FISH PARASITOLOGY - SHORT COMMUNICATION

First report of Myxobolus episquamalis Egusa, Maeno & Sorimachi,

1990 (Myxozoa: Bivalvulida) in Lebranche mullet Mugil liza

Valenciennes, 1836 (Teleostei: Mugiliformes) from Neotropical region

Rayane Duarte

& Bruna Reich Martinatti

& Águida Aparecida de Oliveira

& Jhon Lennon Genovez-Oliveira

Viviane Moreira de Lima

& Rafael de Almeida Tubino

4,5

& Bruno Pereira Berto

& Michelle Daniele Santos-Clapp

Received: 12 January 2020 /Accepted: 6 July 2020

Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract

In the current study, Myxobolus episquamalis Egusa, Maeno & Sorimachi, 1990 (Myxozoa: Bivalvulida) is reported from the

Lebranche mullet Mugil liza Valenciennes, 1836 in the estuarine region of the Maricá Lagoon, State of the Rio de Janeiro,

southeastern Brazil. To date, this myxozoan species was reported in mullets from Asia, Africa, Europe, and Oceania. The

characteristics of M. episquamalis previously reported are similar to the findings of the present study. DNA sequences of the

nuclear small subunit ribosomal DNA (SSU rDNA) had 99.7–100% similarity with the sequences of M. episquamalis from North

Africa and Asia. Therefore, strong morphological and molecular similarities ensure the identification of M. episquamalis in the

current study. Finally, this finding records a new host and locality, revealing the worldwide distribution of this myxozoan species.

Keywords Morphology

Sequencing

Myxozoa

Myxobolus episquamalis

Mugil liza

Maricá Lagoon

Introduction

The Lebranche mullet Mugil liza Valenciennes, 1836 (syn.

M. platanus Günther, 1880) is a catadromous pelagic fish

from tropical and subtropical waters, mainly from estuarine

coastal regions. It is distributed from the USA to Argentina,

occurring in saltwater, freshwater, and brackish water (Froese

and Pauly 2019).

Myxozoans are common parasites of marine and freshwa-

ter fish, rarely of amphibians and reptiles, and exceptionally of

birds and mammals, representing an important pathogenic

group with a worldwide distribution (Lom and Dyková

2006). Among myxozoans, the genus Myxobolus Bütschli,

1882 has the largest known diversity. In a revision of Eiras

et al. (2014), a total of 905 Myxobolus spp. were checked and

listed. Approximately 90 species of myxozoans have been

identified in South America, of which 41 are Myxobolus

spp., most of them from Brazil. To date, there are three reports

of Myxobolus spp. from M. liza:(1)Myxobolus sp. from scales

of M. liza from the coast of Rio de Janeiro State, Brazil (Knoff

and Serra-Freire 1993); (2) Myxobolus platanus Eiras, Abreu,

Robaldo & Pereira Júnior, 2007 described infecting the spleen

of M. liza from lagoons in the State of Rio Grande do Sul,

Brazil (Eiras et al. 2007); and (3) M yxobolus s aladensis

Marcotegui & Martorelli, 2017 which was described parasit-

izing the gills of M. liza from Buenos Aires, Argen tina

(Marcotegui and Martorelli 2017).

In the present study, Myxobolus episquamalis Egusa, Maeno

&Sorimachi,1990, originally described from the flathead gray

mullet Mugil cephalus Linnaeus, 1758 in Japan (Egusa et al.

Section Editor: Astrid Holzer

* Bruno Pereira Berto

berto.ufrrj@gmail.com

Programa de Pós-Graduação em Ciências Veterinárias, Instituto de

Veterinária (IV), Universidade Federal Rural do Rio de Janeiro

(UFRRJ), BR-465 km 7, Seropédica, RJ 23897-000, Brazil

Curso de Graduação em Medicina Veterinária, IV, UFRRJ,

BR-465 km 7, Seropédica, RJ 23897-000, Brazil

Departamento de Microbiologia e Imunologia Veterinária, IV,

UFRRJ, BR-465 km 7, Seropédica, RJ 23897-000, Brazil

Programa de Pós-Graduação em Biologia Animal, Instituto de

Ciências Biológicas e da Saúde (ICBS), UFRRJ, BR-465 km 7,

Seropédica, RJ 23897-000, Brazil

Departamento de Biologia Animal, ICBS, UFRRJ, BR-465 km 7,

Seropédica, RJ 23897-000, Brazil

Parasitology Research

https://doi.org/10.1007/s00436-020-06803-3

1990), was identified from a new host M. liza, from the Maricá

Lagoon in southeastern Brazil, Neotropical region.

Materials and methods

Sample collection

A total of 19 specimens of M. liza (total length 31.9 cm [26–

45 cm]; 328.2 g [178–811 g]) were collected by local fisher-

men in the Maricá Lagoon (22°52′ and 23°00′ S, 43°00′ and

42°45′ W), State of Rio de Janeiro, Brazil. These specimens

were transported to the Laboratório de Biologia e Ecologia de

Parasitos (LABEPAR) of the Universidade Federal Rural do

Rio de Janeiro (UFRRJ) for parasitological analysis.

Morphological analysis

Cyst-like plasmodia on infected scales from freshly collected

fish were carefully removed and squashed for the observation

of myxozoan myxospores. Morphological observations, pho-

tomicrographs, and measurements of live myxospores were

made using an Olympus BX binocular microscope

(Olympus Optical, Tokyo, Japan) coupled to a digital camera

Eurekam 5.0 (BEL Photonics, Monza, Italy).

Histological analysis

For histological analysis, fragments of infected areas were

carefully removed from the scales with a scalpel. These frag-

ments were fixed in 10% neutral buffered formalin and em-

bedded in paraffin blocks. Five mic rometer (μm) sections

were obtained using a manual microtome and stained with

hematoxylin-eosin.

Molecular analysis

DNA was extracted from plasmodia in PBS, using the Qiagen

DNeasy Blood and Tissue Kit (Qiagen, São Paulo, Brazil)

according to the manufacturer’s instructions. The PCR ampli-

fication of the nuclear small subunit ribosomal DNA (SSU

rDNA) was carried out from two samples (two individuals)

as previously described by Özer et al. (2016), using the

primers Henn_Myx_120F (5′ -AATCTGCTCGATTG

TAAGGG-3′ ) and Henn_Myx_2100Rev (5′ -CCGC

TCCCAAGGTATTAT-3′). For amplification, a 25 μlPCR

reaction was prepared using 1 μL of genomic DNA (<

1 μg), 12.5 μL of GoTaq® G2 Hot Start Colorless Master

Mix (Promega) (× 1), 0.25 μL of each primer (0.2 μm), and

11 μL of nuclease free water. The thermal cycling protocol

was as follows: an initial denaturation step at 94 °C for 5 min,

followed by 35 cycles at 94 °C for 30 s, 51 °C for 45 s, and

72 °C for 2 min and 30 s; completed with terminal extension at

72 °C for 5 min; and then stored at 4 °C. The amplicon was

purified using the ReliaPrep™ DNA Clean-up and

Concentration System (Promega Corporation, São Paulo,

Brazil). The PCR product was sequenced using the internal

forward and reverse primers, NS3 (5′ -GCAAGTCT

GGTGCCAGCAGCC-3′ )andNS4(5′ -CTTC

CGTCAATTCCTTTAAG-3′)(Whiteetal.1990), by

Ludwig Biotechnology, were an ABI-Prism 3500 Genetic

Analyzer (Applied Biosystems, Foster City, California) was

used for Sanger sequencing. The results of the sequencing

reactions were analyzed and edited using the program

Chromas 2.6. The sequence of the Myxobolus species obtain-

ed from M. liza was compar ed with se quences o f other

myxozoans available in the GenBank database, using t he

Basic Local Alignment Search Tool (BLAST).

Results and discussion

Nineteen M. liza were examined, and one was infected by a

myxozoan species that was morphologically identified as

M. episquamalis (Fig. 1). The cystic masses were observed

over the scales of the body and on the tail fin, but they were

concentrated on the ventral region of the fish, measuring 9–

10 mm in length and 5–7 mm in width (Fig. 1a). They were

oval or irregularly shaped, spongy, and whitish, located at the

dorsal surface of the scales, mainly near the insertion area in

the integument. A thin and dense collagen capsule was sur-

rounding the plasmodia. In some plasmodia, mature

myxospores were visible. Whitish irregular ellipsoidal and

polysporic plasmodia measuring 50–180 μm in length and

40–110 μm in width were observed in these cysts (Figs. 1b–

c). The mature myxospores (Fig. 1d–k) were broadly ovoid in

front view, tapering toward at the anterior end, measuring

8.2 μm(7.5–9.1) in length, 6.0 μm(5.3–6.9) in width, and

4.8 μm(4.6–5.5) in thickness. Along the sutural edge, several

triangular markings barely discernible were observe d

(Fig. 1f). Two pyriform polar capsules were present, frequent-

ly with unequal sizes, measuring 4.5 μm(3.8–5.3) in length

and 2.2 μm(1.8–2.6) in width. The polar filaments were in-

discernible within polar capsules (Fig. 1d–k). In the minority

of myxospores, a relatively large iodiophilous vacuole could

be observed in the sporoplasm (Fig. 1e–f). In lateral view, the

myxospores were lenticular, and the sutural ridge was broad,

but the sutural line was indistinct (Fig. 1k).

Photomicrographs, histological slide, and cysts in 70% eth-

anol were deposited in the Museu de Zoologia at the UFRRJ,

Brazil, under accession number MZURMTZ2020021.

Photomicrographs were d eposited and available (http://r1.

ufrrj.br/labicoc/colecao.html) in the Parasitology Collection

of the Laboratório de Biologia de Coccídios, at UFRRJ,

under the repository number 104-2020. Photovouchers of

the host specimens were deposited in the same collection.

Parasitol Res

The main distinguishing feature of M. episquamalis is the

tropism for epithelial tissue of scales and tail fin of mullets

(Rothwell et al. 1997). According to Salim and Desser (2000),

myxosporeans have a high degree of specificity, both for host

species and parasitized tissue. In this sense, although

M. episquamalis has been recorded in four host species, the

parasitized tissue remains the same. Furthermore, the

morphological characteristics of M. episquamalis reported

by Egusa et al. (1990) and later by other authors are similar

with the finding of the current study. Myxobolus episquamalis

parasitized hosts of the same family (Mugilidae), with the

same sites of infection and lesion pattern, besides having the

same morphology and morphometry of myxospores. The

differences observed in the current study were the new host

species an d the new locality, which reveal the worldwide

distribution of M. episquamalis. This myxozoan has been

recorded in 12 geographic regions in four continents, Asia,

Africa, Europe, and Oceania. In the current study, we report

this species for the first time from South America. In addition,

we suggest that the Myxobolus sp. reported as M. cephalus

Fig. 1 Cysts, plasmodia, and

myxospores of Myxobolus

episquamalis Egusa, Maeno &

Sorimachi, 1990 from Lebranche

mullet Mugil liza Valenciennes,

1836. a Cysts on the scales

located on the abdomen of the

fish. b Histological section of a

cyst, showing plasmodia and

fragments (arrows) of scales. c

Mature myxospores and other

sporogonic stages in plasmodia.

d–k Fresh myxospores in frontal

view (d–h)andlateralview(i–k)

showing polar capsules (empty

arrowhead), triangular markings

(gray arrowhead), vacuole (black

arrowhead), and the broad sutural

ridge (white arrowhead). Bar =

10 mm (a), 200 μm(b), 20 μm

(c), 3 μm(d–k). H&E stained (b,

Parasitol Res

from Portugal by Menezes (1984)shouldbeM. episquamalis,

since this species is morphologically identical and has been

found on the scales of the mullet.

DNA amplification of the SSU rDNA of the samples of

M. episquamalis from M. liza produced DNA fragments of

1000 bp length. The seque nces of the two samples were 100%

identical to each other and to M. episquamalis (JF810537;

MK012069), reported from gray mullet M. cephalus in Asia

(Kim et al. 2013;Simsek2019), and 99.7% similar to the first

sequence of M. episquamalis (AY129312) deposited in

GenBank, obtained from M. cephalus from Tunisia, northern

Africa (Bahri et al. 2003). A representative sequence of the cur-

rent work was deposited in GenBank under the accession num-

ber MN822014. The high levels of genetic similarity between

our sequence and the sequences of M. episquamalis from North

Africa and Asia confirmed the identification of M. episquamalis

from the Lebranche mullet in southeastern Brazil.

Acknowledgments We thank the fisherman Mr. Lu cielio de Moura

Costa, in the municipality of Maricá, State of Rio de Janeiro, for giving

us the fish that were analyzed in the current study. We are also grateful to

the Laboratório de Histologia e Embriologia of the Departamento de

Biologia Animal of the UFRRJ for the preparation of histological slides.

Funding information This study was supported by a compensatory mea-

sure established by the Chevron Responsibility Adjustment Term, con-

ducted by the Federal Public Ministry, with the implementation of the

Fundo Brasileiro para a Biodiversidade (FUNBIO), in addition to the

support of the Coordenação de Aperfeiçoamento de Pessoal de Nível

Superior (CAPES), Conselho Nacional de Desenvolvimento Científico

e Tecnológico (CNPq), and Fundação Carlos Chagas Filho de Amparo à

Pesquisa do Estado do Rio de Janeiro (FAPERJ). RD and JLG-O have a

scholarship from CAPES (Grant/Award Number: 001). BRM had a

scholarship from CNPq (Grant/Award Number: PIB179/2018). BPB

has a fellowship from CNPq (Grant/Award Number: 302437/2016-9

and 303899/2019-0) and from FAPERJ (Grant/Award Number: E-26/

203.200/2016 and E-26/202.797/2019).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of

interest.

Ethical approval Animal collection complied with the guidelines of the

Conselho Nacional de Controle de Experimentação Animal and were

approved by the Comissão de Ética no uso de Animais (CEUA/ICBS)

of the UFRRJ under protocol no. 005/2019.

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