INTRODUCTION
Sphaerospora dicentrarchi Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1992 is a systemic histozoic parasite of the European seabass, Dicentrarchus labrax (Linnaeus, 1758), and spotted seabass, Dicentrarchus punctatus (Bloch, 1792) (Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1992; Xavier et al. Reference Xavier, Severino, Pérez-Losada, Cable and Harris2013), found infecting the connective tissue of the majority of organs in its hosts, with a particular preference for the gallbladder and the intestine (Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1992). It causes chronic infections and losses in European seabass fish farm production, by making the host fish more susceptible to other pathogenic opportunistic infections (Fioravanti et al. Reference Fioravanti, Caffara, Florio, Gustinelli and Marcer2004). This parasite has a wide distribution in the Mediterranean Sea and the Iberia Peninsula, with high prevalence of infection (38–100%) in the European seabass (Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1992, Reference Sitjà-Bobadilla and Álvarez-Pellitero1993; Santos, Reference Santos1998; Mladineo, Reference Mladineo2003; Fioravanti et al. Reference Fioravanti, Caffara, Florio, Gustinelli and Marcer2004, Reference Fioravanti, Caffara, Florio, Gustinelli and Marcer2006; Merella et al. Reference Merella, Garippa and Salati2006; Mladineo et al. Reference Mladineo, Petrić, Šegvić and Dobričić2010).
Sphaerospora dicentrarchi is a member of the class Myxosporea Bütschli, 1881, a group of microscopic endoparasites whose known life cycles involve alternation between vertebrates, mainly a fish, and invertebrates, mainly an annelid (Lom and Dyková, Reference Lom and Dyková2006; Morris, Reference Morris2012; Yokoyama et al. Reference Yokoyama, Grabner, Shirakashi and Carvalho2012). There is still a remarkable lack of knowledge on the life cycles of the majority of the myxosporean species described, especially of those inhabiting the marine environment, where they are known only for seven species (Køie et al. Reference Køie, Whipps and Kent2004, Reference Køie, Karlsbakk and Nylund2007, Reference Køie, Karlsbakk and Nylund2008, Reference Køie, Karlsbakk, Einen and Nylund2013; Rangel et al. Reference Rangel, Santos, Cech and Székely2009, Reference Rangel, Rocha, Castro, Severino, Casal, Azevedo, Cavaleiro and Santos2015b; Karlsbakk and Køie, Reference Karlsbakk and Køie2012). For the freshwater species, the most usual invertebrate host is an oligochaete (Lom and Dyková, Reference Lom and Dyková2006; Yokoyama et al. Reference Yokoyama, Grabner, Shirakashi and Carvalho2012; Székely et al. Reference Székely, Borkhanuddin, Cech, Kelemen and Molnár2014), but in the marine environment the polychaetes have been reported as invertebrate hosts. The infections by actinospores can be found in marine polychaetes of the families Sabellidae (Køie et al. Reference Køie, Karlsbakk and Nylund2008), Nereididae (Køie, Reference Køie2000; Rangel et al. Reference Rangel, Santos, Cech and Székely2009; Karlsbakk and Køie, Reference Karlsbakk and Køie2012), Serpulidae (Køie, Reference Køie2002; Køie et al. Reference Køie, Karlsbakk and Nylund2007, Reference Køie, Karlsbakk, Einen and Nylund2013), Onuphidae (Rangel et al. Reference Rangel, Cech, Székely and Santos2011) and Capitellidae (Rangel et al. Reference Rangel, Rocha, Castro, Severino, Casal and Santos2015a). The most frequent actinospores in polychaetes, found until now, are tetractinomyxons. The two exceptions are the unicapsulactinomyxon (Rangel et al. Reference Rangel, Cech, Székely and Santos2011) and the echinactinomyxon (Rangel et al. Reference Rangel, Rocha, Castro, Severino, Casal and Santos2015a) actinospores.
Because the annelid form is considered the most vulnerable comprehensive knowledge of the identity and habitat requirements of the annelid host may offer a better management avenue for controlling disease in fish farms, potentially preventing economic losses. In the present study, actinospore stages genetically consistent with S. dicentrarchi were found in a capitellid polychaete invertebrate host.
MATERIAL AND METHODS
Polychaetes were sampled monthly and surveyed for actinospores from February 2013 to March 2014 in two different regions of Portugal. One survey was conducted in a fish farm located in the south of the country, at the Alvor Estuary, Atlantic coast (37°8′N, 8°37′W), Portimão, Algarve, in which polychaetes were collected from the mud of ponds, producing European seabass and gilthead seabream, Sparus aurata (Linnaeus, 1758), as well as from the water outlet ditches, and from an area of the Alvor Estuary that's located downstream to the fish farm. The other survey was conducted about 400 km north in a wild environment, in the Aveiro Estuary, Atlantic coast (40°40′N, 8°45′W). In both sample locations, the water salinity was about 35‰. In estuaries, the mud was collected during the low tide, and in the fish farm tanks the mud was collected either from recently empty tanks or with the help of a core tube in water filled tanks. The mud was brought to the laboratory and the polychaetes were collected by hand with the aid of plastic pipettes from the mud spread over dissection plates. Sphaerospora dicentrarchi myxospores were obtained from cultured European seabass in the Algarve fish farm for morphological study, and from wild seabass specimens (caught in the Aveiro Estuary by professional anglers) for molecular study.
During the parasitological survey, the polychaete worms were examined by cutting off a piece of their posterior body segments and squashed on a microscopy slide. The coelomic fluid and the tissues were screened for myxosporean parasites under an optical microscope with a 200–400× magnification. Specimens of European seabass were dissected and their gallbladder and intestine were screened for S. dicentrarchi.
Free actinospores and myxospores were examined and photographed using a Zeiss Axiophot microscope (Grupo Taper, Sintra, Portugal) with Nomarski differential interference contrast, equipped with a Zeiss AxioCam Icc3 digital camera. AxioVision 4.6.3 software (Grupo Taper) was used for the image analysis. Morphology and morphometry were obtained from fresh material, in accordance to Lom and Arthur (Reference Lom and Arthur1989), Lom et al. (Reference Lom, McGeorge, Feist, Morris and Adams1997) and Rangel et al. (Reference Rangel, Santos, Cech and Székely2009). All measurements included mean values, ±standard deviation (s.d.) and range.
For the molecular analysis, myxospores, actinospores and infected polychaetes were preserved in absolute ethanol. Genomic DNA was extracted using a GenElute™ Mammalian Genomic DNA Miniprep Kit, following the manufacturer's instructions. The parasites’ SSU ribosomal RNA (rRNA) gene was amplified using both universal primers and myxosporean-specific primers (Table 1). For the polychaete host DNA, the universal primers 16sar-L and 16sbr-H (Palumbi et al. Reference Palumbi, Martin, Romano, McMillan, Stice and Grabowski2002) were used (Table 1). PCRs were performed in 50 µL reactions using 0.01 pm of each primer, 10 mm of each dNTP, 2·5 mm MgCl2, 5 µL of 10× Taq polymerase buffer (Finnzymes), 1·5 units of Taq DNA polymerase and approximately 100−150 ng of genomic DNA. The reactions were run on a Hybaid P × E Thermocycler, with initial denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 53 °C for 45 s and 72 °C for 90 s. The final elongation step was performed at 72 °C for 7 min. Five-μL aliquots of the PCR products were electrophoresed through a 1% agarose 1× tris-acetate-EDTA buffer gel stained with ethidium bromide. The sequencing reactions were performed using a BigDye Terminator v1.1 from the Applied Biosystems Kit, and were run on an ABI3700 DNA analyser (Perkin-Elmer, Applied Biosystems).
Primers used for DNA amplification and sequencing the host Capitella sp. and the myxozoa Sphaerospora dicentrarchi
RESULTS
A total of 4275 and 1667 polychaetes worms were examined from the Algarve fish farm and the Aveiro Estuary, respectively. They amounted to about 40 different species grouped in 21 polychaete families. From the Aveiro Estuary, several nereidids Hediste diversicolor (O.F. Müller, 1776) were infect with tetractinomyxon stages consistent with Ellipsomyxa mugilis (Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1993) actinospores. From the Algarve fish farm samples, one capitellid worm was infected by multiple actinospore stages in the coelomic cavity (Fig. 1), showing no external signs of the infection. The actinospores were morphologically identified as belonging to the tetractinomyxon collective group.
Photomicrographs of developmental stages and free actinospores of Sphaerospora dicentrarchi in the coelomic cavity of the capitellid polychaete Capitella sp., from fresh material. (A) Initial binucleated rounded cell. (B) Initial pansporocyst with two internal cells (arrow). (C) Pansporocyst in the gametogamy phase with several inner cells. (D) Set of four spores in different stages of development, released from burst pansporocysts; (E) Pansporocyst in the sporogony stage with eight spores inside. (F) Set of four mature free spores, two of them showing an extension of the valvogenic cells (right side) and other two without it (left side). Scale bars = 10 µm.
Considering only the capitellids worms, 916 and 121 worms were examined from de Algarve fish farm and the Aveiro Estuary, respectively. The infected worm was found only in the sample of capitellids from European seabass ponds, in March 2014. Considering only this location, one worm was infected in 520 (0·2% of prevalence of infection). While considering the entire sample of capitellids from the Algarve fish farm, one worm was infected in 916 (0·1%). From Aveiro Estuary none of the 121 capitellid specimens collected were infected.
The tetractinomyxons were found in a polychaete identified as belonging to the family Capitellidae by morphology. After a BLAST search, the 18S rRNA sequence obtained for the polychaete host (GenBank accession number KT970640) presented a similarity percentage of 99% to Capitella sp. (FN421417) and 98% to Capitella teleta Blake et al. Reference Blake, Grassle and Eckelbarger2009 (U67323) and Capitella capitata (Fabricius, 1780) (JF509728). The 16S rRNA sequence (KT970641) presented a similarity percentage of 91% to C. teleta (JF509722). The other two species do not have 16S rRNA sequences deposited in the GenBank, while the remaining polychaetes with sequences presented a similarity below 80%.
The tetractinomyxons were found developing inside pansporocysts in groups of eight (Fig. 1). The free spores were pyriform with three apical rounded polar capsules (Figs 1F and 2). Spores (n = 12) measured 11·1 ± 0·7 (10·2–12·4) μm in length and 7·2 ± 0·4 (6·9–8·0) μm in width. The spore body measured 6·9 ± 0·2 (6·5–7·1) μm in length. The polar capsules measured 2·4 ± 0·3 (2·0–3·0) μm in diameter. The polar filaments and the secondary cells in the sporoplasms were not visible. Many spores had a small extension of the valvogenic cells while this character was absent in others (Figs 1F and 2).
Schematic illustration of the inferred life cycle of Sphaerospora dicentrarchi.
The myxospores of S. dicentrarchi (n = 14) from European seabass of the Algarve fish farm measured 4·9 ± 0·2 (4·5–5·4) μm in length, 3·4 ± 0·2 (3·1–3·6) μm in width, and 5·3 ± 0·3 (4·9–5·8) μm in thickness. The pyriform polar capsules measured 1·8 ± 0·2 (1·4–2·1) μm in length and 1·3 ± 0·1 (1·1–1·5) μm in width (Fig. 3).
Graphical comparison of the morphometrics of Sphaerospora dicentrarchi myxospores from different sampling locations. Traces = range; Bullets = mean value. Measures in μm (References for Spain: Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1992; Italy: Mladineo, Reference Mladineo2003; Aveiro: Santos, Reference Santos1998; Algarve: Present study).
The sequencing of the 18S rRNA gene of S. dicentrarchi from the myxospores collected from European seabass of the Aveiro Estuary, and from the capitellid tetractinomyxons collected in the Algarve fish farm resulted in a sequence with 1666 bp (KT970638) and 1672 bp (KT970639) respectively. They differ only in one nucleotide position. Taking as reference the myxospore DNA sequence, the nucleotides differ in the position 1448 where a guanine is replaced by an adenine in the actinospore DNA sequence, so both sequences were 99·9% similar. In the GenBank there are three sequences for S. dicentrachi. One sequence from Italy (AY278564) unpublished, with 844 bp, incorporates many errors in the sequence 5′ and 3′ extremities, which make it unreliable for comparison purposes. The other two sequences, from D. labrax (KC516864) and D. punctatus (KC516865) were obtained in the same Algarve fish farm. These two later sequences are smaller (569 bp for both) compared with the sequences of DNA from both myxospore (1666 bp) and actinospore (1672 bp) from this study, and the portion where they align were 100% identical.
DISCUSSION
This study identifies a new marine tetractinomyxon actinospore from a capitellid polychaete, Capitella sp., and presents molecular data which allow inferring a life cycle match between these actinospores stages and the myxospores of S. dicentrarchi. This is the seventh inferred marine life cycle involving polychaetes and this type of actinospore.
From the nine different tetractinomyxon actinospores described in the literature, the pyriform actinospore of S. dicentrarchi is morphologically more similar to E. mugilis (Rangel et al. Reference Rangel, Santos, Cech and Székely2009) and Ellipsomyxa gobii Køie, 2003 (Køie, Reference Køie2000; Køie et al. Reference Køie, Whipps and Kent2004), but with a smaller size in all characters. The others tetractinomyxon morphotypes described have a tetrahedral form (Ikeda, Reference Ikeda1912; Bartholomew et al. Reference Bartholomew, Whipple, Stevens and Fryer1997) or are more spherical or subspherical (Køie, Reference Køie2002; Bartholomew et al. Reference Bartholomew, Atkinson and Hallett2006; Køie et al. Reference Køie, Karlsbakk and Nylund2007, Reference Køie, Karlsbakk, Einen and Nylund2013; Karlsbakk and Køie, Reference Karlsbakk and Køie2012).
All measures of the myxospores of S. dicentrarchi are within the range of the original description (Fig. 3), with the exception of the spore width, which has some variation, but does not diverge significantly. Width and thickness measurements are prone to some variation, according to the spore orientation, especially in these very small spores.
Some doubts were raised about the validity of the S. dicentrarchi 18S rRNA gene sequence deposited in GenBank (AY278564) from Italy (Morris and Adams, Reference Morris and Adams2008), especially because it clustered with the Kudoa species (Eszterbauer and Székely, Reference Eszterbauer and Székely2004; Fiala, Reference Fiala2006), but a posterior study which analysed the 28S rRNA gene and the combined 18S + 28S rRNA data, confirmed the phylogenetic position of S. dicentrarchi (Bartošová et al. Reference Bartošová, Fiala and Hypša2009, Reference Bartošová, Freeman, Yokoyama, Caffara and Fiala2011). A more recent study, which included the 18S and the 28S rRNA genes, also reconfirmed the 18S rRNA sequence for S. dicentrarchi in two different hosts, D. labrax and D. punctatus, from Algarve (Xavier et al. Reference Xavier, Severino, Pérez-Losada, Cable and Harris2013). In the present study, a more extended 18S rRNA sequence for S. dicentrarchi was obtained from spores of both vertebrate and invertebrate hosts, strengthening even more the validity of the molecular data.
Discussion about the position of this species in the myxozoan phylogeny are common in recent phylogenetic studies, as they all place consistently S. dicentrarchi among the Kudoa species clade (Diamant et al. Reference Diamant, Ucko, Paperna, Colorni and Lipshitz2005; Morris and Adams, Reference Morris and Adams2008; Bartošová et al. Reference Bartošová, Fiala and Hypša2009, Reference Bartošová, Freeman, Yokoyama, Caffara and Fiala2011). This led several authors to question the position of S. dicentrarchi among the Sphaerospora (Diamant et al. Reference Diamant, Ucko, Paperna, Colorni and Lipshitz2005; Fiala, Reference Fiala2006; Bartošová et al. Reference Bartošová, Freeman, Yokoyama, Caffara and Fiala2011) due mainly to the unique characters that differentiate them from other Sphaerospora species, like very small spores with binucleate sporoplasm in a histozoic ‘bag-like’ polysporic plasmodia and the ultrastructural detail of overlapping shell valves (Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1992) which is more typical in Kudoa species (Dyková et al. Reference Dyková, Buron, Fiala and Roumillat2009). Therefore, these authors suggested that S. dicentrarchi may be a two-valved Kudoa species.
Sphaerospora dicentrarchi is phylogenetically distant from Sphaerospora species sensu stricto (Bartošová et al. Reference Bartošová, Freeman, Yokoyama, Caffara and Fiala2011), so we cannot generalize that the now inferred life cycle of S. dicentrarchi involving polychaetes and tetractinomyxon actinospores to those other species of Sphaerospora. On the other hand, the close affinity of this species to the Kudoa marine clade (Fiala, Reference Fiala2006) suggests that polychaetes may be candidate hosts for Kudoa species.
The cosmopolitan worm species, C. capitata, has been found to consist of several distinct cryptic species (Grassle and Grassle, Reference Grassle and Grassle1976; Méndez et al. Reference Méndez, Linke-Gamenick and Forbes2000; Blake, Reference Blake2009; Blake et al. Reference Blake, Grassle and Eckelbarger2009). Among them, we find the species C. teleta, recently described by Blake et al. (Reference Blake, Grassle and Eckelbarger2009). These worms are considered to be a group of opportunistic species, inhabiting organically enriched sediment (Tsutsumi, Reference Tsutsumi1990; Tsutsumi et al. Reference Tsutsumi, Fukunaga, Fujita and Sumida1990), like the ones we find in fish farm ponds. The worm found to be infected with S. dicentrarchi in this study, was a Capitella species, most probably belonging to this Capitella species complex. So, for the land fish farm ponds where European seabass are reared, it is natural to find this worm host, possibly perpetuating the S. dicentrarchi infection. Also, this means that estuaries are good environments for these species, and since wild European seabass use this environment for reproduction (Costa, Reference Costa1988; Laffaille et al. Reference Laffaille, Lefeuvre, Schricke and Feunteun2001; Martinho et al. Reference Martinho, Leitão, Neto, Cabral, Lagardère and Pardal2008) both hosts can be easily found in the same location.
The high occurrence of infection of S. dicentrarchi in the fish host (Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1992, Reference Sitjà-Bobadilla and Álvarez-Pellitero1993; Santos, Reference Santos1998; Mladineo, Reference Mladineo2003; Fioravanti et al. Reference Fioravanti, Caffara, Florio, Gustinelli and Marcer2004, Reference Fioravanti, Caffara, Florio, Gustinelli and Marcer2006; Merella et al. Reference Merella, Garippa and Salati2006; Mladineo et al. Reference Mladineo, Petrić, Šegvić and Dobričić2010) contrast with the low prevalence of infection in the invertebrate host. However, this difference of prevalences between the vertebrate and the invertebrate hosts is common to the other myxosporean species. For instance, E. mugilis can reach a level of infection of 70% in mugilids fishes (Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1993); however, only 0·5% of the polychaete worms are infected (Rangel et al. Reference Rangel, Santos, Cech and Székely2009). Generally, prevalence of infection of actinospores in the invertebrate hosts is very low, ranging from 0·1 to 4% (Yokoyama et al. Reference Yokoyama, Grabner, Shirakashi and Carvalho2012). Polychaetes in particular, have prevalences more variable, ranging from 0·3 to 17% (Rangel et al. Reference Rangel, Cech, Székely and Santos2011; Karlsbakk and Køie, Reference Karlsbakk and Køie2012), but they are more frequently about 1–8% (Køie et al. Reference Køie, Whipps and Kent2004, Reference Køie, Karlsbakk and Nylund2008, Reference Køie, Karlsbakk, Einen and Nylund2013; Køie, Reference Køie2005; Rangel et al. Reference Rangel, Santos, Cech and Székely2009, Reference Rangel, Rocha, Castro, Severino, Casal and Santos2015a). Nevertheless, because the tetractinomyxons in this study were found in a host belonging to a species complex, the prevalence of infection may also be underestimated, since not all Capitella species in this species complex may serve as host for these actinospores.
Sphaerospora dicentrarchi tetractinomyxons develop in the polychaetes coelomic cavity. This is the most typical site of infection in the invertebrate host for this type of actinospore (Ikeda, Reference Ikeda1912; Køie, Reference Køie2000, Reference Køie2002; Bartholomew et al. Reference Bartholomew, Atkinson and Hallett2006; Rangel et al. Reference Rangel, Santos, Cech and Székely2009; Karlsbakk and Køie, Reference Karlsbakk and Køie2012; Køie et al. Reference Køie, Karlsbakk, Einen and Nylund2013), with the exception of Ceratonova shasta (Noble, 1950) tetractinomyxons which develops in the epidermal tissue (Bartholomew et al. Reference Bartholomew, Whipple, Stevens and Fryer1997). However, the infection was underestimated, since only the posterior part of the worms was surveyed for parasite infection.
It is not clear how the coelozoic tetractinomyxons are released from the invertebrate coelomic cavity to infect the vertebrate host. Several processes have been suggested (Ikeda, Reference Ikeda1912; Køie, Reference Køie2000, Reference Køie2002; Bartholomew et al. Reference Bartholomew, Atkinson and Hallett2006; Rangel et al. Reference Rangel, Santos, Cech and Székely2009). One possible way is through the pores in the invertebrate tegument, like the gonopores (Køie, Reference Køie2002; Bartholomew et al. Reference Bartholomew, Atkinson and Hallett2006) or the nephridiopores (Ikeda, Reference Ikeda1912). One other possible way is the release of the actinospores after the death of the worm host either for reproduction (Rangel et al. Reference Rangel, Santos, Cech and Székely2009) or by natural death (Ikeda, Reference Ikeda1912; Køie, Reference Køie2000). Finally, it was also suggested that the vertebrate host may be infected by ingesting infected worms (Køie, Reference Køie2000). The tetractinomyxons of S. dicentrarchi are smaller compared with the spores of E. gobii (Køie, Reference Køie2000) and E. mugilis (Rangel et al. Reference Rangel, Santos, Cech and Székely2009) and have a closer size to other tetractinomyxons with a tethraedric or spherical form. Their smaller size turns them more suitable for gonopore or nephridiopore release mechanisms. The Capitella species complex worms in spite of having a short lifespan, approximately 1 year (Warren, Reference Warren1976; Martin and Bastida, Reference Martin and Bastida2006), reproduce several times during the year (Warren, Reference Warren1976; Martin and Grémare, Reference Martin and Grémare1997; Martin and Bastida, Reference Martin and Bastida2006) which can facilitate the exit of tetractinomyxons through the polychaete gonopores. Another possible mechanism of infection for S. dicentrarchi can be the ingestion of infected worms. It was suggested that S. dicentrarchi can infect young fish orally, first infecting hematopoietic organs and then spread via the bloodstream and became a chronic infection in older fishes (Sitjà-Bobadilla and Álvarez-Pellitero, Reference Sitjà-Bobadilla and Álvarez-Pellitero1993). The crustaceans are the main food items for juvenile European seabass, while annelids are a very small portion on their diet (Pickett and Pawson, Reference Pickett and Pawson1994; Cabral and Costa, Reference Cabral and Costa2001; Laffaille et al. Reference Laffaille, Lefeuvre, Schricke and Feunteun2001; Hampel et al. Reference Hampel, Cattrijsse and Elliott2005). However, in some localities the annelids, mainly polychaetes like capitellids and nereidids can be important (Pickett and Pawson, Reference Pickett and Pawson1994; Hampel et al. Reference Hampel, Cattrijsse and Elliott2005; Martinho et al. Reference Martinho, Leitão, Neto, Cabral, Lagardère and Pardal2008), due to the opportunistic character of this fish (Pickett and Pawson, Reference Pickett and Pawson1994). In adult European seabass, the use of annelids as prey is considered negligible (Spitz et al. Reference Spitz, Chouvelon, Cardinaud, Kostecki and Lorance2013).
Diamant (Reference Diamant1997) suggested that in the marine environment, fish-to-fish transmission of myxosporean parasites could be the model, nevertheless the recent findings of actinospores matching genetically with fish myxospores (Køie et al. Reference Køie, Whipps and Kent2004, Reference Køie, Karlsbakk and Nylund2007, Reference Køie, Karlsbakk and Nylund2008, Reference Køie, Karlsbakk, Einen and Nylund2013; Rangel et al. Reference Rangel, Santos, Cech and Székely2009, Reference Rangel, Rocha, Castro, Severino, Casal, Azevedo, Cavaleiro and Santos2015b; Karlsbakk and Køie, Reference Karlsbakk and Køie2012) and the present data allow us to infer that two host life cycles may also be an important mechanism of transmission for marine myxosporeans. However, there are many thousands of marine myxosporeans species known so far and the life cycles have only been inferred for fewer than 10 species.
ACKNOWLEDGEMENTS
The authors are grateful to the anonymous reviewers whose suggestions helped to improve the original version of this paper.
FINANCIAL SUPPORT
This research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE – Operational Competitiveness Programme and national funds through FCT – Foundation for Science and Technology, under the project ‘PEst-C/MAR/LA0015/2013’ and the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT-PTDC/MAR/116838/2010, both to M. J. S., and the Ph.D. fellowship grant SFRH/BD/82237/2011 attributed to L. Rangel and the Ph.D. fellowship grant SFRH/BD/92661/2013 attributed to S. Rocha through the programme POPH/FSE QREN, and the fellowship grants attributed to F. Cavaleiro and R. Castro through the project DIRDAMyx, reference FCOMP-01-0124-FEDER-020726/FCT-PTDC/MAR/116838/2010. This work complies with the current laws of the country where it was performed.
