IDENTIFICATION AND CHARACTERIZATION OF MYXOSPOREAN
SPECIES INFECTING FRESHWATER FISHES IN A NEW AREA OF
STUDY – ALTO RABAGÃO RESERVOIR (PORTUGAL)
Dissertation for Application to the Masters Degree in
Marine Sciences – Marine Resources, Specialization
in Aquaculture and Fisheries, submitted to the Institute
of Biomedical Sciences Abel Salazar of the University
of Porto.
Supervisor – Doctor Graça Maria Figueiredo Casal
Category – Assistant Professor
Affiliation – University Institute of Health Sciences,
Gandra, Portugal
Co-Supervisor – Master Sónia Raquel Oliveira Rocha
Category – Master
Affiliation – Institute of Biomedical Sciences Abel
Salazar, University of Porto, Porto, Portugal
To Professor Graça Casal, my supervisor, who always supported my project, helping me and giving me confidence along the study. Your guidance and motivation gave me courage to overcome the barriers that were emerging.
To Master Sónia Rocha, my co-supervisor, who daily accompanied me at the laboratory, patiently teaching me all of the techniques and procedures. She was by my side every single time, and transmitted me crucial knowledge. Your attention and patience were essentials for me to fulfill all of the subjects of this work. You always comforted me and helped me in the tiredness moments. And the thing I really appreciate was when you gave me comfort when everything was not collaborating or when I was nervous. For all the moments that you spent with me, I am truly grateful.
To Professor Carlos Azevedo, director of the research group, for the honor and opportunity to join your team work. I would like to express my gratitude for your dedication and knowledge transmitted.
To Professor Alexandre Lobo, director of Microscopy department, for granting me the laboratory of Cell Biology to accomplish my project.
To Elsa Oliveira, Ângela Alves and Ana Paula, technicians from Laboratory of Cell Biology, for all the support and good disposition during all the time shared with me. I am grateful for your patience, attention, transmitted knowledge and for you had helped me always that I needed.
To Doctor José Calheiros, and all the members from the company Quinta do Salmão – Comércio de Peixe Lda., for the collaboration and for believing in my project. Without you, this work would never been possible. He gave me the opportunity to know the aquaculture facility and provide fish specimens for my study, gave me the best attention and helped me in all my requests. I also really thank Domingos and João
To Doctor Carlos Antunes, and Aquamuseu do Rio Minho for the collaboration and for all the support about the dredge and the fishing nets. I am really thankful for your availability to help me and for the best attention that you always gave me.
To Professor António Paulo Carvalho and Professor Nuno Formigo, for the clarifications and the transmitted knowledge on wild fish.
To local fisherman, for the wild fish provided. I am very grateful for your willingness to help me and for the sympathy shown.
To the forest officers from Destacamento da Guarda Nacional Republicana of Chaves, for the assistance support during wild fish sampling, showing a great disposition to cooperate in my project.
To Joaquim Fernandes and Manuela Fernandes, my father and my mother, for all contribute and support, not only during this study but in my entire life, unconditionally giving me all the love and strength to fulfill all of my dreams. I am grateful for everything that you make for me.
To Joaquim Fernandes, my brother, for all the dedication and enthusiasm along this study. His unconditional support was essential for me to never give up. He traveled with me to Montalegre in every single trip listening all the stuff about parasites and fishes, and help me in some decisions to my study. He was a fundamental partner showing a great disposition, even when we faced cold and hard rainy days. You know how important you were to me.
mind for this project. You were crucial for me in every time, having patience for listen me to talk about parasites, polar capsules and their evolution. I am heartily grateful for everything that you make for me and for you had helped me unhesitatingly. You was a stronger pillar that I had and a comfortable port I can count on. Thank you for believe in me and to taught me to believe in myself.
To my friends, Tiago Lopes for the enthusiasm showed in my study and for the demonstrated interests to know the latest results, and Maria João Xavier, Sara Martins and Nicole Pires for the affection and attention. I would like to show my gratitude to For Lia Henriques, Sílvia Coelho, Sofia Abrunheiro, Diana Almeida, Epeli Logan, Miguel Pinto, Zé Pedro Ribeiro, Filipe Silva and Gaspar Lobo for these two amazing years sharing with me good disposition, great study moments, and hard work shared.
Myxosporeans are the group of parasites more abundant in nature, being widely distributed infecting freshwater and marine fish hosts. In most documented cases, myxosporean infections are apparently harmless, causing little damage to the fish host tissues. However, there are some myxosporean species extremely pathogenic, responsible for high levels of morbidity and mortality in fish populations. These species have severe negative impact in aquatic ecosystems, and may also undermine the viability and sustainability of aquacultures and fisheries. Recognizing that several myxosporean species continue to emerge, the description of myxosporean biodiversity from novel aquatic environments is crucial. The main goal of this work is to explore myxosporeans species in a new study area, with the finality of increasing the knowledge on these parasites, namely in terms of fish host selection in the Alto Rabagão Reservoir, site of infection, and distribution.
For this study, 30 cultured Oncorhynchus mykiss specimens and 77 wild fish specimens were sampled from the Alto Rabagão Reservoir: 49 specimens of Rutilus rutilus, 27 specimens of Lepomis gibbosus, and one specimen of Sander lucioperca. In addition, oligochaete specimens were sampled from the reservoir sediment. During the parasitological surveys, 14 fish organs per fish and 1127 oligochaetes were observed using light microscopy in order to determine myxosporean infections. A combination of methodologies were then used for the identification and characterization of the parasites found. Infected tissues were photographed and prepared for transmission electron microscopy, and for molecular biology.
Four myxosporean species were recorded. While the parasitological survey of cultured O. mykiss revealed no myxosporean infections, the parasitological survey of wild fish revealed one myxosporean parasite infecting R. rutilus, and three infecting L. gibbosus. No myxosporean infection was detected in the sole specimen of S. lucioperca analyzed. Regarding the parasitological survey of oligochaetes, myxosporean infections were not observed.
In R. rutilus, a Myxobolus sp. was detected in the spleen with a prevalence of infection of 65.5%. The morphology and molecular analysis of the SSU rRNA gene revealed significant similarity to Myxobolus pseudodispar, a species previously described from the musculature of R. rutilus. Therefore, the parasite was identified as
a Myxobolus sp. that was further present in the spleen, with an overall prevalence of infection of 18.5%. In this host, a Myxobilatus sp. was also observed infecting the urinary bladder, with a prevalence of 3.7%. Taking into account the morphological, ultrastructural and biological characteristics presented by the Sphaerospora sp., this study determined the possibility of this species being a member of the Sphaerospora sensu stricto clade. However, the acquisition of molecular data is needed to confirm this hypothesis. Regarding the Myxobolus sp. and Myxobilatus sp., ultrastructural observations were not performed due to the low intensity of infection. Nevertheless, molecular procedures were performed for sequencing of the SSU rRNA genes, and phylogenetic analysis showed both these species clustering within the main myxobolids clade: the Myxobolus sp. within a subclade of other perciformes-infecting myxobolids, and the Myxobilatus sp. within the excretory system subclade. Having contributed with novel data regarding the these myxosporean parasites, this study further provided new information on the fish fauna of the Alto Rabagão Reservoir, by reporting the occurrence of R. rutilus, L. gibbosus and S. lucioperca.
Overall, this study showed that exploiting the occurrence of myxosporeans species in new study areas constitutes an important effort, for the construction of a basis that in the future will allow better understanding of these parasites distribution and pathology.
Os mixosporídeos são o grupo de parasitas mais abundantes na natureza, estando amplamente distribuídos em peixes marinhos e de água doce. Na maior parte dos casos, as infeções por mixosporídeos são aparentemente inofensivas, causando poucos danos nos tecidos do peixe hospedeiro. No entanto, existem algumas espécies extremamente patogénicas, responsáveis por altos níveis de morbidade e mortalidade em população de peixes. Estas espécies de parasitas causam graves impactes negativos em ecossistemas aquáticos, e podem também comprometer a viabilidade e a sustentabilidade das aquaculturas e pescas. Reconhecendo que diferentes espécies de myxosporídeos continuam a emergir, é essencial a descrição da sua diversidade em ambientes aquáticos ainda não amostrados. O principal objetivo deste trabalho é explorar espécies de myxosporídeos numa nova área de estudo com a finalidade de aumentar o conhecimento sobre estas espécies, principalmente em relação a peixes selecionados como hospedeiros na Albufeira do Alto Rabagão, tecidos de infeção e a sua distribuição.
Para este estudo, 30 Oncorhynchus mykiss de aquacultura e 77 peixes selvagens foram amostrados Albufeira do Alto Rabagão: 49 espécimes de Rutilus rutilus, 27 espécimes de Lepomis gibbosus e um espécime de Sander lucioperca. Além disso, foram amostradas oligoquetas a partir do sedimento da albufeira. Durante a análise parasitológica, 14 órgãos por peixe e 1127 oligoquetas foram observados usando microscopia de luz para determinar infeções por mixosporídeos. Neste estudo, foi usada uma combinação de metodologias para identificar e caracterizar todos os mixosporídeos encontrados. Os tecidos infetados foram fotografados e preparados para a microscopia eletrónica de transmissão e para biologia molecular.
No presente estudo, 4 espécies de mixosporídeos foram registadas. Enquanto a análise parasitológica aos peixes de aquacultura não revelou infeções por mixosporídeos, a análise parasitológica aos peixes selvagens revelou uma espécie de myxosporídeo em R. rutilus e 3 espécies em L. gibbosus. Em S. lucioperca, não foram observadas infeções por mixosporídeos. Em relação à análise parasitológica às oligoquetas, também não foram observadas infeções.
Em R. rutilus, foi detetado Myxobolus sp. no baço com uma prevalência de 65.5%. As análises morfológicas e moleculares do gene SSU rRNA revelaram
pseudodispar. Em L. gibbosus, foi observado Sphaerospora sp. no rim com uma prevalência de 33.3%, e em alguns casos foi exibida coinfecção com Myxobolus sp., que esteve também presente no baço, com uma prevalência geral de infeção de 18.5%. Neste hospedeiro, também foi observado Myxobilatus sp. na bexiga urinária com uma prevalência de 3.7%. Tendo em conta as características morfológicas, ulta-estruturais e biológicas de Sphaerospora sp., este estudo determinou a possibilidade de esta espécie ser um membro da clade Sphaerospora sensu stricto. No entanto, a aquisição de dados moleculares são necessários para confirmar esta hipótese. Em relação ao Myxobolus sp. e Myxobilatus sp., as observações ultra-estruturais não foram realizadas devido à baixa intensidade de infeção. Apesar disto, os procedimentos moleculares foram realizados para sequenciar os genes SSU rRNA, e as análises filogenéticas mostraram que ambas as espécies agruparam dentro da clade principal dos mixobolídeos: Myxobolus sp. foi agrupado na subclade dos perciformes, e Myxobilatus sp. foi agrupado na subclade do sistema excretor. Tendo contribuído com novos dados referentes a estes mixosporídeos, este estudo também forneceu novas informações sobre a fauna piscícola da Albufeira do Alto Rabagão, reportando a ocorrência de R. rutilus, L. gibbosus e S. lucioperca.
De uma forma geral, este estudo mostrou que explorar a ocorrência de espécies de mixosporídeos em novas áreas de estudo constitui um esforço importante para a construção de uma base que no futuro permitirá uma melhor compreensão da distribuição destes parasitas e patologias associadas.
LIST OF TABLES ... V LIST OF FIGURES... VII LIST OF ABBREVIATIONS ... IX C h a p t e r I 1. INTRODUCTION 1.1. MOTIVATION ... 3 1.2. GOALS... 4 1.3. STRUCTURE OF THESIS... 5 1.4. REFERENCES ... 6 C h a p t e r I I 2. STATE OF ART 2.1. MYXOSPOREANS ... 11 2.1.1. DISTRIBUTION ... 12 2.1.2. PATHOLOGIES ... 13 2.1.3. LIFE CYCLE ... 15
2.1.4. STRUCTURES AND MORPHOLOGY ... 17
2.1.5. POSITION AND TAXONOMIC CLASSIFICATION ... 19
2.1.6. MOLECULAR AND PHYLOGENETIC STUDIES ... 21
2.2. REFERENCES ... 23
C h a p t e r I I I 3. STUDY AREA 3.1. STUDY AREA... 33
C h a p t e r I V 4. MATERIALS AND METHODS
4.1. SAMPLED AREA ... 39
4.2. SAMPLING... 39
4.2.1. AQUACUTURE FISH ... 39
4.2.2. WILD FISH... 40
4.2.3. OLIGOCHAETES ... 42
4.3. PARASITOLOGICAL SURVEYS AND PARASITE COLLECTION ... 42
4.4. MORPHOLOGICAL ANALYSIS OF MYXOSPORES... 43
4.4.1. LIGHT MICROSCOPY ... 43
4.4.2. TRANSMISSION ELECTRON MICROSCOPY ... 44
4.5. MOLECULAR ANALYSIS... 45 4.5.1. DNA EXTRACTION ... 45 4.5.2. PCR AMPLIFICATION ... 45 4.5.3. ELECTROPHORESIS... 48 4.5.4. SEQUENCING ... 48 4.6. PHYLOGENETIC ANALYSIS ... 49 4.7. REFERENCES ... 50 C h a p t e r V 5. RESULTS 5.1. AQUACULTURE FISH... 53 5.1.1. PARASITOLOGICAL SURVEY... 53 5.2. WILD FISH... 53 5.2.1. PARASITOLOGICAL SURVEY... 53 5.2.2. MORPHOLOGICAL ANALYSIS ... 54 5.2.3. MOLECULAR ANALYSIS ... 62 5.3. OLIGOCHAETES... 67 5.3.1. PARASITOLOGICAL SURVEY... 67 5.4. REFERENCES ... 68
C h a p t e r V I 6. DISCUSSION
6.1. AQUACULTURE FISH... 71
6.2. WILD FISH ... 73
6.2.1. METODOLOGICAL CONSIDERATIONS ... 73
6.2.2 CAPTURED WILD FISH... 73
6.2.3. MYXOSPOREAN PARASITES ... 75 6.2.3.1. SPHAEROSPORID ... 75 6.2.3.2. MYXOBOLIDS ... 78 6.2.3.3. MYXOBILATID ... 82 6.3. OLIGOCHAETES... 84 6.4. REFERENCES ... 85 C h a p t e r V I I 7. CONCLUSIONS ... 93 C h a p t e r V I I I 8. APPENDIX 8.1. APPENDIX A ... 97 8.2. APPENDIX B ... 99 8.3. REFERENCES ... 115
Chapter IV – Materials and Methods Page
Table 1 Primers used in PCR amplifications. 45
Table 2 Pairs of primers used to amplify the SSU rRNA gene of each
myxosporean species. 46
Chapter V – Results
Table 3 Myxosporean species observed infecting wild fish from Alto Rabagão Reservoir.
53 Table 4 Pairwise distances in percentage between Myxobolus sp.
infecting Rutilus rutilus SSU rRNA sequence and 12 Myxobolus spp. sequences deposited in the GenBank.
64
Table 5 Pairwise distances in percentage between Myxobolus sp. infecting Lepomis gibbosus SSU rRNA sequence and 10 Myxobolus spp. deposited in the GenBank.
66
Chapter VIII – Appendix B
Table 6 Features of myxosporean parasites related to Oncorhynchus mykiss, site of infection and locality.
99
Table 7 Morphometric data and features of Sphaerospora spp. 100
Table 8 Morphometric data and features of Myxobolus pseudodispar. 107 Table 9 Morphometric data and features of Myxobolus spp. infecting
Rutilus rutilus.
109 Table 10 Morphometric data and features of Myxobolus spp. infecting
Lepomis gibbosus. 111
Table 11 Morphometric data and features of Myxobilatus spp. infecting Lepomis gibbosus.
Chapter II – State of Art Page Figure 1. Schematic drawing showing the life cycle of myxosporeans. 16 Figure 2. Schematic drawings of common myxospore morphotypes that
occur in freshwater environments.
18
Chapter III – Study Area
Figure 3. Maps illustrating the geography of the study area. 34
Chapter IV – Materials and Methods
Figure 4. Oncorhynchus mykiss grow-out cages in the company Quinta do Salmão – Comércio de Peixe, Lda. in ARR.
39 Figure 5. Trammel net casting in ARR aided by a boat and collaboration
of the company Quinta do Salmão – Comércio de Peixe, Lda. 40
Figure 6. Specimen of Rutilus rutilus from ARR. 41
Figure 7. Specimen of Lepomis gibbosus from ARR. 41
Figure 8. Specimen of Sander lucioperca from ARR. 41
Chapter V – Results
Figure 9. Light micrographs showing several free fresh mature myxospores of Myxobolus sp. infecting the spleen of Rutilus rutilus.
54
Figure 10. TEM micrographs of the Myxobolus sp. infecting the spleen of
Rutilus rutilus. 56
Figure 11. Light micrographs of the myxosporean parasites infecting
different tissues of Lepomis gibbosus. 58
Figure 12. TEM micrographs of Sphaerospora sp. from the kidney of
Lepomis gibbosus. 59
Figure 14. PCR products of myxobolids and myxobilatid. 63 Figure 15. Phylogenetic tree showing the relations of myxosporean
species found in the present study.
65
Chapter VIII – Appendix A
ARR Alto Rabagão Reservoir
BLAST Basic Local Alignment Search Tool
bp Base Pair
CPN Portuguese Freshwater Fish Database
DNA Deoxyribonucleic Acid
EPON Epoxy Resin Solution
GC Guanine-Cytosine
ICNF Instituto da Conservação da Natureza e das Florestas
Lda Limitada
LM Light Microscopy
LSU rRNA Large Subunit Ribosomal
ML Maximum Likelihood
NCBI National Center for Biotechnology Information
PCR Polymerase Chain Reaction
PGD Proliferative Gill Disease
SEM Scanning Electron Microscopy
SSU rRNA Small Subunit Ribosomal
TEM Transmission Electron Microscopy
1.1. M O T I V A T I O N
Myxosporeans are endoparasites commonly found in aquatic organisms (Kent et al., 2001; Lom and Dyková, 2006). Parasitological surveys have reported the occurrence of these parasites in several aquatic environments worldwide (Fiala et al., 2015). In most documented cases, myxosporean infections are apparently harmless, causing little damage to the fish host tissues (Gunter et al., 2009; Okamura et al., 2015; Rocha et al., 2015; Schmidt–Posthaus and Wahli, 2015). However, some are extremely pathogenic, causing high levels of morbidity and mortality in wild and cultivated fish populations (Rocha et al., 2015). The problematics associated with this class of parasites relate to its severe negative impact in the viability and sustainability of aquacultures and fisheries (Hutson et al., 2007), as well as in the aquatic ecosystems (Jones et al., 2015). Environmental parameters, such as temperature and precipitation rates, are also important factors influencing the emergence of parasitic diseases due to alterations in myxosporean–host dynamics (Ray et al., 2015).
Acknowledging the importance of myxosporean parasites, many studies have been developed in aquacultures and natural environments in order to describe new species, and to understand hosts–myxosporean relationships, life cycles, hosts infection sites, morphology and phylogenetic position, and to develop effective control methodologies (Alama–Bermejo et al., 2011; Azevedo et al., 2014; Gleeson and Adlard, 2012; Hemananda et al., 2009; Jones et al., 2012; Rangel et al., 2014; Rangel et al., 2015; Rocha et al., 2015; Shin et al., 2014; Yokoyama and Fukuda, 2001). Despite the increasing knowledge available, some subjects still lack information (Okamura et al., 2015). The threat that these parasites pose to economically relevant fish gives an additional incentive for studies aiming to fill in these gaps (Lom and Dyková, 2006; Okamura et al., 2015).
The motivation of the present master thesis dissertation is to contribute with new knowledge for this group of parasites in Portuguese waters, by performing parasitological surveys in wild and cultured fishes sampled from a study area that is strongly altered by human activities and that has never been addressed before. Bibliographic revision shows the importance to approach new study areas in order to face the lack of information about myxosporean species. Taking into account that numerous myxosporean distributions are undocumented (Bartholomew and Kerans,
2015), there are the necessity of a continued exploration in unsampled regions making known their distribution, dissemination and in parallel their specific diversity, the fish species used as hosts and infection sites.
In Portugal, the majority of studies performed in concern estuaries and marine coastal areas (Cruz et al., 2000; Rangel et al., 2015; Rocha et al., 2013; Rocha et al., 2015). Therefore, I had interest in developing my study in the countryside and in a freshwater artificial reservoir with socio–economic value, containing an aquaculture facility and ample recreational activities. As such, the aim of my work was to provide knowledge about present myxosporean diversity in Alto Rabagão Reservoir.
1.2. G O A L S
The main goal of this work is to explore myxosporean diversity in a new study area with the finality of increasing the knowledge regarding the parasites distribution, selection of hosts, and sites of infection. In order to identify and characterize the myxosporean species observed infecting fish hosts sampled from Alto Rabagão Reservoir, several tasks were proposed:
• To sample cultivated and wild fish, as well as oligochaetes from the reservoir sediment;
• To identify fish species infected by myxosporean parasites, and the sites of infection selected;
• To characterize and classify the myxosporean species found infecting the sampled fishes and oligochaetes using a combination of morphological, ultrastructural and molecular methodologies:
o Determination of myxosporean genera by morphological data using light
microscopy and description of myxosporean spores by ultrastructural data using transmission electron microscopy;
o To establish the position and phylogenetic relationships of the species
analyzed in comparison to myxosporeans already documented, with basis on molecular data obtained from ribosomal genes.
1.3. S T R U C T U R E O F T H E S I S
This master dissertation is divided into 8 chapters:
In Chapter I – Introduction, it is presented the motivation, main goals and
structure of this master thesis dissertation;
In Chapter II – State of Art, a brief review of literature is presented within
the framework of the class Myxosporea, and includes, general introduction, distribution, pathologies, life cycle, structures and morphology, classification and taxonomic position, molecular and phylogenetic studies;
In Chapter III – Study Area, the study area is described and georeferenced;
In Chapter IV – Materials and Methods, the materials and methods used
to reach the results are described. This chapter is subdivided in sampled area, studied fish sampling, parasitological surveys and parasite collections, morphologic analyses of myxospores, molecular and phylogenetic analyses;
In Chapter V – Results, the results obtained are presented;
In Chapter VI – Discussion, the results are discussed and compared with
other studies with the same subject;
In Chapter VII – Conclusion, a synthesis of the main findings are presented;
1.4. R E F E R E N C E S
Alama–Bermejo, G., Raga, J. A., & Holzer, A. S. (2011). Host–parasite relationship of Ceratomyxa puntazzi n. sp. (Myxozoa: Myxosporea) and sharpsnout seabream Diplodus puntazzo (Walbaum, 1792) from the Mediterranean with first data on ceratomyxid host specificity in sparids. Veterinary Parasitology, (182) 181– 192.
Azevedo, C., Rocha, S., Matos, P., Matos, E., Oliveira, E., Al–Quraishy, S., & Casal, G. (2014). Morphology and phylogeny of Henneguya jocu n. sp. (Myxosporea, Myxobolidae), infecting the gills of the marine fish Lutjanus jocu. European Journal of Protistology, (50) 185–193.
Bartholomew, J. L., & Kerans, B. (2015). Risk Assessments and Approaches for Evaluating Myxozoan Disease Impacts. In: B. Okamura, Gruhl, A. & Bartholomew, J. L. (Eds.), Myxozoan Evolution, Ecology and Development. Springer, pp. 379–395.
Cruz, C., Saraiva, A., & Ferreira, S. (2000). Preliminary observations on Myxobolus sp. from cyprinid fish in Portugal. Bulletin of the European Association of Fish Pathologists, (20) 65–69.
Fiala, I., Bartošová–Sojková, P., Okamura, B., & Hartikainen, H. (2015). Adaptive Radiation and Evolution Within the Myxozoa. In: B. Okamura, Gruhl, A. & Bartholomew, J. L. (Eds.), Myxozoan Evolution, Ecology and Development. Springer, pp. 69–84.
Gleeson, R. J., & Adlard, R. D. (2012). Phylogenetic relationships amongst
Chloromyxum Mingazzini, 1890 (Myxozoa: Myxosporea), and the description of six novel species from Australian elasmobranchs. Parasitology
International, (61) 267–274.
Gunter, N. L., Whipps, C. M., & Adlard, R. D. (2009). Ceratomyxa (Myxozoa:
Bivalvulida): Robust taxon or genus of convenience? International Journal for Parasitology, (39) 1395–1405.
Hemananda, T., Mohilal, N., Bandyopadhyay, P. K., & Mitra, A. K. (2009). Two new Myxosporidia (Myxozoa: Myxosporea) of the genus Myxobolus Butschli, 1882 from cornea of Clarias batrachus (Linnaeus, 1758) caught from a fish farm in India. North–Western Journal of Zoology, (5) 165–169.
Hutson, K. S., Ernst, I., & Whittington, I. D. (2007). Risk assessment for metazoan parasites of yellowtail kingfish Seriola lalandi (Perciformes: Carangidae) in South Australian sea–cage aquaculture. Aquaculture, (271) 85–99.
Jones, S. R. M., Bartholomew, J. L., & Zhang, J. Y. (2015). Mitigating Myxozoan Disease Impacts on Wild Fish Populations. In: B. Okamura, Gruhl, A. & Bartholomew, J. L. (Eds.), Myxozoan Evolution, Ecology and Development. Springer, pp. 397–413.
Jones, S. R. M., Forster, I., Liao, X., & Ikonomou, M. G. (2012). Dietary nicarbazin reduces prevalence and severity of Kudoa thyrsites (Myxosporea:
Multivalvulida) in Atlantic salmon Salmo salar post–smolts. Aquaculture 1–6.
Kent, M. L., Andree, K. B., Bartholomew, J. L., El‐Matbouli, M., Desser, S. S., Devlin, R.
H., Feist, S. W., Hedrick, R. P., Hoffmann, R. W., & Khattra, J. (2001). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic
Lom, J., & Dyková, I. (2006). Myxozoan genera: definition and notes on taxonomy, life–cycle terminology and pathogenic species. Folia Parasitologica, (53) 1–36. Okamura, B., Gruhl, A., & Bartholomew, J. L. (2015). An Introduction to Myxozoan
Evolution, Ecology and Development. In: B. Okamura, Gruhl, A. &
Bartholomew, J. L. (Eds.), Myxozoan Evolution, Ecology and Development. Springer, pp. 1–20.
Rangel, L. F., Rocha, S., Borkhanuddin, M. H., Cech, G., Castro, R., Casal, G., Azevedo, C., Severino, R., Székely, C., & Santos, M. J. (2014). Ortholinea auratae n. sp. (Myxozoa, Ortholineidae) infecting the urinary bladder of the gilthead seabream Sparus aurata (Teleostei, Sparidae), in a Portuguese fish farm. Parasitology Research, (113) 3427–3437.
Rangel, L. F., Rocha, S., Castro, R., Severino, R., Casal, G., Azevedo, C., Cavaleiro, F., & Santos, M. J. (2015). The life cycle of Ortholinea auratae (Myxozoa:
Ortholineidae) involves an actinospore of the triactinomyxon morphotype infecting a marine oligochaete. Parasitology Research 1–8.
Ray, R. A., Alexander, J. D., De Leenheer, P., & Bartholomew, J. L. (2015). Modeling the Effects of Climate Change on Disease Severity: A Case Study of
Ceratonova (syn Ceratomyxa) shasta in the Klamath River. In: B. Okamura, Gruhl, A. & Bartholomew, J. L. (Eds.), Myxozoan Evolution, Ecology and Development. Springer, pp. 363–378.
Rocha, S., Casal, G., Al–Quraishy, S., & Azevedo, C. (2013). Morphological and
molecular characterization of a new myxozoan species (Myxosporea) infecting the gall bladder of Raja clavata (Chondrichthyes), from the Portuguese
Atlantic coast. Journal of Parasitology, (99) 307–317.
Rocha, S., Casal, G., Rangel, L. F., Castro, R., Severino, R., Azevedo, C., & Santos, M. J. (2015). Ultrastructure and phylogeny of Ceratomyxa auratae n. sp.
(Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae). Parasitology International, (64) 305–313. Schmidt–Posthaus, H., & Wahli, T. (2015). Host and Environmental Influences on
Development of Disease. In: B. Okamura, Gruhl, A. & Bartholomew, J. L. (Eds.), Myxozoan Evolution, Ecology and Development. Springer, pp. 281–293.
Shin, S. P., Nguyen, V. G., Jeong, J. M., Jun, J. W., Kim, J. H., Han, J. E., Baeck, G. W., & Park, S. C. (2014). The phylogenetic study on Thelohanellus species
(Myxosporea) in relation to host specificity and infection site tropism. Molecular Phylogenetics and Evolution, (72) 31–34.
Yokoyama, H., & Fukuda, Y. (2001). Ceratomyxa seriolae n. sp. and C. buri n. sp. (Myxozoa: Myxosporea) from the gall–bladder of cultured yellowtail Seriola quinqueradiata. Systematic Parasitology, (48) 125–130.
2.1. M Y X O S P O R E A N S 3.
Fisheries and aquaculture industries provide fish products that are recognized by their high nutritional values for human consumption (Thilsted et al., 2016). According to World Bank (2009), the income resulting from the international trade of these products exceeds that of all other food products of animal origin. Consequently, many researchers have been exploring indicators for environmental management (Fezzardi et al., 2013), and determining strategies for the detection and control of threats, such as diseases, poor water quality, and parasitic infections that may undermine the viability of the sector (Fontes et al., 2015; Okamura et al., 2015). Given the current information available, myxosporean species are often protagonist of fish diseases, as they constitute the group of parasites that is most abundant in nature (Gómez et al., 2014). Parasitic infections may generate severe diseases causing histopathological damages that increase morbidity and mortality. According to the degree of their pathogenicity, some parasites are of concern to economical relevant fishes (Palenzuela et al., 1997; Rocha et al., 2015).
The class Myxosporea Bütschli, 1881 (phylum Myxozoa Grassé, 1970) is a diverse group of obligate endoparasites that are multicellular, microscopic and spore– forming (Gómez et al., 2014; Lom and Dyková, 2006; Okamura and Gruhl, 2015; Siddall et al., 1995). These parasites are commonly found infecting freshwater and marine fishes, aquatic annelids such as oligochaetes and polychaetes, and less frequently, mollusks (Yokoyama and Masuda, 2001), small mammals, amphibians, birds and reptiles (Lom and Dyková, 2006; Okamura et al., 2015).
Myxosporeans were first reported as potential threats for fish hosts in 1825, upon the observation of cysts proliferating in the musculature of Coregonus fera from Lake Geneva (Jurine, 1825). Since then, myxosporeans have become subject of interest and concern for many scientists (Mladineo et al., 2010), whose work aims to increase the amount of knowledge available for these parasites. Despite the already relatively large number of myxosporean species nowadays recognized, new species continue to emerge from both the freshwater and the marine environment. Currently, myxosporean identification is performed on the basis of biological, morphological and molecular data (Fiala et al., 2015b; Okamura et al., 2015). The increasing knowledge has allowed a better understanding of their biology, morphology,
evolution, myxosporean–hosts relationship and disease ecology (Okamura et al., 2015).
2.1.1. D I S T R I B U T I O N
Parasitological studies have reported the occurrence of myxosporeans from North and South America to Europe, Africa, Asia and Australia; the only exception being Antarctica (Hallett et al., 2015a). Myxosporeans are widely distributed in several marine (Bartošová–Sojková et al., 2015) and freshwater aquatic environments, including rivers (Fiala et al., 2015c), estuaries (Rocha et al., 2015), lakes (Székely et al., 2001), and reservoirs (Macconnell and Peterson, 1992). They have also been observed in non–natural and modified environments, infecting cultivated fish in
aquaculture facilities with intensive (Holzer et al., 2010; Sitjà‐Bobadilla and Alvarez‐
Pellitero, 2001), semi–intensive (Rocha et al., 2015; Seo et al., 2012) and semi offshore (Mladineo et al., 2010; Yokoyama and Fukuda, 2001) systems.
The dissemination of myxosporeans is associated with natural and anthropogenic factors. The natural dissemination relates to their life cycle, with both waterborne myxospore and actinospore stages dispersing in the water column, or as a result of the migration of the vertebrate host while carrying vegetative stages (Hallett et al., 2015b). Anthropogenic dissemination mainly relates to human activities, such as aquaculture and mariculture, fishing (both commercial and recreational), traffic of ornamental species, and transport of live and dead specimens (Okamura et al., 2015), being responsible for dispersal at a bigger geographic scale.
Studies focusing on this subject have mainly considered the myxosporean species displaying major impact in cultivated and wild fish populations (Hallett et al., 2015b). For instance, the pathogenic agent causing Whirling Disease (WD) in salmonids (Hoffman and Snieszko, 1970), Myxobolus cerebralis, which originated in Europe and was later introduced in North America, as well as in at least 25 other countries, as a direct consequence of human activities (Bartholomew and Reno, 2002). Other examples are Thelohanellus nikolskii, which spread from Asia to the center of the European continent, causing severe losses in the production of cyprinids, namely carps (Molnár, 2002), and Myxobolus pavlovskii, which was
introduced to Europe from Asian Hypophthalmichthys molitrix and H. nobilis fishes (Molnár, 1979).
2.1.2. P A T H O L O G I E S
The pathologies caused by myxosporean species often do not present external disease signs (Hallett et al., 2015b), or may be asymptomatic. In most cases, this is commonly associated with low severity infections (Schmidt–Posthaus and Wahli, 2015). However, some species are extremely pathogenic, causing serious diseases that damage the organs and affect growth, flesh quality and reproduction (Gómez et al., 2014), often leading to the fish death (Fontes et al., 2015).
The pathogenicity of the infectious agent can be related to several host and environmental factors. Schmidt–Posthaus and Wahli (2015) suggested that host strain, age, size, weight, gonadal maturation, and water temperature, eutrophication and nutrition may influence the pathogenicity of myxosporean infections. In fact, temperature has been shown to constitute one of the most relevant environmental factors influencing myxosporean development and pathogenicity. According to Baldwin et al. (2000), the pathological effects may be aggravated by temperature increase because it enhances the development of the parasite, while accelerating the fish immune system.
Several myxosporean species are recognized as agents capable to develop serious diseases in both cultured and wild economically important fish (Lom and Dyková, 2006). The most well documented pathogenic species infecting freshwater farmed and wild fish populations is M. cerebralis which causes serious pathologies in salmonids of North America, Europe, Asia and Far East (El–Matbouli et al., 1992). The development of this parasite in the host’s cartilage induces the disintegration of this tissue, leading to severe neural damages, and cranial and skeleton deformities (Markiw, 1992), thus reducing the fish marketability. The parasites Parvicapsula minibicornis and Ceratonova shasta are species native from North America that threaten salmonid conservation (Alexander et al., 2015). Parvicapsula minibicornis causes hyperplasia and austere inflammation of the gill lamellae, particularly in Oncorhynchus nerka (Bradford et al., 2010), while C. shasta causes inflammation of
the intestinal tissues (denominated as ceratomyxosis) of several salmonids; being that the severity of both these species has been correlated with water temperatures (Bartholomew, 1998). Another myxosporean species capable of causing significant pathological damages belongs to the genus Henneguya Thélohan, 1892 (Batueva et al., 2013). For instance, H. ictaluri causes Proliferative Gill Disease (PGD) in cultured Ictalurus punctatus, this being considered one of the most relevant diseases caused by myxosporeans (Griffin et al., 2010), with the hosts developing anorexia (Wise et al., 2004). In austere outbreaks of PGD, the mortalities of I. punctatus populations can surpass 50%. The parasite causes lesions in the gill filament cartilage, triggering a severe inflammatory response (Griffin et al., 2010; Lovy et al., 2011). The genus Thelohanellus Kudo, 1933, has attracted the attention of European pathologists due to the devastating diseases that it can cause in some species of cultured cyprinids from Central Europe (e.g. ornamental common carp industries). For instance, T. nikolskii causes visible external infections with several plasmodia in the cartilaginous fin rays of fry Cyprinus carpio resulting in moderate deformations (Molnár, 2002); while T. kitauei causes intestinal giant cystic disease, almost blocking the intestinal lumen (Egusa and Nakajima, 1981), as well as the formation of several large cysts in the skin (Zhai et al., 2016). Both theses pathogens species may kill their hosts and create considerable economic impacts in Asia (Zhai et al., 2016). Another genus that represents a concern for freshwater cultived and wild fish is Sphaerospora Thélohan, 1892, specially the species S. cristata, S. truttae, and S. dykovae [reviewed by El– Matbouli et al. (1992)]. The pathologies induced by these three species result from the serious histopathological damages that they cause in the kidney. Myxospores and other developmental stages of S. cristata have been reported from damaged renal corpuscles and glomeruli in Lota lota. Sphaerospora truttae severely affects Salmo trutta by causing disintegration of the glomerular capillaries, extensive vacuolization of the tubules’ epithelium and substantial infection of the Bowman’s capsules (El– Matbouli et al., 1992). In addition, Holzer et al. (2003) also reported S. truttae having negative impact in hatcheries of Salmo salar, with the parasite developing entirely within the renal tubules. Finally, S. dykovae has been recognized as a problematic pathogen in European intensive fish farms of carps (El–Matbouli et al., 1992), since it causes significant histological damages to the renal tubuli epithelial cells, as well as enlargement of the trunk kidney (Dyková and Lom, 1982).
The pathologies caused by myxosporean parasites have mainly been documented from infections in commercially valuable fish (Schmidt–Posthaus and Wahli, 2015). Consequently, the pathogenicity of many species remains unknown, not only due to the low economic value of its fish hosts (Gómez et al., 2014), but also to logistic and legal issues (Fontes et al., 2015).
2.1.3. L I F E C Y C L E
The first life cycle of Myxosporea was described by Wolf and Markiw (1984). The authors observed that Myxobolus cerebralis, the parasite causing WD in salmonid fish, infects oligochaete worms in order to complete its life cycle. Since then, several efforts have been performed to uncover the life cycle of myxosporean species, aiming to promote a better understanding of their biology (Okamura et al., 2015). There are about 2300 recognized species of myxosporeans, but only about 40 have their life cycle described (Morris, 2012; Rangel et al., 2015). The acquisition of information regarding transmission processes and myxosporean–host relationships is essential to fully understand the development and dissemination of myxosporean diseases.
Most myxosporeans have been proven to have an indirect life cycle (Morris, 2012) with two alternate hosts: an invertebrate – definitive host – and a vertebrate – intermediate host (Okamura et al., 2015). The vertebrate host is commonly a fish, marine or freshwater, and less frequently amphibians, reptiles, waterfowl and small mammals (Hallett et al., 2015a). The invertebrate host appears to differ according to the environment. While marine myxosporeans utilize polychaetes, freswaher myxosporeans utilizes oligochaetes (Alexander et al., 2015). On the other hand, some myxosporean species appear to have direct life cycles. For instance, parasites of the genus Enteromyxum Palenzuela, Redondo and Alvarez–Pellitero, 2002, appear to be transmissible fish–to–fish, requiring only vertebrate hosts (Eszterbauer et al., 2015).
Waterborne myxospores are released into the environment through the host decomposition (Hedrick et al., 2002) or through excretion and urination (Hallett et al., 2015a). In the sediment, they infect the annelid via ingestion. The parasite then releases its infective sporoplasms and develops intracellularly in the intestinal
epithelium or in the coelom cavity. The actinospores development and proliferation comprises three main stages: schizogony, gametogony and sporogony, ultimately producing waterborne actinospores (Eszterbauer et al., 2015). These are released into
Figure 1. Schematic drawing showing the life cycle of myxosporeans. A. Fish hosts B.
Waterborne myxospores C. Annelid hosts D. Waterborne actinospores
the environment through the digestive system or secretory pores in the host epidermis, and rise in the water column by osmotically inflating the valve cells in opposite directions (Fiala et al., 2015a; Kallert et al., 2015). When actinospores reach the fish host, their polar filaments are discharged, attaching to the skin, buccal cavity or gills epithelium (Kallert et al., 2015) (Figure 1). Simultaneously, the suture along the apical valves opens and creates a connection for the passage of the infectious agent – the sporoplasms. Within the fish host, the sporoplasms deteriorate, and the enclosed secondary cells are released, migrating to reach the specific organs or tissues of infection. Migration may take place through blood stages (for example, several Sphaerospora spp.) or occur intracellularly (Kallert et al., 2015). The parasite’s
development is coelozoic if it occurs within body and organ cavities, or histozoic if it develops intracellularly, or intercellularly between the host tissue cells (within plasmodia or pseudoplasmodia) (Lom and Dyková, 2006).
2.1.4. S T R U C T U R E S A N D M O R P H O L O G Y
Throughout their evolution, myxosporeans lost typical tissues and structures of the phylum Cnidaria, such as the gastrovascular system, in order to adapt to their endoparasitic lifestyle, as well as typical characteristics of Metazoa like nerve cells, sensory systems and epithelia (Foox and Siddall, 2015). Simplification, morphological differentiation among myxospores and actinospores, and size reduction are evolutionary traits that myxosporeans acquired in order to adapt to parasitism, to infect different hosts, and to successfully complete their complex life cycles (Atkinson et al., 2015). The production of distinct spores in the same life cycle shows significant morphological plasticity related with potential of transmission to their vertebrate and invertebrate hosts (Fiala et al., 2015a).
In order to fully characterize myxosporean species, and consequently define reliable taxonomic keys, the precise description of the morphological and biological structures of these microparasites is crucial (Fiala et al., 2015b). Morphologically, myxospores are constituted by two to several valves linked along a suture line, and enclosing one or more sporoplasms, and one to many polar capsules (Lom and Dykova, 1992). Morphological and structural characteristics used for myxosporean classification at the order and suborder level are the shape of the valves (elongated, fusiform, ellipsoidal, spherical, ovoid, elongated, and among others), the number of polar capsules and its arrangement to the sutural plane. In turn, the features allowing classification at the family–level are the presence or absence of caudal appendages, the particularities of the polar filament, and the shape of the suture line. At the species–level, the characteristics used include the dimensions of the myxospores and polar capsules, as well as other structural specificities, for example: striations of the myxospore valves, number of coils of the polar filament, and presence or absence of a mucous envelope (Fiala et al., 2015b; Lom and Dyková, 2006; Rocha et al., 2013). The sporoplasm may also provide some features that can be used as morphologic
criteria (number and organization of sporoplasm cells, for instance) (Feist et al., 2015), as in the case of the two main Sphaerospora sensu stricto lineages, which are distinguishable through the presence of one binucleate or 2–12 uninucleate sporoplasms (Bartošová et al., 2013).
These parasites are an economically important group with about 2300 species (Lom and Dyková, 2006) distributed among 60 genera, each of the latter corresponding to a different morphotype (Carriero et al., 2013; Fiala and Bartošová, 2010; Özer et al., 2016). The first most common myxospore morphotype is that of the genus Myxobolus Bütschli, 1882, which comprises over 900 histozoic species (Liu et al., 2013), and the second most common is that of the genus Henneguya, which comprehends around 198 species mainly infecting freshwater fish (Eiras, 2002; Eiras and Adriano, 2012). However, there are many other morphotypes considerably common, such as those of Thelohanellus, Chloromyxum Mingazzini, 1890, Myxidium Bütschli, 1882, Sphaerospora, Zschokkella Auerbach, 1910, and Sphaeromyxa Thélohan, 1892 (Fiala et al., 2015a; Lom and Dyková, 2006), as can be seen in the figure below (Figure 2). The rare morphotypes include, for example, Acauda Whipps, 2011, Myxobilatus Davis, 1944, and Agarella Dunkerly, 1915 (Lom and Dyková, 2006; Whipps, 2011).
Figure 2. Schematic drawings of common myxospore morphotypes that occur in freshwater
environments. A. Henneguya B. Sphaeromyxa C. Thelohanellus D. Chloromyxum E. Myxobolus
F. Myxidium G. Sphaerospora H. Zschokkella
Despite morphological characteristics being useful to classify myxosporeans, sometimes it is not possible to discriminate closely related species, such as
Myxobolus species, because the ambiguities of similar shared characteristics (Atkinson et al., 2015). Therefore, many researchers have combined other characteristics, for example infection sites, hosts and morphology of vegetative stages (Atkinson et al., 2015; Lom and Dyková, 2006), in order to get new criteria to identify and distinguish close myxosporean species. Even so, due to the simplicity of these parasites and difficulty in finding truly indicative features, most authors use a combination of microscopic and molecular resources. The optic microscope is used for measurements and external morphology, the transmission electron microscope (TEM) for ultrastructural analysis, and molecular procedures for the amplification and sequencing of gene markers, namely of the small subunit ribosomal DNA (SSU rRNA) gene. These methodologies are commonly used by several authors, for example Capodifoglio et al. (2016), Zatti et al. (2015), Rocha et al. (2015), Azevedo et al. (2014), Holzer et al. (2013b), and Alama–Bermejo et al. (2011). Less commonly, the scanning electron microscope (SEM) is also used in combination with other technics above referred for obtaining details on the external ultrastructure (Al–Jufaili et al., 2016; Kristmundsson and Freeman, 2014; Liu et al., 2014; Rocha et al., 2013).
Studies based on the SSU rRNA gene revealed discrepancies between molecular phylogeny and classification based on myxospore morphology. Thus, since the implementation of molecular procedures to the study of myxosporeans, several taxa have been suppressed, resurrected and re–established (Fiala et al., 2015b). For instance, Myxidium leei was reclassified within the genus Enteromyxum Palenzuela, Redondo and Alvarez–Pellitero, 2002. Nonetheless, the phylogenetic relationship between some genera remained consistent with phylogeny based on morphologic taxonomies, showing that the morphological characters adopted are taxonomically informative; it is the case of the genera Parvicapsula Shulman, 1953, Enteromyxum and Sphaeromyxa (Fiala, 2006; Nylund et al., 2005; Palenzuela et al., 2002).
2.1.5. P O S I T I O N A N D T A X O N O M I C C L A S S I F I C A T I O N
Myxozoans were reported for the first time by Jurine (1825). In 1881, Bütschli formalized the taxonomy of this parasitic group, assigning the phylum Myxosporidia within the Sporozoa Leuckart, 1879 [reviewed by Foox and Siddall (2015)] alongside a
diverse group of unicellular organisms and spore–forming parasites (Okamura and Gruhl, 2015). Considering that myxozoans present tubular mitochondrial cristae in some taxa, but lack cryptomitosis and centrioles, these organisms were classified as protists (Okamura and Gruhl, 2015). Years later, Štolc (1899) recognized the multicellular nature of actinosporeans (class Actinosporea, order Actinomyxidia), thus proposing the reclassification of myxozoans as Metazoans [reviewed by Foox and Siddall (2015)]. Moreover, Weill (1938) noted that the intracellular organelles presented by myxozoans (polar capsules) and by cnidarians (nematocysts) are homologous, leading to the suggestion that myxozoans could be cnidarians [reviewed by Okamura and Gruhl (2015)]. Despite several authors recognizing the multicellularity of its spores, myxozoans remained as protists for some time, simply because there was not enough information that could be used to associate them to another lineage (Foox and Siddall, 2015).
In 1984, Wolf and Markiw described the first myxozoan life cycle. These authors demonstrated that the life cycle of M. cerebralis (causative agent of WD) requires the parasite to develop and form spores of the actinosporean type within an annelid host, Tubifex tubifex, thus yielding two different spore types. Up until that point, myxozoans and actinosporeans belonged to different taxa of the class Cnidosporidia Doflein, 1901: Myxozoa and Actinosporea, respectively [reviewed by Okamura and Gruhl (2015)]. Thus, the evidence shown by these authors lead to the suppression of the Class Actinosporea, with its species becoming life–cycle counterparts of myxozoans (Okamura and Gruhl, 2015). The implementation of molecular procedures to the study of myxozoans, confirmed them as metazoans (Siddall et al., 1995; Smothers et al., 1994), further triggering the revision of its overall taxonomic setting (Foox and Siddall, 2015).
Since 1995, there is some controversy about the phylogenetic position of Myxozoa (Foox and Siddall, 2015), with two hypothesis emerging (Chang et al., 2015; Evans et al., 2010; Feng et al., 2014; Foox and Siddall, 2015). While Smothers et al. (1994) verified that myxozoans possibly were a sister–group to nematodes grouped within Bilateria, Siddall et al. (1995) verified that myxozoans possibly were a sister– group of Polypodium hydriforme grouped within the Cnidaria. Feng et al. (2014) corroborated the hypothesis of Myxozoa forming a sister–group to Medusozoa within
the Cnidaria, by showing the robust affinity between proto–mesodermal genes of Thelohanellus kitauei and Hydra magnipapillata.
Nowadays, the phylum Cnidarian is divided in three subphylums: Anthozoa, Medusozoa and Myxozoa (Okamura et al., 2015). The subphylum Myxozoa is composed by two classes containing over 2000 species: Myxosporea and Malacosporea (Lom and Dyková, 2006). The major difference between these classes is their definitive host and hardness of the valves. While for Malacosporea the definitive hosts are bryozoans and the spores have soft valves (Canning and Okamura, 2004), the definitive hosts of Myxosporea are annelids and the spores have hardened valves (Fiala et al., 2015a).
2.1.6. M O L E C U L A R A N D P H Y L O G E N E T I C S T U D I E S
The taxonomic classification of myxosporeans solely according to
morphological and structural features of myxospores has been shown to be artificial (Lom and Dyková, 2006), since molecular markers clearly reveal significant discrepancies between morphology and phylogeny (Atkinson et al., 2015). Recognizing this, many researchers now base their studies on the combination of morphological characteristics with information acquired from the sequencing of gene markers, such as the SSU rRNA gene, in order to infer phylogenetic relationships, analyze myxosporean lineages and discriminate species (Atkinson et al., 2015; Fiala et al., 2015b). Other molecular markers are the large subunit ribosomal RNA (LSU rRNA) gene, the elongation factor 2 (EF2 – it is less informative comparatively to rRNA markers) gene, the internal transcribed spacer region 1 (ITS–1 – allows to discriminate differences at the intraspecific level, and is also used in phylogeographical studies), and the internal transcribed spacer region 2 (ITS–2 – used together with ITS–1to uncover cryptic species of amphibian–infecting myxosporeans) [reviewed by Atkinson et al. (2015)]. Nonetheless, the SSU rRNA gene still remains the first choice when seeking myxozoan phylogeny, because there is more available data in the GenBank, being broadly used in phylogenetic studies and descriptions of myxosporean species [e.g. Özer et al. (2016), Liu et al. (2016), Capodifoglio et al. (2016), and Rocha et al. (2015)].
Phylogenetic studies of the class Myxosporea initially revealed the existence of two main clades dividing the group according to the aquatic habitat occupied by their hosts (marine or freshwater) (Fiala, 2006; Kent et al., 2001). Nowadays, three well– supported lineages are recognized, with basis on the SSU rRNA gene: a marine myxosporean lineage, a freshwater myxosporean lineage (Fiala et al., 2015b), and a third Sphaerospora sensu stricto lineage that occurs in both habitats above referred (Bartošová et al., 2013). Within each clade, other phylogenetic studies also suggest that myxosporean species of different genera cluster according to their site of infection (Holzer et al., 2004).
Fiala et al. (2015b) suggested the sub–division of the freshwater lineage into five sublineages: histozoic species of the family Myxobolidae; coelozoic species of the genera Sphaeromyxa and Zschokkella; species infecting the urinary system (including Chloromyxum, Hoferellus Berg, 1898, Ortholinea Shulman, 1962, Acauda, Zschokkella, Myxobilatus, and Myxidium genera); Myxidium lieberkuehni clade (includes species infecting the urinary tract of freshwater fish); and a Chloromyxum clade (includes Chloromyxum species). The Sphaerospora s. s. lineage is divided in two sublineages: sublineage A and sublineage B. The first comprises marine Sphaerospora spp., while the second relates to host group and habitat, consequently
splitting into three subclades: freshwater fish hosts, freshwater–brackish–
anadromous fish hosts, and brackish–fish hosts (Bartošová et al., 2013).
The use of phylogenetic analyses has been elucidating the evolution and the relationships of myxosporeans (Kent et al., 2000). These analyses have shown that myxospores do not cluster in monophyletic branches according to the morphology (Fiala, 2006; Holzer et al., 2004). Recognizing the artificiality of traditional taxonomic criteria to resolve the evolutionary events of myxosporean parasites, studies now resort to molecular data for the phylogenetic positioning of new species (Capodifoglio et al., 2016; Liu et al., 2016; Rocha et al., 2015), the identification of cryptic species assemblages (Bartošová and Fiala, 2011; Holzer et al., 2013a), as well as for the development of diagnostic tools to determine myxosporean infections (Nylund et al., 2005; St–Hilaire et al., 1997).
2.2. R E F E R E N C E S
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