Health Status of Mytilus chilensis from Intensive Culture Areas in Chile Assessed by Molecular, Microbiological, and Histological Analyses

  Citation: Santibáñez, P.; Romalde, J.; Fuentes, D.; Figueras, A.; Figueroa, J. Health Status of Mytilus chilensis from Intensive Culture Areas in Chile Assessed by Molecular, Microbiological, and Histological Analyses. Pathogens 2022,11, 494. https://doi.org/10.3390/ pathogens11050494 Academic Editor: Lawrence S. Young Received: 18 November 2021 Accepted: 11 January 2022 Published: 21 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pathogens Article Health Status of Mytilus chilensis from Intensive Culture Areas in Chile Assessed by Molecular, Microbiological, and Histological Analyses Pablo Santibáñez 1,2,*, Jesús Romalde 3, Derie Fuentes 4, Antonio Figueras 5and Jaime Figueroa 2,6 1Programa de Doctorado en Ciencias de la Acuicultura, Facultad de Ciencias, Universidad Austral de Chile, Los Pinos s/n, Balneario Pelluco, Puerto Montt 5110566, Chile 2Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, Bío-Bío 4030000, Chile; [email protected] 3Department of Microbiology and Parasitology, CRETUS & CIBUS-Faculty of Biology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; [email protected] 4 Bio-Computing and Applied Genetics Division, Center for Systems Biotechnology, Fraunhofer Chile Research Foundation, Santiago 8580704, Chile; [email protected] 5 Institute of Marine Research (IIM), National Research Council (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain; [email protected] 6 Department of Biochemistry and Microbiology, Faculty of Biochemistry, University Austral of Chile, Valdivia, Los Ríos 5091000, Chile *Correspondence: pabloandr[email protected] Abstract: Shellfish farming is a relevant economic activity in Chile, where the inner sea in Chiloé island concentrates 99% of the production of the mussel Mytilus chilensis. This area is characterized by the presence of numerous human activities, which could harm the quality of seawater. Additionally, the presence of potentially pathogenic microorganisms can influence the health status of mussels, which must be constantly monitored. To have a clear viewpoint of the health status of M. chilensis and to study its potential as a host species for exotic diseases, microbiological, molecular, and histological analyses were performed. This study was carried out in October 2018, where M. chilensis gut were studied for: presence of food-borne bacteria (Vibrio parahaemolyticus,Escherichia coli,Salmonella spp.), exotic bacteria (“Candidatus Xenohaliotis californiensis”), viruses (abalone and Ostreid herpes virus), and protozoa (Marteilia spp., Perkinsus spp. and Bonamia spp.). Additionally, 18S rDNA metabarcoding and histology analyses were included to have a complete evaluation of the health status of M. chilensis. Overall, despite the presence of risk factors, abnormal mortality rates were not reported during the monitoring period and the histological examination did not reveal significant lesions. Pathogens of mandatory notification to World Organization for Animal Health (OIE) and the Chilean National Fisheries and Aquaculture Service (SERNAPESCA) were not detected, which confirms that M. chilensis have a good health status, highlighting the importance of an integrated vision of different disciplines to ensure the sustainability of this important mussel industry in Chile. Keywords: mussel; aquaculture; pathogens; eukaryotic communities; 18S rDNA 1. Introduction Increasing social apprehension about the environmental quality and vulnerability of biodiversity of the marine coastal areas have been observed in recent years, both on a global and local scale [ 1 , 2 ]. Mussels of the genus Mytilus, are widely distributed throughout the oceans and are typically found in cold waters in both hemispheres, following an antitropical distribution pattern [ 3 ]. They are important components of coastal ecosystems [ 4 ] and objects of research as cosmopolitan inhabitants of high-latitude coastal marine ecosystems. In addition to their ecological significance, they are commercially important in the global Pathogens 2022,11, 494. https://doi.org/10.3390/pathogens11050494 https://www.mdpi.com/journal/pathogens Pathogens 2022,11, 494 2 of 16 scenario with the Chilean mussel (M. chilensis) ranking in the top five mollusk species intended for aquaculture production worldwide [5]. Mytilus chilensis (Hupé1854) belongs to the group of marine mollusks included in the Bivalvia class. It recently acquired the definitive status of a species recognized and accepted in the World Register of Marine Species (WoRMS) with the AphiaID: 397,041 [ 6 ]. It is distributed along approximately 1900 km of the Chilean coast from the Gulf of Arauco (35 ◦ S) in the north to Cape Horn (55 ◦ S) in the south, inhabiting intertidal and subtidal environments down to 10 m in depth [ 7 ]. Currently, 99% of the total commercial production of M. chilensis comes from suspended cultures in large-scale mussel farms located mainly between 41 ◦ 30” S and 43 ◦ 30” S, within the protected Chiloéinner sea, in southern Chile. The Chilean mussel industry has grown exponentially in the last 20 years, with current production at 338,000 tons/year, which makes M. chilensis the second most important product of Chilean aquaculture, after Atlantic salmon [8]. Harvesting of Mytilus spp. mussels throughout the world is based on the exploitation of both wild and cultivated populations [ 9 ]. Mussel farming is based on long-line systems suspended either at the surface or sub-surface, in coastal or open waters. The Chilean mussel farms are in highly anthropized areas in the inner sea of Chiloé, characterized by intensive aquaculture and tourist activities. Although these guarantee a good supply of nutrients for mussels breeding, they could represent a problem due to the introduction of contaminants that can weaken the immune system of the mussels. The presence of biotic and abiotic stressors agents can lead to a critical situation, depending on the seasonality or the performance of human activities at specific sites. Although no mass mortality events associated to pathogens have been reported in Chile, events have been reported in mussels in other parts of the world [10]. To continue previous studies [ 11 ] and contribute to the definition of the health status of the Chilean mussel, we report the results of a multidisciplinary study, evaluating pathogens that can affect the welfare of marine species with economic importance, as well as public health, using M. chilensis as a sentinel species. The target microorganisms are defined in the list of exotic diseases of the World Organisation for Animal Health (OIE) and Chilean National Fisheries and Aquaculture Service (SERNAPESCA), supplemented with 18S rDNA metabarcoding, histology, and foodborne pathogens analyses. This study provides a new and comprehensive perspective on the health status of M. chilensis to provide baseline data for future reference in the mussel aquaculture regions of Patagonia. 2. Results 2.1. Microbiological Analysis A total of three mussel farms (S1–S3) were sampled for microbiological, histological, and molecular analysis (Figure 1, Table 1). Analysis of foodborne pathogens in this study corroborated previous findings from our laboratory, where Salmonella spp. and V. parahaemolyticus were not detected in mussel farms. The E. coli bacterium was detected in all the samples with the most probable number analysis (MPN), all with contamination levels <100 MNP/100 g (Table 2). This finding indicates that the mussels are of a good quality for harvesting, all mussel farms being classified as category A. Table 1. Coordinates and other characteristics of the sampling locations. Site Location Latitude (◦S) Longitude (◦W) Season PH Salinity T ◦C O2Dissolved Site 1 Huelmo Bay 41.677730 73.048031 Spring 8.2 32.5 10.1 8.1 Site 2 Codihue Bay 41.778566 73.373335 Spring 7.7 30.8 9.9 8.4 Site 3 Quinchao Island 42.487768 73.527073 Spring 7.7 31.3 10.2 7.3 Site 4 Calfuco 39.789.288 73.391.704 Spring 7.8 32.5 12.2 7.4 Pathogens 2022,11, 494 3 of 16 Pathogens 2022, 11, x FOR PEER REVIEW 3 of 17 Figure 1. Collection sites for Mytilus chilensis samples in southern Chile. Sampling coordinates are indicated in Table 1. Sites 1–3 have mussel farms and Site 4 is free of them. Table 1. Coordinates and other characteristics of the sampling locations. Site Location Latitude (°S) Longitude (°W) Season PH Salinity T °C O2 Dissolved Site 1 Huelmo Bay 41.677730 73.048031 Spring 8.2 32.5 10.1 8.1 Site 2 Codihue Bay 41.778566 73.373335 Spring 7.7 30.8 9.9 8.4 Site 3 Quinchao Island 42.487768 73.527073 Spring 7.7 31.3 10.2 7.3 Site 4 Calfuco 39.789.288 73.391.704 Spring 7.8 32.5 12.2 7.4 Table 2. Microbiological analysis. Site Sample E. coli MPN/100 g Salmonella spp. P/A 25 g V. parahaemolyticus MPN/100 g Site 1 1 80 Absence <0.3 2 50 Absence <0.3 3 90 Absence <0.3 4 60 Absence <0.3 5 80 Absence <0.3 Site 2 1 80 Absence <0.3 2 70 Absence <0.3 3 60 Absence <0.3 4 80 Absence <0.3 5 70 Absence <0.3 Site 3 1 20 Absence <0.3 2 10 Absence <0.3 3 <0.3 Absence <0.3 4 10 Absence <0.3 5 <0.3 Absence <0.3 Figure 1. Collection sites for Mytilus chilensis samples in southern Chile. Sampling coordinates are indicated in Table 1. Sites 1–3 have mussel farms and Site 4 is free of them. Table 2. Microbiological analysis. Site Sample E. coli MPN/100 g Salmonella spp. P/A 25 g V. parahaemolyticus MPN/100 g Site 1 1 80 Absence <0.3 2 50 Absence <0.3 3 90 Absence <0.3 4 60 Absence <0.3 5 80 Absence <0.3 Site 2 1 80 Absence <0.3 2 70 Absence <0.3 3 60 Absence <0.3 4 80 Absence <0.3 5 70 Absence <0.3 Site 3 1 20 Absence <0.3 2 10 Absence <0.3 3 <0.3 Absence <0.3 4 10 Absence <0.3 5 <0.3 Absence <0.3 MPN: most probable number. P/A: Presence/Absence. 2.2. Pathogens Evaluation and Histological Analyses No mass mortality events were observed during this study. Samples from sites S1–S3 , are in good external condition. All samples were PCR negative for abalone (AbHV) and Ostreid herpesvirus 1 (OsHV-1), parasites (Bonamia spp., Marteilia refringens and Perkinsus spp.) and bacteria (Candidatus Xenohaliotis californiensis) (Table 3). Histological Pathogens 2022,11, 494 4 of 16 analysis described that the common finding in sites S1-S3 was the presence of intracellular bacteria in the digestive tract in low to moderate intensity (prevalence S1: 20%, S2: 36.7% and S3: 33.3%). Additionally, it was observed that intracellular bacteria did not cause alteration in the digestive tract that could affect its correct functioning, observing no structural anomaly (data not shown). These histological results were consistent with the field observations and the PCR results described above. Table 3. Pathogens DNA detection results. Site Pathogen Samples Analyzed PCR Detection * S1 Bonamia spp. 30 n.d. M. refringens n.d. AbHV n.d. X. californiensis n.d. OsHV-1 n.d. Perkinsus spp. n.d. S2 Bonamia spp. 30 n.d. M. refringens n.d. AbHV n.d. X. californiensis n.d. OsHV-1 n.d. Perkinsus spp. n.d. S3 Bonamia spp. 30 n.d. M. refringens n.d. AbHV n.d. X. californiensis n.d. OsHV-1 n.d. Perkinsus spp. n.d. *n.d: No detected. 2.3. General Sequencing Results All samples obtained from gut tissue (n = 10) failed the PCR amplification [ 12 – 14 ]. From the gut content samples (n = 10), DNA was successfully obtained in five of them, three from mussels collected in site 3 (MF group) and two from natural habitat from site 4 (Wild Type group, WT). A total of 5,017,389 raw data reads were generated by Illumina MiSeq sequencing. 2,818,830 high-quality reads remained after trimming and filtering, with a range between 506,051 and 629,065 of non-chimeric reads per sample (Table 4). The rarefaction curves show the number of species as a function of the number of reads, initially growing rapidly as the most common species are found and then stabilizing as only the less frequent species remain. All rarefaction curves of observed species richness reached saturation (Figure 2). Differences in sequencing efficiency were observed in the samples analyzed. The classification against SILVA reference database assigned the reads to 79 families and revealed the presence of 116 genera, where approximately 20% of the sequences could not be assigned to any eukaryote genus. A Tukey’s HSD (Honestlysignificant-difference) test showed that the WT group were significantly different from the MF group (p< 0.05). Table 4. Characteristics of 18S rDNA metagenomic libraries. Site Sample * Input Reads Filtered Reads Merged Non Chimeras No. of Genera No. of Family Shannon-Wiener Index Simpson Index 3 MF1 1,038,053 828,653 737,414 566,455 27 26 3.11 0.95 3 MF2 942,997 789,981 788,394 629,065 14 11 3.00 0.94 3 MF3 994,332 820,426 817,042 596,117 31 27 3.18 0.95 4 WT1 999,049 788,556 782,046 506,051 63 49 3.46 0.96 4 WT2 1,042,958 826,767 821,658 521,142 66 42 3.41 0.96 * WT = Wild Type, MF = Mussel Farm. Pathogens 2022,11, 494 5 of 16 Pathogens 2022, 11, x FOR PEER REVIEW 5 of 17 Table 4. Characteristics of 18S rDNA metagenomic libraries. Site Sample * Input Reads Filtered Reads Merged Non Chimeras No. of Genera No. of Family ShannonWiener Index Simpson Index 3 MF1 1,038,053 828,653 737,414 566,455 27 26 3.11 0.95 3 MF2 942,997 789,981 788,394 629,065 14 11 3.00 0.94 3 MF3 994,332 820,426 817,042 596,117 31 27 3.18 0.95 4 WT1 999,049 788,556 782,046 506,051 63 49 3.46 0.96 4 WT2 1,042,958 826,767 821,658 521,142 66 42 3.41 0.96 * WT = Wild Type, MF = Mussel Farm. Figure 2. Rarefaction curves showing observed species richness in samples from WT (Wild Type) and MF groups. The MF1–MF3 samples were collected from Site 3 and WT1 and, WT2 from Site 4. 2.4. Comparison between 18S rDNA Gut Eukaryotic Communities from WT and MF Groups of M. chilensis A high proportion of host DNA sequences were detected in the samples, highlighting the importance of having specific nucleic acid extraction protocols to reduce contamination with the host species. The analysis of the eukaryotic communities showed that the Simpson index was close to 1 in all samples analyzed, showing high community diversity (Table 4). Figure 3 shows the relative abundance of the 10 most-represented eukaryotic organisms at the genus level found in the mussels gut. All samples have a significant abundance of 18S rDNA sequences from host tissue, ranging from 68.8% to 79.02% of the total sequences. The number of unidentified 18S rDNA reads, representing new species, remained high for all samples tested, close to 20%. The relative abundance of sequences Figure 2. Rarefaction curves showing observed species richness in samples from WT (Wild Type) and MF groups. The MF1–MF3 samples were collected from Site 3 and WT1 and, WT2 from Site 4. 2.4. Comparison between 18S rDNA Gut Eukaryotic Communities from WT and MF Groups of M. chilensis A high proportion of host DNA sequences were detected in the samples, highlighting the importance of having specific nucleic acid extraction protocols to reduce contamination with the host species. The analysis of the eukaryotic communities showed that the Simpson index was close to 1 in all samples analyzed, showing high community diversity (Table 4). Figure 3shows the relative abundance of the 10 most-represented eukaryotic organisms at the genus level found in the mussels gut. All samples have a significant abundance of 18S rDNA sequences from host tissue, ranging from 68.8% to 79.02% of the total sequences. The number of unidentified 18S rDNA reads, representing new species, remained high for all samples tested, close to 20%. The relative abundance of sequences that have been assigned to eukaryotic organisms ranges from 0.2% to 13.95% (Supplementary Tables S3 and S4). Pathogens 2022,11, 494 6 of 16 In general, the eukaryotic composition in both groups was dominated by few genera, which represents the majority of reads in the analyzed samples. Dominant genera were different between the WT and MF groups, showing differences in their relative frequencies as shown in a heat-map (Figure 4). From the sequences that could be assigned, the Peridiniales family have a greater abundance than the other families of the WT group, not being detected in individuals from the MF group. Despite the low abundances obtained in most from the eukaryotic organisms detected, this study was able to determine the presence of 116 different genera that are part of the gut eukaryotic community of M. chilensis. This is influenced by the site-dependent feeding of the samples and provides preliminary information on the possible composition of the surrounding plankton. Further studies are needed to establish the relationship of plankton with the eukaryotic composition of the gut of mussels. Pathogens 2022, 11, x FOR PEER REVIEW 6 of 17 that have been assigned to eukaryotic organisms ranges from 0.2% to 13.95% (Supplementary Tables S3 and S4). Figure 3. Relative abundances of eukaryotic organisms found in gut of M. chilensis by 18S rDNA gene-based profiling analysis. Relative abundance of the top 10 most represented taxa at genus level. In general, the eukaryotic composition in both groups was dominated by few genera, which represents the majority of reads in the analyzed samples. Dominant genera were different between the WT and MF groups, showing differences in their relative frequencies as shown in a heat-map (Figure 4). From the sequences that could be assigned, the Peridiniales family have a greater abundance than the other families of the WT group, not being detected in individuals from the MF group. Despite the low abundances obtained in most from the eukaryotic organisms detected, this study was able to determine the presence of 116 different genera that are part of the gut eukaryotic community of M. chilensis. This is influenced by the site-dependent feeding of the samples and provides preliminary information on the possible composition of the surrounding plankton. Further studies are needed to establish the relationship of plankton with the eukaryotic composition of the gut of mussels. Figure 3. Relative abundances of eukaryotic organisms found in gut of M. chilensis by 18S rDNA gene-based profiling analysis. Relative abundance of the top 10 most represented taxa at genus level. The NMDS plot showed that the MF and WT groups could be differentiated by their gut 18S rDNA eukaryotic composition (Figure 5). The distinct eukaryotic communities were evident from the beta analyses, showing that the composition was similar within each group, but different between them. In general, bivalve samples from mussel farms showed lower diversity and richness of the associated eukaryotic communities compared to samples from natural habitats. Although the two groups share many species, the WT group shows more different genera than the MF group (Figure 6), having 34 genera exclusive to this group, the genus Heterocapsa being the most important. Pathogens 2022,11, 494 7 of 16 Pathogens 2022, 11, x FOR PEER REVIEW 7 of 17 Figure 4. Heat-map (threshold ≥ 0.1%) of eukaryote community at genus level observed in the gut mussel. The change in relative abundance (%) within the community of each phylum is shown by colour intensity. White indicates extremely low abundance and yellow high abundance. The host DNA sequences were removed for visual purposes. The NMDS plot showed that the MF and WT groups could be differentiated by their gut 18S rDNA eukaryotic composition (Figure 5). The distinct eukaryotic communities were evident from the beta analyses, showing that the composition was similar within each group, but different between them. In general, bivalve samples from mussel farms Abundance % Figure 4. Heat-map (threshold ≥ 0.1%) of eukaryote community at genus level observed in the gut mussel. The change in relative abundance (%) within the community of each phylum is shown by colour intensity. White indicates extremely low abundance and yellow high abundance. The host DNA sequences were removed for visual purposes. Pathogens 2022,11, 494 8 of 16 Pathogens 2022, 11, x FOR PEER REVIEW 8 of 17 showed lower diversity and richness of the associated eukaryotic communities compared to samples from natural habitats. Although the two groups share many species, the WT group shows more different genera than the MF group (Figure 6), having 34 genera exclusive to this group, the genus Heterocapsa being the most important. Figure 5. 2D NMDS plot of beta diversity for gut samples from M. chilensis calculated on Bray-Curtis distance matrix. Figure 5. 2D NMDS plot of beta diversity for gut samples from M. chilensis calculated on Bray-Curtis distance matrix. Pathogens 2022, 11, x FOR PEER REVIEW 8 of 17 showed lower diversity and richness of the associated eukaryotic communities compared to samples from natural habitats. Although the two groups share many species, the WT group shows more different genera than the MF group (Figure 6), having 34 genera exclusive to this group, the genus Heterocapsa being the most important. Figure 5. 2D NMDS plot of beta diversity for gut samples from M. chilensis calculated on Bray-Curtis distance matrix. Figure 6. Venn diagram showing the unique, and shared eukaryotic families ( A ) and genera (B) among Mytilus gut samples. 3. Discussion Regarding histological analysis, intracellular prokaryotic inclusions were broadly distributed in the analyzed mussels, with the highest prevalence in Site S2 (36,7%). This finding was not related to any alteration in the digestive epithelium affecting its normal functioning. Additionally, no mass mortalities were recorded in the period of this study. The occurrence of prokaryotic inclusions in the epithelial cells of the gills and digestive gland tubules is widespread among mollusks [ 15 – 17 ]. Some histopathologic effects caused by prokaryotic inclusions have been reported previously in bivalves [ 15 ]. In M. chilensis from southern Chile, the multiplication of the bacteria caused hypertrophy of the infected gill epithelium cells, although low values of prevalence and intensity of infection were recorded [18]. Pathogens 2022,11, 494 9 of 16 The comparative analyses of three geographical sites characterized by the presence the mussel farms (Sites 1–3) showed that M. chilensis had an adequate health status, without the presence of exotic pathogens, significant histological findings, or critical counts of microorganisms dangerous to public health. Protozoa parasites of the genera Perkinsus, Marteilia and Bonamia were recognized as the main challenges for populations of natural and cultured bivalves [ 19 ]. However, since their identification, the publications and studies have not reflected the high environmental and economic impact that they have [ 20 ]. These species can infect abalones, oysters, clams, and mussels. For this reason, they are currently under mandatory notification to the OIE [ 21 ]. Since the first description of Marteilia (Paramyxea) in the flat oyster Ostrea edulis in 1968 in the Aber Wrach, Brittany (France), the life cycle of this parasite has remained unknown. M. refringens is one of the most significant pathogens of bivalve mollusks, with two species “O” and “M” based on polymorphisms in the internal transcribed spacer region of the ribosomal RNA genes [ 22 ]. Marteilia was detected with low prevalence in M. galloprovincialis and M. edulis [ 23 – 25 ], having a significant negative effect on the growth rate and length in M. galloprovincialis [ 26 ]. On the other hand, during a study on the mussel M. galloprovincialis in Tokyo Bay, infection by the protozoan parasite Perkinsus beihaiensis and P. olseni was found by histological examination and PCR analyses [ 27 ]. However, no mass mortality on account of Perkinsus has been described in mussels, where Mytilus plasma probably has a protecting role [ 28 ]. Finally, Bonamia sp. has not been detected in species of the genus Mytilus [ 29 ]; however, in Chile, it was diagnosed in the Ostrea chilensis oyster [ 30 , 31 ]. The possible relationship with other putative intermediate hosts such as M. chilensis is relevant. In this study, presence of M. refringens ,Perkinsus spp., and Bonamia spp. parasites was not detected in the samples analyzed by PCR. Furthermore, no histopathological abnormalities were detected in the individuals studied, and 18S rDNA reads were not taxonomically assigned to these parasitic genera. Interestingly, 18S rDNA metabarcoding identified the genus Parvilucifera [ 32 ] of the family Perkinsidae in one sample from WT group (Supplementary Table S4), Parvilucifera being an alveolate that parasite dinoflagellates [33]. The AbHV and OsHV-1 are important pathogens in abalones and oysters, respectively [ 34 , 35 ]. The genus Mytilus can harbor the OsHV-1, being considered a reservoir or host for this virus without histological abnormalities [ 36 – 40 ]. In this study, the OsHV1 and AbHV viruses were not detected in the samples analyzed by PCR. Furthermore, no histopathological abnormalities were detected in the individuals studied. To date, to our knowledge there are no reports of AbHV and OsHV-1 in M. chilensis, in accord with our results. Regarding bacterial analysis, withering syndrome is a fatal disease in abalones, caused by a Rickettsiales-like “Candidatus Xenohaliotis californiensis”. However, as infected abalones have been transported to Chile and others countries [ 41 ], X. californiensis was surveyed in this study. DNA of this bacterium was not detected in the individuals analyzed. However, a previous study using 16S rDNA metabarcoding found sequences from Xenohaliotis in the gut of mussels collected from mussel farms in Chile [ 11 ]. Futher studies should be carried out to clarify the relationship between M. chilensis and Xenohaliotis. High-throughput sequencing enables microbial community structure to be analyzed with higher taxonomic resolution. The most popular method for high-throughput sequencing is PCR amplicon sequencing of genetic markers, such as 18S rRNA genes for eukaryotes [ 42 , 43 ]. This approach contributes to our general understanding of environmental eukaryotic diversity and distribution [ 44 ]. In this study, 20% of the sequences could not be taxonomically assigned to any family, which reveals a great research opportunity to discover new eukaryotic species in the waters of southern Chile. In general, the eukaryotic composition in mussels was dominated by few genera, showing differences in their relative frequencies according to the origin of the individuals. The family Peridiniales have a greater abundance than the other families in the WT group, not being detected in individuals from the MF group (Site 3). This family of dinoflagellate organisms are numerous in plankton, where the genus Heterocapsa detected in this work, was previously associated with algal Pathogens 2022,11, 494 16 of 16 47. 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