Veterinary Parasitology 185 (2012) 110–120
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Veterinary Parasitology
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Phylogenetic and host–parasite relationship analysis of Henneguya
multiplasmodialis n. sp. infecting Pseudoplatystoma spp. in Brazilian
Pantanal wetland夽
E.A. Adriano a,c,∗,1 , M.M. Carriero b,2 , A.A.M. Maia b , M.R.M. Silva b , J. Naldoni c,3 ,
P.S. Ceccarelli d , S. Arana e
a
Departamento de Ciências Biológicas, Universidade Federal de São Paulo (Unifesp), Rua Professor Artur Riedel, 275, Jardim Eldorado, CEP 09972-270
Diadema, SP, Brazil
b
Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo (USP), Rua Duque de Caxias Norte, 225,
CEP 13635-900 Pirassununga, SP, Brazil
c
Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6109, CEP 13083-970 Campinas, SP,
Brazil
d
Centro Nacional de Pesquisa e Conservação de Peixes Continentais (CEPTA), Instituto Chico Mendes de Conservação da Biodiversidade (ICMbio), Rod. SP
201, Km 6.5, Caixa Postal 64, CEP 13630-970 Pirassununga, SP, Brazil
e
Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6109, CEP 13083-970
Campinas, SP, Brazil
a r t i c l e
i n f o
Article history:
Received 7 February 2011
Received in revised form 18 August 2011
Accepted 11 October 2011
Keywords:
Myxozoa
Club cells
Henneguya corruscans
18S rDNA
Pseudoplatystoma corruscans
Pseudoplatystoma reticulatum
a b s t r a c t
A new species of the genus Henneguya (Henneguya multiplasmodialis n. sp.) was found infecting the gills of three of 89 specimens (3.3%) of Pseudoplatystoma corruscans and two of
79 specimens (2.6%) of Pseudoplatystoma reticulatum from rivers in the Pantanal wetland,
Brazil. Partial sequencing of the 18S rDNA gene of the spores obtained from one plasmodium from the gills of P. corruscans and other one from the gills of P. reticulatum, respectively,
resulted in a total of 1560 and 1147 base pairs. As the spores of H. multiplasmodialis n. sp.
resemble those of Henneguya corruscans, which is also a parasite of P. corruscans, sequencing of the 18S rDNA gene of the spores of H. corruscans found on P. corruscans caught in the
Brazilian Pantanal wetland was also provided to avoid any taxonomic pendency between
these two species, resulting in 1913 base pairs. The sequences of H. multiplasmodialis n.
sp. parasite of P. corruscans and P. reticulatum and H. corruscans did not match any of the
Myxozoa available in the GenBank. The similarity of H. multiplasmodialis n. sp. obtained
from P. corruscans to that from P. reticulatum was of 99.7%. Phylogeny revealed a strong
tendency among Henneguya species to form clades based on the order and/or family of
the host fish. H. multiplasmodialis n. sp. clustered in a clade with Henneguya eirasi and H.
corruscans, which are also parasites of siluriforms of the family Pimelodidae and, together
© 2011 Elsevier B.V. Open access under the Elsevier OA license.
夽 Worksupported by FAPESP (Proc. no. 06/59075-6) and CEPTA/ICMBio.
∗ Corresponding author at: Departamento de Ciências Biológicas, Universidade Federal de São Paulo (Unifesp), Rua Professor Artur Riedel, 275, Jardim
Eldorado, CEP 09972-270 Diadema, SP, Brazil. Tel.: +55 11 3319 3300; fax: +55 11 4043 6428.
E-mail address: edapadriano@gmail.com (E.A. Adriano).
1
Research productivity grant from the Brazilian Fostering Agency CNPq.
2
Master’s student supported by FAPESP scholarship.
3
Master’s student supported by CAPES scholarship.
0304-4017 © 2011 Elsevier B.V. Open access under the Elsevier OA license.
doi:10.1016/j.vetpar.2011.10.008
E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
111
with the clade composed of Henneguya spp. parasites of siluriforms of the family Ictaluridae,
formed a monophyletic clade of parasites of siluriform hosts. The histological study revealed
that the wall of the plasmodia of H. multiplasmodialis n. sp. were covered with a stratified
epithelium rich in club cells and supported by a layer of connective tissue. The interior of the
plasmodia had a network of septa that divided the plasmodia into numerous compartments.
The septa were composed of connective tissue also covered on both sides with a stratified
epithelium rich in club cells. Inflammatory infiltrate was found in the tissue surrounding
the plasmodia as well as in the septa.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Fish of the genus Pseudoplatystoma, which includes
species of Pimelodus that attain large sizes, are found in
the main river basins in South America (Lundberg and
Littmann, 2003). The species Pseudoplatystoma corruscans
Spix and Agassiz, 1829 (popularly known in Brazil as
“pintado” or “surubim”; English name = spotted sorubim)
and Pseudoplatystoma reticulatum Eigenmann e Eigenmann, 1889 (known as “cachara”; English name = barred
sorubim) are found in the Prata River (Resende, 2003).
These carnivorous, migratory fish attain large sizes, with
P. corruscans reaching more than 100 kg and P. reticulatum reaching 20 kg (Campos, 2005). These species play an
important role in the fishing economy of the regions in
which they occur and are among the most important freshwater species in Brazil due to the quality of their meat
(Campos, 2005). Moreover, their rapid growth and high
market value have led to increased interest on the part
of fish farmers (Campos, 2005). On Brazilian fish farms,
the production of these fish reached 1,094,000 kg in 2006
(Ibama, 2008), supplying both domestic and international
markets (Mar and Terra, 2010).
In many parts of the world, wild and cultivated fish
are infected by Henneguya spp. There are more than 200
such species (Lom and Dyková, 2006), many of which
have pathogenic importance and cause economic impacts
on fish farm activities (Feist and Longshaw, 2006; Feist,
2008). Thirty-nine species of Henneguya species have been
reported in South American fish (Azevedo et al., 2009), with
Henneguya corruscans (Eiras et al., 2009), Henneguya pseudoplatystoma (Naldoni et al., 2009) and Henneguya eirasi
(Naldoni et al., 2011), described infecting fish of the genus
Pseudoplatystoma (Eiras et al., 2009; Naldoni et al., 2009,
2011).
Different cells and mechanisms in the immune system
are related to host–parasite interactions in fish (SitjáBobadilla, 2008a). Despite the widespread occurrence
of Myxozoa, host–parasite interactions remain poorly
understood. The typical histopathological finding is the
encapsulation of the parasite by a connective, fibrous and
epithelioid layer (Martyn et al., 2002; Adriano et al., 2005;
Sitjà-Bobadilla, 2008b; Naldoni et al., 2011). In some cases,
different cell types, such as macrophages, lymphocytes and
granulocytes, are also involved (Martyn et al., 2002).
Club cells commonly occur in the skin of fish of
the super-order Ostariophysi (Halbgewachs et al., 2009;
Chivers et al., 2007) and are often called alarm cells (Pollock
and Chivers, 2004), which are known to play a role in predator/prey interactions (Chivers and Smith, 1998; Pollock and
Chivers, 2004). More recently, studies have also demonstrated that club cells are part of the innate immune
system, suggesting that the alarm function evolved secondarily (Chivers et al., 2007; Halbgewachs et al., 2009).
However, the exact role that club cells play in defending
against pathogens and their integration within the complex
immune system cascade in fish is unknown.
The aim of the present study was to perform histopathologic and molecular analyses of new Henneguya species
found infecting wild specimens of P. corruscans and P. reticulatum from the Pantanal wetland (Brazil). The role of
epithelial cells, such as club cells, in the interaction with
the parasite is also discussed.
2. Materials and methods
Eighty-nine wild juvenile and adult specimens of
P. corruscans (ranging from 34 to 131 cm in length)
and 76 of P. reticulatum (ranging from 42 to 114 cm
in length) were collected from rivers in the Pantanal
wetland: Aquidauna River (20◦ 29 19 S/55◦ 46 49 W),
Miranda River (20◦ 11 27 S/56◦ 30 19 W), Paraguay
River (17◦ 54 58 S/57◦ 28 01 W) and Cuiaba River
(17◦ 50 32 S/57◦ 23 46 W). Sampling was performed
in the rainy season (spring 2001, 2002, 2003, 2004, 2009,
2010) and dry season (autumn 2003, 2004, 2005 and
2008).
Immediately after capture, the fish were transported
alive to the nearby field laboratory for measurement and
necropsy. Measurements of the spores (38 from P. corruscans and 41 from P. reticulatum) were performed based
on Lom and Arthur (1989). Spore dimensions (in ␮m) are
expressed as mean ± standard deviation (SD).
For the molecular study, the content of the plasmodium was collected in a 1.5 ml microcentrifuge tube and
DNA was extracted using a Wizard® Genomic DNA Purification kit (Promega, Madison, WI, USA), following the
manufacturer’s instructions. DNA content was determined
using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA) at 260 nm. The polymerase
chain reaction (PCR) was carried out in a final volume
of 25 ␮l, containing 10–50 ng of extracted DNA, 1× Taq
DNA Polymerase buffer (Invitrogen By Life Technologies,
Brasil), 0.2 mmol of dNTP, 1.5 mmol of MgCl2 , 0.2 pmol
of each primer, 0.25 ␮l (1.25 U) of Taq DNA polymerase
(Invitrogen By Life Technologies, MD, USA) and ultrapure
water (Barnstead/Thermolyne, Dubuque, IA, USA). The PCR
was performed in an AG 22331 Hamburg Thermocycler
(Eppendorf, Hamburg, Germany). Fragments of ∼1600 bp
of the SSU rDNA gene were amplified using the primers
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E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
Fig. 1. Plasmodium and spores of Henneguya multiplasmodialis n. sp. from gills of Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum: (A)
formalin-fixed plasmodium covering part of gill surface of Pseudoplatystoma reticulatum; GA = gill arch; GF = gill filaments; note appearance of plasmodium
divided into compartments (arrow), scale bar = 1 cm; (B) scanning electron image of spores; note prominent rim around spores (arrow), scale bar = 10 ␮m;
(C) light photomicrographs of mature fresh spore in frontal (black arrow) and lateral (white arrow) view, scale bar = 10 ␮m.
MX5–MX3 (Andree et al., 1999), fragments of ∼1000 bp
were amplified using the primers ERIB1–ACT1R and fragments of ∼1200 bp using the primers MYXGEN4f–ERIB10
(Barta et al., 1997; Andree et al., 1997; Hallett and Diamant,
2001; Diamant et al., 2004). Initial denaturation was carried
out at 95 ◦ C for 5 min, followed by 35 cycles of denaturation (95 ◦ C for 60 s), annealing (62 ◦ C for 60 s) and extension
(72 ◦ C for 120 s) and a final extended elongation step at
72 ◦ C for 5 min. PCR products were electrophoresed in 1.0%
agarose gel (BioAmerica, Miami, FL, USA) in a TBE buffer
(0.045 M Tris-borate, 0.001 M EDTA, pH 8.0), stained with
ethidium bromide and analyzed in a FLA-3000 (Fuji Photo
Film, Tokyo, Japan) scanner. Size of the amplified fragments
was estimated by comparisons with the 1 kb DNA Ladder
(Invitrogen By Life Technologies, CA, USA).
Purified PCR products of the samples of H. multiplasmodialis n. sp. were sequenced using the primer pair
MX5–MX3 and the primer pairs MC5–MC3 and MB5–MB3
(Eszterbauer, 2004). PCR products obtained from H. corruscans were sequenced using the primer pairs ERIB1–ACT1R
and MYXGEN4f–ERIB10. Sequencing was performed with
the BigDye® Terminator v3.1 cycle sequencing kit (Applied
Biosystems Inc., CA, USA) in an ABI 3730 DNA sequencing
analyzer (Applied Biosystems).
A standard nucleotide–nucleotide BLAST (blastn) search
was conducted (Altschul et al., 1997). The Bioedit (Hall,
1999) program was used to align the sequence studied
herein and for comparisons with those found in the GenBank. Phylogenetic analyses using Ceratomyxa sparusaurati
as the outgroup were conducted with the MEGA 5.0 program (Tamura et al., 2011), employing the neighbor-joining
(NJ) and maximum likelihood (ML) phylogenetic methods. The Kimura two-parameter (K2P) evolution sequence
model was used in the analysis, with gaps treated with
complete deletion. Bootstrap analysis (500 replicates [ML]
and 1000 replicates [NJ]) was employed to assess the relative robustness of the branches of the trees. The distance
analyses were performed using a p-distance model conducted with the MEGA 5.0 program.
For histological analysis, fragments of infected organs
were fixed in 10% buffered formalin and embedded in
paraffin. Serial sections 4 ␮m in thickness were stained
with Sirius Red, which was developed by Montes and
Junqueira (1991).
Table 1
Measurements, infection sites and geographic regions of Henneguya spp. compared with Henneguya multiplasmodialis n. sp., parasite of Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum; PCL = polar
capsule length; PCW = polar capsule width; NFC = number of polar filament coils; dash = no data.
Total
length
Spore
length
Spore
width
Thickness
PCL
PCW
Tail length
NFC
Infection site
and host
Locality
Reference
H. multiplasmodialis n.
sp
H. multiplasmodialis n.
sp
H. pseudoplatystoma
30.8 ± 1.3 ␮m
14.7 ± 0.5 ␮m
5.2 ± 0.3 ␮m
4.4 ± 0.1 ␮m
6.1 ± 0.1 ␮m
1.4 ± 0.1 ␮m
15.4 ± 1.3 ␮m
6–7
14.5 ± 0.4 ␮m
5.2 ± 0.2 ␮m
4.2 ± 0.3 ␮m
6.2 ± 0.2 ␮m
1.5 ± 0.2 ␮m
14.8 ± 1.4 ␮m
6–7
33.2 ± 1.9
10.4 ± 0.6
3.4 ± 0.4
–
3.3 ± 0.4
1.0 ± 0.4
22.7 ± 1.7
6–7
H. corruscans
14.3(13–15)
5.0
–
6.8(6–7)
2.0
13.7(12–15)
5–6
H. corruscans
27.6
(25–29)
26.9 ± 1.6
14.4 ± 0.4
4.7 ± 0.3
3.1 ± 0.4
5.5 ± 0.3
1.6 ± 0.1
13.5 ± 1.5
5–6
H. eirasi
37.1 ± 1.8
12.9 ± 0.8
3.4 ± 0.3
3.1 ± 0.1
5.4 ± 0.5
0.7 ± 0.1
24.6 ± 2.2
12–13
Brazilian
Pantanal
wetland
Brazilian
Pantanal
wetland
Fish farms in
states of São
Paulo and Mato
Grosso do Sul,
Brazil
State of Paraná,
Brazil
Brazilian
Pantanal
Wetlend
Brazilian
Pantanal
wetland
This study
30.6 ± 1.2 ␮m
Large cists in
the gills of P.
corruscans
Large cists in
the gills of P.
reticulatum
Gills of hybrid
of the genus
Pseudoplatistoma
H. chydadea
17.6–20.0
8.8–11.2
3.2–5.6
3.6–4.0
3.2–4.4
1.2–1.6
8.0–9.6
9–10
H. schizodon
27–30
12–14
3–4
–
5–6
1–1.5
15–17
8–10
H. tchangi
27.1
(24.4–30.8)
10.9
(9.2–11.6)
7.6
(7.2–7.6)
77(7–8)
4.9(3.6–5.6)
3.2(2.4–4.0)
16.2(15.6–19.2)
–
H. hemibagri
26.2(24.8–28.4) 13.2(12.8–14.0) 4.3
(4.0–6.4)
3.1(2.8–3.7)
4.0(2.8–4.8)
1.9(1.6–2.4)
13(12–14.4)
–
Gills of P.
corruscans
Gills of P.
corruscans
Gill filaments o
P. corruscans
and P.
reticulatum
Gills of
Astyanax
altiparanae
Kidney of
Schizodon
fasciatum
Urinary
Bladder of
Schizotorax
davidi
Gills of Mystus
macropterus
State of São
Paulo, Brazil
Brazil
This study
Naldoni
et al.
(2009)
Eiras et al.
(2009)
This study
Naldoni
et al.
(2011)
Barassa
et al.
(2003)
Eiras et al.
(2004)
China
Eiras
(2002)
China
Eiras
(2002)
E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
Species
113
114
E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
Fig. 2. Histological sections of gill arch from Pseudoplatystoma reticulatum infected by Henneguya multiplasmodialis n. sp.: (A) development of plasmodium
on gill arch; note expansion of tissues from gill arch (white arrow) forming wall of plasmodium (black arrow) and septa (thin arrows) that separate the
multiple compartments (cp); (B) other extremity of plasmodium showing growth on gill filaments (black arrow); note in (A) and (B), plasmodium with
multiple compartments (cp) separated by septa (thin arrows) (B = born tissue, ct = connective tissue); Sirius red stain, scale bar = 500 ␮m.
For scanning electron microscopy, histological sections of plasmodia fixed and embedded using routine
histological procedures were cut into sections of 10 ␮m,
deposited on a coverslip coated with albumin, dried in an
oven for 48 h, deparaffinized with xylol, re-dehydrated,
post-fixed with 1% OsO4 in cacodylate buffer for 30 min
at 4 ◦ C, washed in the same buffer, dehydrated in ethanol,
critical-point dried in CO2 , covered with metallic gold and
examined in a Joel JMS 35 microscope operated at 10 kV
(Adriano et al., 2002).
3. Results
Three of the 89 specimens of P. corruscans (3.3%) and two
of the 76 specimens of P. reticulatum (2.6%) had plasmodia
of an unknown Henneguya species infecting the gills.
3.1. Description of Henneguya multiplasmodialis n. sp.
(Figs. 1–5)
The plasmodia were white and large, measuring up to
2.5 cm, and were found covering part of the gill surface.
The plasmodia were divided into compartments (Fig. 1A),
which contained spores in different stages of development
(Fig. 1B and C). The histological study revealed that the gill
arch was the site of parasite development, with the plasmodium growing toward the gill filaments and covering
part of these filaments (Figs. 1A and 2B). Externally, the wall
of the plasmodium was enveloped by a stratified epithelium (continuation of the epithelium that covers the gill
arch), which was composed by several cell types, with a
predominance of mucous and club cells (Figs. 2A and 3A).
Just below this stratified epithelium, there was a layer
of connective tissue, which was internally coated with
a layer of stratified epithelium also rich in club cells
(Fig. 3). Internally, the plasmodium had a network of septa
formed by connective tissue, dividing the plasmodium into
compartments (Figs. 2–4). The connective tissue of the
walls of the septa was covered with stratified epithelium
on both sides and numerous club cells were observed
(Figs. 3 and 4). Inflammatory infiltrate was found in the
tissue surrounding the plasmodium as well as in the septa
(Fig. 4B).
The network of septa gave rise to compartments of different sizes – some completely individualized and others
connected to one another (Figs. 2 and 3). In the tissue of
the walls of the septa, small compartments were found,
containing only initial development stages (Fig. 3).
E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
Fig. 3. Histological sections of plasmodium of Henneguya multiplasmodialis n. sp. from gills of Pseudoplatystoma reticulatum: (A) typical stratified
squamous epithelium similar to tissues of gill arch, with mucous (large
white arrow) and cells club cells (arrowheads) supported by layer of
connective tissue (ct) covering plasmodium; internally, network of septa
(sp) composed of connective tissue (ct) covered by stratified epithelium
(black arrow) and numerous club cells (arrowhead), dividing plasmodia
into compartments (cp); Sirius red stain, scale bar = 100 ␮m; (B) detail
of plasmodium showing septum composed of connective tissue (ct) and
stratified epithelium (black arrows) with several club cells (arrowheads)
delimiting small compartments (cp) containing spores in initial developmental stages (ids); Sirius red stain, scale bar = 20 ␮m.
Mature spores were ellipsoidal in the frontal view and
biconvex laterally, with a prominent rim surrounding the
spores at the point of junction of the two valves and
the tail bifurcated only in the end. The polar capsules
were elongated, of equal size, occupied only the anterior
half of the spores and the polar filaments had 6–7 turns
(Figs. 1B, C and 5). The measurements of the spores and
polar capsules are displayed in Table 1.
The sequencing of the 18S rDNA gene from the spores
of H. multiplasmodialis n. sp. obtained from gills of P. corruscans and P. reticulatum resulted in a total of 1560 and
1147 base pairs, respectively. As the spores of H. multiplasmodialis n. sp. resemble those of H. corruscans, sequencing
of the 18S rDNA gene of the spores of H. corruscans parasite
of P. corruscans caught in the Brazilian Pantanal wetland
was provided to avoid any taxonomic pendency between
the species, resulting in 1913 base pairs.
The comparison between the sequence from H. multiplasmodialis n. sp. parasite of P. corruscans with that
of H. multiplasmodialis n. sp. parasite of P. reticulatum
115
Fig. 4. Histological sections of plasmodium of Henneguya multiplasmodialis n. sp. from gills of Pseudoplatystoma reticulatum: (A) detail of septum
(sp) showing squamous epithelial cells (black arrows) in contact with plasmodium compartment (cp), club cells (white arrows) below this cell layer
and connective tissue (*); Sirius red stain, scale bar = 20 ␮m; (B) plasmodium compartments (cp) containing mature spores (ms), spores in initial
developmental stages (ids), inflammatory process () and club cells (white
arrow) in septum (sp); Sirius red stain, scale bar = 50 ␮m.
demonstrated 99.7% similarity, with only four distinct
bases. In the position 116 occurred one transversion (adenine for P. corruscans and cytosine for P. reticulatum).
Transitions occurred in the position 328 (guanine for P. corruscans and adenine P. reticulatum) and in the positions 335
and 435 (adenine for P. corruscans and guanine for P. reticulatum). Moreover, 95.8% similarity was found between the
sequence from H. corruscans and that from H. multiplasmodialis n. sp. parasite of P. reticulatum and 93.0% similarity
was found between the sequence from H. corruscans and
that from H. multiplasmodialis n. sp. parasite of P. corruscans.
These sequences were not identical to any of the Myxozoa
available in the GenBank.
Both phylogenetic methods (NJ and ML), with 550 informative characters and complete deletion, revealed that the
Henneguya species clustered into six main monophyletic
clades (Clades A–F) (Figs. 6 and 7). Clade A was formed
by Henneguya spp. parasites of siluriform hosts and was
further divided into Clades A1 and A2. Clade A1 was composed of Henneguya spp. parasites of siluriform hosts of
the family Ictaluridae. Clade A2 was composed of H. eirasi,
116
E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
Fig. 5. Schematic representation of mature spore of Henneguya multiplasmodialis n. sp., scale bar = 5 ␮m.
H. corruscans and H. multiplasmodialis n. sp., all parasites of
fish of the genus Pseudoplatystoma (Siluriformes: Pimelodidae). Clade B was composed of two parasites of percids, two
parasites of esocids and Henneguya weishanensis, for which
there are no data regarding host order/family. Clades C–E
were formed by Henneguya spp. parasites of fish belonging
to different families of the order Perciformes. Clade F was
formed by parasites of salmonids (F1) cyprinids (F2) and
siluriforms of the family Bagridae (F3) (Figs. 6 and 7).
4. Discussion
H. multiplasmodialis n. sp. was compared with South
American Henneguya spp. described parasitizing freshwater fish (Eiras et al., 2008, 2009; Naldoni et al., 2009, 2011;
Azevedo et al., 2011). Among the approximately 40 species
described thus far in South American fish, the spores of H.
corruscans infecting the gills of P. corruscans demonstrate
the greatest similarity to the spores of H. multiplasmodialis
n. sp. However, the spores of other species, such as Henneguya chydadea Barassa, Arana et Cordeiro, 2003, parasite
of the gills of Astyanax altiparanae (Characidae) and Henneguya schizodon Eiras, Malta, Varela et Pavanelli, 2004,
parasite of the kidney of Schizodon fasciatum (Anastomidae), also resemble those of H. multiplasmodialis n. sp.
Although H. multiplasmodialis n. sp. infects the same
host and its spore morphology is similar to that of H. corruscans, there are differences in the morphology of the
plasmodia and development sites. H. corruscans produces
small plasmodia in the gill lamellae, while H. multiplasmodialis n. sp. forms large plasmodia on the gills. Moreover,
there are small differences in the morphometric aspects
of the spores. H. multiplasmodialis n. sp. has a larger spore
body (30.8 ± 1.3 and 30.6 ± 1.2 ␮m in that infecting P. corruscans and P. reticulatum, respectively, in comparison
to 27.6 ␮m [25–29] in H. corruscans). H. multiplasmodialis n. sp. has a longer caudal process (15.4 ± 1.3 and
14.8 ± 1.4 ␮m in that infecting P. corruscans and P. reticulatum, respectively, in comparison to 13.7 ␮m [12–15] in
H. corruscans). H. multiplasmodialis n. sp. has smaller polar
capsules (6.1 ± 0.1 and 6.2 ± 0.2 ␮m in that infecting P. corruscans and P. reticulatum, respectively, in comparison to 6.8
[6–7] in H. corruscans). H. corruscans has wider polar capsules (2 ␮m) in comparison to H. multiplasmodialis n. sp.
(1.4 and 1.5 ␮m in that infecting P. corruscans and P. reticulatum, respectively). H. multiplasmodialis n. sp. has thicker
spores (4.4 ± 0.1 and 4.2 ± 0.3 ␮m in that infecting P. corruscans and P. reticulatum, respectively, in comparison to 4 ␮m
in H. corruscans). H. multiplasmodialis n. sp. has more coils
in the polar filaments (6–7 in comparison to 5–6 in H. corruscans). Moreover, there is a smaller proportion between
polar capsule size and spore size in H. multiplasmodialis n.
sp in comparison to H. corruscans.
With regard to H. chydadea, the differences are the host
species, aspects of the plasmodia, the smaller total length of
the spores (17.6–20 ␮m), smaller body length of the spores
(8.8–11.2 ␮m) and smaller polar capsule size (3.2–4.4 ␮m)
in comparison to H. multiplasmodialis n. sp. Moreover, H.
schizodon differs from H. multiplasmodialis n. sp. in the
smaller body length of the spores (3.3 ␮m), smaller polar
capsule (5.4 ␮m), infection site and host species.
When the features of H. multiplasmodialis n. sp. are compared with nearly all Henneguya species known so far,
morphometric similarity in the total length of the spores
is observed in Henneguya tchangi Ma, 1988, parasite of
the urinary bladder of Schizothorax davidi (Cyprinidae), and
in Henneguya hemibagri Tchang and Ma, 1993, parasite of
the gills of Mystus macropterus (= Hemibagrus macropterus)
(Bagridae), both of which are from China (Eiras, 2002).
However, differences were observed in other morphometric values and the hosts are phylogenetically very
different.
The BLAST search using the 18S rDNA of H. multiplasmodialis n. sp. and H. corruscans revealed that neither
species is identical to any of the Myxozoa available in the
GenBank. In the comparison between H. corruscans and H.
multiplasmodialis n. sp., in addition to the morphologic differences pointed out above, the sequencing of the 18S rDNA
gene from spores of H. corruscans revealed a difference
of 7.0% and 4.2% when compared to the sequences from
spores of H. multiplasmodialis n. sp., parasite of P. corruscans and P. reticulatum, respectively. These data lead us to
E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
117
Fig. 6. Neighbor-joining tree showing relationship between Henneguya multiplasmodialis n. sp. and other Henneguya spp. based on partial 18S rDNA
sequences; GenBank accession numbers and host taxa given in front of species names (S = Siluriformes, P = Perciformes, E = Esociformes C = Cypriniformes
and SL = Salmoniformes), based on Froese and Pauly (2009); numbers above nodes indicate bootstrap confidence levels.
consider H. corruscans and H. multiplasmodialis n. sp. as
distinct species.
Both phylogenetic methods revealed a strong tendency
among Henneguya species to form clades based on the order
and, more specifically, family of the host fish, corroborating the observation offered by Ferguson et al. (2008). This
was quite evident in Clade A, which appears as a monophyletic unit composed of nine Henneguya parasites of fish
of the order Siluriformes in both the neighbor-joining and
the maximum likelihood topologies. Six of these species
are parasites of ictalurid hosts in North America (Clade A1)
and three (H. eirasi, H. corruscans and H. multiplasmodialis
n. sp.) are parasites of pimelodid hosts in South America (Clade A2). This tendency toward clustering parasites,
especially according to the family of the host fish, was seen
in the other clades. Clade B clustered two Henneguya parasites of fish of the family Esocidae, two species parasites
of perciforms of the family Percidae plus H. weishanensis,
for which there is no data regarding the host. Clades C–E
exclusively clustered Henneguya species parasites of perciforms, but involving hosts from different families. Although
exhibiting a slightly different topology in the two trees,
Clade F clustered Henneguya species parasites of salmonids
(F1), cyprinids (F2) and, surprisingly, again parasites of siluriforms, however, now with Henneguya parasites of the
family Bagridae (F3).
The present investigation and that carried out by
Naldoni et al. (2011) are the first phylogenetic studies on
Henneguya parasites of pimelodid hosts. Future phylogenetic studies will demonstrate the accurate position of
Henneguya species as well as other myxosporean genera
that are parasites of pimelodid hosts in relation to parasites
of other families of fish and in other geographic regions.
It is important to point out that, in the present analyses,
all Henneguya species parasites of hosts from the same family were clustered together and that, in Clades A, D, E and
F (F1 and F2), there was a clear tendency toward the clustering of Henneguya species according to order of the host.
118
E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
Fig. 7. Maximum likelihood phylogenetic tree showing relationship between Henneguya multiplasmodialis n. sp. and other Henneguya spp. based on
partial 18S rDNA sequences; GenBank accession numbers and host taxa given in front of species names (S = Siluriformes, P = Perciformes, E = Esociformes
C = Cypriniformes and SL = Salmoniformes), based on Froese and Pauly (2009); numbers above nodes indicate bootstrap confidence level.
However, in some cases, the topology of the trees seems to
contradict these data, as seen in Clade B, in which parasites
of esociforms (Henneguya psorospermica and Henneguya
lobosa) clustered with parasites of perciforms (Henneguya
creplini and Henneguya doori), and Clade F, in which parasites of Hemibagrus nemurus and an Asiatic siluriform
of the family Bragridae (Henneguya basifilamentalis and
Henneguya mystusia) clustered in a monophyletic clade
together with parasites of cyprinids (Henneguya doneci and
Henneguya cutanea) and parasites of salmonids (Henneguya
nuesslini, Henneguya salmonicola and Henneguya zschokkei).
However, both Clades B and F further divided to form clades
in which there was a clear tendency toward clustering
based on the family of the host.
This tendency observed herein does not confirm coevolution between hosts and their myxosporean parasites.
However, the topology of the trees allows one to speculate that, in most cases, the ancestors of current hosts were
infected by the ancestors of current parasites.
The specimens of H. multiplasmodialis found infecting
P. corruscans and P. reticulatum exhibited 99.7% similarity.
There is no specific value for determining how much difference in 18S rDNA is necessary for the inter-specific and/or
intra-specific differentiation of Myxozoa. In a sample of
E.A. Adriano et al. / Veterinary Parasitology 185 (2012) 110–120
Myxobolus cerebralis from different hosts and geographic
areas, Andree et al. (1997) found 99.2–99.3% similarity.
Hervio et al. (1997) compared Kudoa thyrsites, a parasite of
the Atlantic salmon and tubesnout and found 99.93% similarity between isolates. These findings were considered
intra-specific differences (Andree et al., 1997; Hervio et al.,
1997). Easy et al. (2005) found 97.9% similarity between
intracellular and intercellular plasmodia of Myxobolus procerus, parasite of Percopsis omiscomaycus, and the results
led to the designation of a new species (Myxobolus intramusculi) for the intracellular form. Thus, the high degree of
molecular similarity (99.7%.) and the morphological data
on the samples of H. multiplasmodialis found infecting P.
corruscans and P. reticulatum led to considering these samples as belonging to a single species.
The histological analyses revealed that the plasmodia
were coated by a layer of connective tissue, which is a
common characteristic of myxosporean infection (SitjáBobadilla, 2008a). However, this connective tissue was
covered by stratified epithelium rich in club cells on both
the internal and the external faces. Another peculiar feature of this infection was the presence of septa, formed
by connective tissue covered by stratified epithelium on
both faces, also containing numerous club cells. These septa
are believed to be result of the host reaction to control
the infection. However, the parasite apparently used the
septa as substrate to continue its development, which gives
this parasite an uncommon and interesting host–parasite
relationship.
Club cells, which atypically appeared distributed in
epithelial tissue of the external plasmodium wall and walls
of the septa in the present study, commonly occur in the
skin of fish of the super-order Ostariophysi (Halbgewachs
et al., 2009; Chivers et al., 2007). For some time, club
cells were often called alarm cells (Pollock and Chivers,
2004) with a role in predator/prey interactions (Chivers and
Smith, 1998; Pollock and Chivers, 2004). However, recent
studies have demonstrated that club cells play a role in the
immune system of fish. Considering the action of stressors,
Iger et al. (1994) state that, in addition to the production
and release of alarm substances, club cells influence the cell
kinetics of filament cells in skin epithelium and are engaged
in the elimination of leukocytes. Chivers et al. (2007) found
a significant increase in club cell numbers in Pimephales
promelas exposed to parasites, suggesting that club cells
may serve as a first line of defense for the protection of
underlying tissues and that the alarm functions may have
evolved secondarily. Halbgewachs et al. (2009) found that
P. promelas exposed to cortisol (a known immunosuppressant) had a suppressed immune system and significantly
lower number of club cells, indicating that the club cells
of Ostariophysan fish are part of the innate immune system and also suggesting that the alarm function evolved
secondarily.
In many myxosporean species, encapsulation of the
plasmodia by connective, fibrous and/or epithelioid tissue
layers is a common effort to isolate the parasite and prevent
its dispersal to adjacent tissues (Sitjà-Bobadilla, 2008b).
The situation described in the present study, with numerous club cells in the epithelial tissue of the plasmodial
wall and septa of H. multiplasmodialis may be related to
119
the immunological function of these cells, as suggested
by Chivers et al. (2007) and Halbgewachs et al. (2009).
Thus, the involvement of club cells represents an uncommon immunological response to myxosporean infection
and may be important to the understanding of the function of these cells. However, definitive knowledge on the
immunologic role club cells play in the immune system in
fish remains a challenge and the involvement of these cells
in this kind of infection needs to be further explored in
order to cast light on host–parasite interactions involving
infection by Myxosporea.
The present study was performed on wild fish and the
impact of infection by H. multiplasmodialis on farmed P. corruscans and P. reticulatum is unknown. However, the large
size of the plasmodia may make this parasite an important
pathogen to these fish species in fish farms and the presence and dispersal of this organism needs to be monitored
closely by commercial fish farmers.
Acknowledgments
The authors are grateful to Ricardo Afonso Torres de
Oliveira (CEPTA/ICMBio) for help in dissecting the fish, Dr.
Laerte Batista de Oliveira Alves, manager of the National
Center for Research and Conservation of Continental Fish
(CEPTA/ICMBio), for support during the field work and Dr.
José Augusto Ferraz de Lima, manager of Pantanal National
Park, for providing the site for the field laboratory and hosting during the fieldwork.
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