MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 105(6) September 2010
PAGES: 762-769 DOI: 10.1590/S0074-02762010000600006 Full paper
Phylogeny, ultrastructure, histopathology and prevalence ofMyxobolus oliveirai sp. nov., a parasite of Brycon hilarii (Characidae) in the Pantanal wetland, Brazil

Tiago MilaninI; Jorge C EirasII; Sarah AranaIII; Antônio AM MaiaIV; Anderson L AlvesV; Márcia RM SilvaIV; Mateus M CarrieroIV; Paulo S CeccarelliVI; Edson A AdrianoVII, +

IDepartamento de Biologia Animal
IIDepartamento de Biologia e Centro Interdisciplinar de Investigação Marinha e Ambiental, Faculdade de Ciências, Universidade do Porto, Porto, Porto, Portugal
IIIDepartamento de Histologia e Embriologia, Universidade Estadual de Campinas, Campinas, SP, Brasil
IVDepartamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brasil
VDepartamento de Biologia, Universidade Estadual Paulista Julio de Mesquita Filho, Rio Claro, SP, Brasil
VICentro Nacional de Pesquisa e Conservação de Peixes Continentais, Instituto Chico Mendes de Conservação da Biodiversidade, Pirassununga, SP, Brasil
VIIDepartamento de Ciências Biológicas, Universidade Federal de São Paulo, Rua Prof. Artur Riedel 275, 09972-270 Jardim Eldorado, Diadema, SP, Brasil

Abstract

This paper presents the morphological, histological and ultrastructural characteristics of Myxobolus oliveirai sp. nov., a parasite of the gill filaments in Brycon hilarii from the Brazilian Pantanal. Out of 216 B. hilariispecimens examined (126 wild and 90 cultivated), 38.1% of wild specimens (n = 48) were infected. The parasites form elongated plasmodia primarily in the tip of gill filaments, reaching about 3 mm in length. A thorough comparison with all the Myxobolus species described from South American hosts, as well as nearly all the Myxobolus species described so far is provided. Partial sequencing of the 18S rDNA gene revealed a total of 1,527 bp. The Myxobolus species parasite of B. hilarii did not match any of the Myxozoa available in GenBank. In the phylogenetic analysis, M. oliveirai sp. nov. composed a monophyletic group with eight other species: five species of Myxobolus parasites of mugilid fishes, two parasites of pangasiid and one of centrarchid. Infection prevalence values of the parasite revealed no significant differences between wet and dry seasons or between males and females. The importance of the infection to the farming of the host species is emphasized.

Brycon hilarii (Valenciennes, 1950) (Syn. Brycon microlepis Perugia, 1897) is an endemic characid species of the upper Paraguay Basin (Resende 2003), popularly known as piraputanga. It is highly appreciated in central Brazil (especially in the states of Mato Grosso and Mato Grosso do Sul) (Resende 2003) and is a very valuable species, fetching high market prices. B. hilariihas recently been introduced into aquaculture in several regions of Brazil, with a production of 633,000 kg on fish farms in 2006 (IBAMA 2008), demonstrating the considerable potential of this species to this activity.

Myxosporean parasites are among the most important fish pathogens (Schmahl et al. 1989) and more than 2,300 species have been reported to infect fish (marine and/or freshwater fish) in either their natural environment or on farms (Adriano et al. 2006, 2009a, b, Feist & Longshaw 2006, Eiras et al. 2008, Azevedo et al. 2009). The Myxobolus Butschli, 1882, is the genus with the greatest number of species (approximately 790 valid species) (Eiras et al. 2005, Lom & Dyková 2006) and many of these species are reported as pathogenic to fish (Kent et al. 2001, Feist & Longshaw 2006). In South America, 30 Myxobolus species have been reported (Azevedo et al. 2010, Eiras et al. 2010), but none have been reported to infect fish from the genus Brycon.

As part of ongoing research on the characteristics of myxosporea parasites of freshwater fish in Brazil, the present paper describes a new species of Myxobolus found to infect wild specimens of piraputanga in the Brazilian Pantanal wetland using morphological, histological, ultrastructural and molecular phylogeny analyses.

 

MATERIALS AND METHODS

A total of 126 specimens of wild B. hilarii caught in the Pantanal Mato-Grossense in central Brazil and 90 farmed specimens from three fish farms in the state of São Paulo (SP) were examined. The wild fish were examined in two seasons: spring (flood period) of 2001-2004 (n = 45) and autumn (drought period) of 2003-2005 and 2008 (n = 81). The fish were captured at three locations: in the Aquidauana, Miranda and Paraguay Rivers in the southern region of the Pantanal (n = 34), in rivers and lakes of the Pantanal National Park in the central region (n = 60) and in the Cuiabá and Manso Rivers in the municipality of Nobres in the northern region (n = 32). The fish from the Miranda, Aquidauana and Paraguay Rivers were caught in the spring of 2001 and 2002 as well as the autumn of 2003, whereas those from the Pantanal National Park and the municipality of Nobres were caught in the spring of 2003 and 2004 as well as the autumn of 2004, 2005 and 2008. The cultivated specimens were examined in the summer and winter of 2008. Immediately after collection, the live fish were transported to the field laboratory mounted nearby, where they were measured, weighed and necropzied. Cysts were removed from the gill filaments of different piraputanga specimens and examined under a light microscope. Spore dimensions (?m) were expressed as mean ± standard deviation. Smears containing free spores were stained with Giemsa solution and mounted in a low-viscosity mounting medium as permanent slides. For histological analysis, fragments of infected organs were fixed in 10% buffered formalin, embedded in paraffin, cut into serial sections (4 ?m in thickness) and stained with haematoxylin/eosin and Sirius red. For transmission electron microscopy, plasmodia were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 12 h, washed in glucose-saline solution for 2 h and post-fixed in OsO4, all at 4ºC. After dehydration in an acetone series, the material was embedded in EMbed 812 resin. Ultrathin sections, double stained with uranyl acetate and lead citrate, were examined in an LEO 906 electron microscope operated at 60 kV.

For molecular analysis, plasmodia were removed from the host tissue and fixed in ethanol. After rupturing the plasmodia with the aid of a needle, the contents were collected in a 1.5 mL microcentrifuge tube. DNA was extracted using the Wizard® Genomic DNA Purification kit (Promega, USA), following the manufacturer's instructions. DNA content was determined using the NanoDrop 2000 spectrophotometer (Thermo Scientific) at 260 nm. Polymerase chain reaction (PCR) was carried out in a final volume of 25 ?L, which contained 10-50 ng of extracted DNA, 1X Taq DNA polymerase buffer (Invitrogen), 0.2 mmol of dNTP (Invitrogen), 1.5 mmol of MgCl2, 0.2 pmol of each primer (Invitrogen), 0.25 ?L (1.25 U) of Taq DNA polymerase (Invitrogen) and ultra pure (MilliQ) water. The Eppendorf AG 22331 Hamburg Thermocycler was used. An ~1600 bp 5' fragment of the SSU rDNA gene was amplified using the primers MX5-MX3 (Andree et al. 1999) in the following manner: an initial denaturation step at 95ºC for 5 min followed by 35 cycles of denaturation (95ºC for 60 s), annealing (62ºC for 60 s) and extension (72ºC for 120 s), finished with an extended elongation step at 72ºC for 5 min. PCR products were submitted to electrophoresis on 1% agarose gel (BioAmerica) in a Tris-Borate-EDTA buffer (0.045 MTris-borate, 0.001 M EDTA pH 8.0), stained with ethidium bromide and analyzed in a FLA-3000 (Fugi) scanner. The size of the amplified fragments was estimated by comparisons with the 1 kb DNA Ladder (Invitrogen).

Purified PCR products were cloned in pCR®4-TOPO® vectors from the TOPO-TA Cloning® kit for sequencing (Invitrogen). A single clone was sequenced using MX5-MX3 and MC5-MC3 (Eszterbauer 2004) primer pairs with the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™) in an ABI 3730 DNA sequencing analyzer (Applied Biosystems™).

A standard nucleotide-nucleotide Basic Local Alignment Search Tool (BLAST) (blastn) search was conducted (Altschul et al. 1997). The sequence of the Myxobolus species obtained from B. hilarii was aligned with sequences obtained in the GenBank using DAMBE (Xia & Xie 2001). To evaluate the position of the Myxobolus species obtained from B. hilarii in relation to other Myxobolus spp, phylogenetic analyses were conducted using the phylogenetic methods neighbor-joining (NJ) and maximum parsimony (MP) [MEGA 4.0 programme (Tamura et al. 2007)] and maximum likelihood (ML) [MEGA 5.0 (programme in test phase)]. The Kimura 2-parameter (K2P) evolution sequence model was used in the analysis. Bootstrap analysis (1,000 replicates) was employed to assess the relative robustness of the branches of the NJ, MP and ML trees using the MEGA 4.0 and 5.0 programmes. The distance analyses were performed using the K2P model conducted using the MEGA 4.0 programme (Tamura et al. 2007). The species Ceratomyxa seriolae and Ceratomyxa shasta were used as outgroups in the phylogenetic analyses.

The effects of season, study location and sex of the host on the prevalence of the parasite were assessed using the ?2 test, with the level of significance set at p < 0.05.

 

RESULTS

Among the 126 wild and 90 cultivated specimens of piraputanga (a total of 216) examined in the present study, 22.2% (n = 48) had plasmodia of an unknown parasite from the genus Myxobolus. The prevalence was of 38.1% in the wild specimens and 0% in the cultivated specimens. In the wild specimens, the prevalence of the parasite varied significantly with regard to study location (?22 = 28.79; p = 0.00), with the lowest prevalence found in southern region of the Pantanal (2.9%). No significant difference was found between the central (43.3%) and northern regions (65.6%) (?21 = 4.15; p = 0.04). The parasite was found in both seasons studied (prevalence of 33.3% in spring and 40.7% in autumn) and the variation between seasons was not significant (?21 = 0.63; p = 0.41). Regarding the sex of the host, the prevalence was 32.2% for female specimens and 16.2% for males; this difference was also not significant (?21 = 3.07; p = 0.07). Sex was not defined in seven infected specimens.

Myxobolus oliveirai sp. nov.

(Figs 1-5)

 


 

 


 

 


 

 


 

 

 

Plasmodia elongated in shape and measuring up to 3 mm were found primarily in the distal extremity of the gill filaments (Fig. 1A). Histological and ultrastructural analysis revealed that the development of the plasmodia induced the expansion of the gill filaments and the compression caused thinning of the tissue of the distal extremity, which exhibited only a delicate layer of cells (Fig. 1C). In the proximal extremity, growth of the plasmodia occurred by invaginations in the filament tissue (Figs 1C, 2A). The plasmodia were surrounded by a well-defined capsule of host connective tissue, with distinct delicate and interlaced collagen fibres (Figs 2B, 3A). The plasmodium wall consisted of a single layer that was continuous with pinocytic canals extending into the granular layer of the plasmodium ectoplasm (Fig. 3A, C).

The plasmodia exhibited development with different sporogenic stages, such as generative cells and disporoblastic pansporoblasts (Fig. 3C) along the periphery of the plasmodia (occurring in a thin layer of not more than 40 ?m), followed by a layer containing immature spores and mature spores occurring in the internal region (Figs 2-4). The spores were pear-shaped in the frontal view (Figs 1B, 5A), measuring 11.2 ± 0.4 ?m in length, 7.4 ± 0.5 ?m in width and 4.6 ± 0.6 ?m in thickness. The polar capsules were elongated in shape and equal in size, measuring 5.6 ± 0.2 ?m in length and 2.3 ± 0.2 ?m in width. The anterior ends of polar capsules were close to one another and the polar filaments exhibited 6-8 turns arranged perpendicular to the longitudinal axis of the capsules (Figs 4C, 5A). A few, small sporoplasmosomes were found in the sporoplasm (Fig. 4A) and two nuclei were discernible when stained with Giemsa (Fig. 5A).

In the molecular analysis, the specific primer pair MX5-MX3 successfully amplified an approximately 1,600-bp fragment of the 18S rDNA gene in the spores obtained from plasmodia found infecting the gill filaments of B. hilarii. The BLAST search using the partial 18S rDNA sequence data (1,527 bp) of the Myxobolus species parasite of B. hilarii did not match any of the Myxozoa available in the GenBank.

In the phylogenetic analyses, the Myxobolus species clustered into three distinct lineages. Myxobolus spirosulcatus represented the basal group and Myxobolus acanthogobii clustered as the sister group of the remaining species of Myxobolus (Fig. 6). These remaining species clustered in a monophyletic group composed of numerous species divided into four smaller clades (A-D) (Fig. 6). M. oliveirai sp. nov. formed a monophyletic unit with eight other Myxobolus species in Clade C: five species parasites of hosts from the family Mugilidae; two parasites of Pangasiidae hosts and one parasite of Centrarchidae. Mean genetic divergence within Clade C (Table) was 20.4%. In this clade, the smallest distance in reference to M. oliveirai sp. nov. was with Myxobolus hakyi (16.6%) and the largest was with Myxobolus osburni (28.6%).

 

 

Type host - B. hilarii (Valenciennes, 1850) (Characidae, Bryconinae).

Etymology - The specific epithet name is in homage to Ricardo Afonso Torres de Oliveira, research assistant of the laboratory of Saúde e Bem Estar dos Peixes of the Centro Nacional de Pesquisa e Conservação de Peixes Continentais of Instituto Chico Mendes de Conservação da Biodiversidade, who, in recent years, has provided great support to the fieldstrip of our research group.

Type data and depository - Pantanal Mato-Grossense (Aquidauana, Cuiabá, Miranda and Paraguay Rivers) in the Paraguay basin, Brazil.

Specimens deposited - Slides with stained spores (syntype) have been deposited in the collection of the Museum of Natural History, Institute of Biology, State University of Campinas, SP, Brazil (accession ZUEC 28). The 18S rDNA sequence was deposited in GenBank under the accession HM754633.

Prevalence - Fourty-eight of 216 B. hilarii examined (22.2%) had plasmodia of an unknown parasite from the genus Myxobolus - 48/126 wild specimens (38.1%) and 0/90 (0%) cultivated specimens.

Site of infection - Distal extremity of the gill filaments.

 

DISCUSSION

M. oliveirai sp. nov. was compared with all Myxobolus species previously reported in fish of South America (Azevedo et al. 2010, Eiras et al. 2010) and other geographic regions (Eiras et al. 2005). Among the South American species, M. oliveirai sp. nov. resembles the body shape of Myxobolus cordeiroi (Adriano et al. 2009a), a parasite of Zungaro jahu; Myxobolus chondrophilus (Nemeczek, 1926), reported to infect Sardinella anchovina; Myxobolus associatus (Nemeczek, 1926), a parasite of Leporinus mormyrops; Myxobolus cunhai (Penido, 1927), a parasite of Pygocentris piraya; Myxobolus serrasalmi (Walliker 1969), a parasite of Serrasalmus rhobeus; Myxobolus maculatus (Casal et al. 2002), a parasite of Metynnis maculates; Myxobolus sp. (Walliker 1969), a parasite of Colossoma bidens, and Myxobolus sp. (Walliker 1969), a parasite of Serrasalmus sp. Among all these species, however, only M. cordeiroi and the Myxobolus sp. parasite of Serrasalmus have a similar size to that of M. oliveirai sp. nov. Although the description of Myxobolus sp. parasite of Serrasalmus sp. (Walliker 1969) is poor, a superficial comparison reveals that the anterior end of the spores of Myxobolus sp. are more pointed than in M. oliveirai sp. nov. Furthermore, the infection site (kidney) and host (Serrasalmus sp.) of the parasite described by Walliker (1969) differ from those of M. oliveirai sp. nov., which infects gill filaments in B. hilarii. Regarding M. cordeiroi, the differences were in the number of polar filament turns, infection sites (connective tissue of several organs) and host (a pimelodid fish). The spore dimensions of the different species do not fit the values found in the present material. Therefore, our specimens cannot be identified with any of the aforementioned species.

When comparing M. oliveirai sp. nov. with species from other continents (Eiras et al. 2005), we selected those species that most resembled our material in their sets of characteristics. Thus, we included Myxobolus bellus infecting the integument of Carpioides carpio in the USA (Kudo 1934), Myxobolus bramaeformis described from the kidneys and gut of Hypophthalmichthys molitrix in the Amur Basin (Akhmerov 1960), Myxobolus fahmii infecting the gills of Barbus bynni in Egypt (Ali et al. 2002) and Myxobolus pseudosquamae parasitizing the gills and kidney of Sinocyclochilus grahami tingi in China (Ma & Zhao 1993). Despite some similarities, the features of our specimens do not match the characteristics of any of the aforementioned species. Moreover, their hosts are phylogenetically very different and their geographic locations are also quite different. In particular, only a very few number of species have as small a spore thickness (4.6 ± 0.6 ?m) as that described for M. oliveirai sp. nov. Considering the 744 nominal species reported by Eiras et al. (2005), only 21 have a similar spore thickness. In these cases, however, the other spore features are very different. Furthermore, this is the first report of a myxosporean species infecting fish from the genus Brycon and the BLAST search using the partial 18S rDNA sequence of M. oliveirai sp. nov. did not match any of the Myxozoa available in GenBank.

The molecular phylogenetic analysis was based on the comparative analyses of the 18S rDNA gene in 62 species of Myxobolus (approximately 1,500 bp) obtained from GenBank with M. oliveirai sp. nov. To enhance the accuracy of the analysis, smaller species were not used. The results, with high bootstrap values, showed the Myxobolus species clustering as a monophyletic unit. M. spirosulcatus was the most divergent species of the genus in this analysis and M. acanthogobii appears to be the sister group of the large monophyletic clade composed by the remaining Myxobolus species. This large clade further divided into four monophyletic units (A-D), with unsolved groups, despite the high bootstrap values supporting the relationships.

M. oliveirai sp. nov. clustered in Clade C together with eight other species, forming a monophyletic unit, where it appears as sister group of the clade formed by M. pangasii and M. hakyi, which are parasites of Pangasianodon hypophthalmus (Pangasiidae: Siluriformes) and, together, these three species compose the sister group of the clade formed by five species (Myxobolus exiguus, Myxobolus spinacurvatura, Myxobolus ichkeulensis, Myxobolus bizerti and Myxobolus episquamalis), parasites of mugilid hosts. M. osburni, a parasite of Lepomis gibbosus, which is a Perciformes from the family Centrarchidae, appears as the basal unit of Clade C.

The mean genetic divergence estimated in Clade C was 20.4%. With regard to the new species, the smallest divergence was with M. hakyi (16.6%), a sister taxa of M. oliveirai sp. nov., whereas the largest divergence was with M. osburni (28.6%) (Table) which is the basal species for Clade C.

The phylogenetic tree obtained here is in accordance with results described by Ferguson et al. (2008) and Liu et al. (2010), who report M. acanthogobii as a species with the greatest divergence among Myxobolus spp as well as in relation to the clustering of M. exiguus, M. spinacurvatura, M. ichkeulensis, M. bizerti, M. episquamalis, M. pangasii and M. hakyi in a monophyletic unit.

The results of the present study show the phylogenetic relation of the new species with other Myxobolus species available in GenBank. This is the first phylogenetic study on a Myxobolus species parasite of a South American characid host considering Myxozoa parasites of fresh water fish. From this region, only the 18S rDNA gene sequence of M. cordeiroi (Adriano et al. 2009a), with around 500 bp, has been deposited in GenBank. Thus, additional molecular and phylogenetic studies of Myxobolus spp parasites of this fish family are needed to identify the true position of the Myxobolus parasites of characid hosts in relation to the Myxobolus species parasites of other families of fish.

There was a much lower prevalence of M. oliveirai sp. nov. in the southern region of the Pantanal than that found in the central and northern areas, demonstrating that environmental characteristics may influence the distribution of this myxosporean species in the Brazilian Pantanal. Environmental parameters (water temperature and water flow) have been reported to influence the prevalence of Myxobolus cerebralis (Baldwin et al. 2000, Hallett & Bartholomew 2008). The Brazilian Pantanal is the largest floodplain area in the world, with approximately 150,000 km2 (Silva 1986, Ceccarelli et al. 2007), and this very large ecosystem has areas with heterogeneous features, which may have contributed toward the lower prevalence of M. oliveirai sp. nov. in the southern region. However, the nature of the parameters that influence the prevalence of infection is unknown.

Regarding the seasons studied, the variation in prevalence was non-significant, revealing that season had no influence over infection by M. oliveirai sp. nov. in the present study. This finding is in contrast to results described by Gbankoto et al. (2001), who report the occurrence of significant seasonal variations in Myxobolus sp. and Myxobolus zillii parasites of Sarotherodon melanotheron melanotheron and Tilapia zillii, respectively, and Gbankoto et al. (2003), who report seasonal differences in the infection of S. melanotheron melanotheron by Myxobolus heterospora. However, the results of the present study are in agreement with those described by Gbankoto et al. (2003), who report no seasonal variation in the infection of T. zillii by M. heterospora.

The comparison of infection between female and male hosts revealed no influence of sex on the prevalence of M. oliveirai sp. nov. This finding is in agreement with accounts describing some Myxozoa species (Gbankoto et al. 2001, Viozzi & Flores 2003), but contrasts with others (Muzzall 1995, Gbankoto et al. 2003) that reported a significant difference between females and males regarding the prevalence of infection.

The histological analysis of piraputanga gills infected with M. oliveirai sp. nov. revealed numerous large cysts primarily in the distal area of the gill filaments, but no pronounced inflammatory response was found in the infection site, which is similar to findings described for other South American myxoporean species (Barassa et al. 2003, Eiras et al. 2008, 2009, Adriano et al. 2009a, b). However, the development of the plasmodia caused expansion and compression of the gill filaments, producing a thinning of the tissue at the distal extremity, whereas the growth of the plasmodia in proximal extremity occurred through invaginations, pervading and occupying the filament tissue. These structural alterations in the gills are similar to structural changes reported for other myxosporean species (Feist & Longshaw 2006).

The ultrastructural analysis revealed that sporogenesis in M. oliveirai sp. nov. followed the general pattern of other Myxobolus species (Current et al. 1979, Casal et al. 1996, 2002, Adriano et al. 2006) and the plasmodium wall consisted of a single membrane, which was continuous with pinocytic canals extending to the ectoplasm, as seen in some Myxobolus species (Current et al. 1979), but differed from others delimited by a double membrane (Casal et al. 2002, 2006).

Gill infections by Myxoporea in farmed fish can cause significant tissue damage (Martins et al. 1997, 1999, Adriano et al. 2005a, b, 2006, Feist & Longshaw 2006) and occasionally death (Martins et al. 1999, Feist & Longshaw 2006). Although the histopathological changes produced by M. oliveirai sp. nov. impaired the function of the gill surface, the infection apparently did not significantly affect the general health of the fish. However, the histopathological analysis only addressed the wild host specimens (farmed fish were not infected) and we do not know the impact that infection by M. oliveirai sp. nov. may have on farmed specimens, especially in intensive farming. Thus, the presence and dispersion of M. oliveirai sp. nov. needs to be monitored closely by commercial fish farmers.

 

ACKNOWLEDGEMENTS

To Ricardo Afonso Torres de Oliveira (CEPTA/ICMBio), for help in dissecting the fish, and to Dr Laerte Batista de Oliveira Alves, manager of the National Center for Research and Conservation of Continental Fishes (CEPTA/ICMBio), and Dr José Augusto Ferraz de Lima, the manager of Pantanal National Park, for support during the fieldwork.

 

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Received 15 December 2009
Accepted 13 July 2010
Financial support: FAPESP (06/59075-6), CEPTA/ICMBio, CAPES (to TM), FAPESP (to MMC)

+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.

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