cercariae, which caused gonadal castration, were found in most localities with a prevalence ranging between 0.8 and 1.9%. Sanguinicolidae gen. sp. was recorded only in CAR, exclusively affecting the gills, and Spionidae gen. sp. was recorded only in MEH. The prevalence and mean intensity of the most common symbionts showed significant variations among localities (Table II and Fig. 5). Prevalence of P. mesodesmatis did not vary among localities (G = 2.97; df = 2; P = 0.22, but Paravortex sp. was most prevalent in CUC (G = 129.79; df = 3; P < 0.01).
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Over the entire study area, only the prevalence of P. mesodematis (r = 0.72; P < 0.001; n = 42) showed positive correlations with host shell length. Similarly, the intensity of infection of this copepod was positively correlated with host length (r = 0.63; P < 0.01; n = 502), whereas the intensity of Monorchiidae gen. sp. (metacercaria) was negatively correlated with host length (r = -0.26; P < 0.001; n = 312). For each locality, the correlation coefficients between host size and prevalence and intensity are shown in Table III. The sex of the host was not associated with the prevalence (all P > 0.140) or with the intensity of the symbionts (all P > 0.263). Discriminant analysis showed significant variations in parasites among localities (Wilks' Lambda: 0.312; F (20,1712) = 36.014 p
