Architecture of Paradiplozoon homoion: A diplozoid monogenean exhibiting highly-developed equipment for ectoparasitism
Architecture of Paradiplozoon homoion: A diplozoid monogenean exhibiting highly- developed equipment for ectoparasitism
Iveta HodovaÂ 0 1
Radim Sonnek 0 1
Milan Gelnar 0 1
Andrea ValigurovaÂ ☯ 0 1
☯ These authors contributed equally to this work. 0 1
0 Department of Botany and Zoology, Faculty of Science, Masaryk University , KotlaÂřsk aÂ 2, Brno , Czech Republic
1 Editor: Claude Prigent, Institut de Genetique et Developpement de Rennes , FRANCE
Diplozoidae (Monogenea) are blood-feeding freshwater fish gill ectoparasites with extraordinary body architecture and a unique sexual behaviour in which two larval worms fuse and transform into one functioning individual. In this study, we describe the body organisation of Paradiplozoon homoion adult stage using a combined approach of confocal laser scanning and electron microscopy, with emphasis on the forebody and hindbody. Special attention is given to structures involved in functional adaptation to ectoparasitism, i.e. host searching, attachment and feeding/metabolism. Our observations indicate clear adaptations for blood sucking, with a well-innervated mouth opening surrounded by sensory structures, prominent muscular buccal suckers and a pharynx. The buccal cavity surface is covered with numerous tegumentary digitations that increase the area in contact with host tissue and, subsequently, with its blood. The buccal suckers and the well-innervated haptor (with sclerotised clamps controlled by noticeable musculature) cooperate in attaching to and moving over the host. Putative gland cells accumulate in the region of apical circular structures, pharynx area and in the haptor middle region. Paired club-shaped sacs lying laterally to the pharynx might serve as secretory reservoirs. Furthermore, we were able to visualise the body wall musculature, including peripheral innervation, the distribution of uniciliated sensory structures essential for reception of external environmental information, and flame cells involved in excretion. Our results confirm in detail that P. homoion displays a range of sophisticated adaptations to an ectoparasitic life style, characteristic for diplozoid monogeneans.
Data Availability Statement: All relevant data are
within the paper.
Funding: We acknowledge the financial support
from Czech Science Foundation Project No. P505/
12/G112 (ECIP - European Centre of
Ichthyoparasitology). The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Authors acknowledge support from the
Department of Botany and Zoology of the Faculty
Monogenea Bychowsky 1937 are among the most species-rich groups of fish parasites [
Monogenean parasites display a direct life cycle, lacking alternation of generations or hosts.
Host specificity in the group is well defined, with morphological adaptations to the attachment
organs often restricting species to a particular host and/or a very narrow niche [
Blood-feeding freshwater fish gill ectoparasites of the family Diplozoidae occupy a unique position
of Science at Masaryk University towards the
preparation of this manuscript.
Competing interests: The authors have declared
that no competing interests exist.
amongst monogenean taxa as they exhibit extraordinary body morphology and have a life
cycle involving permanent fusion of two larval worms that subsequently transform into a
single individual. As such, they represent an attractive model for evolutionary and morphological
studies. The first morphological studies on diplozoids were published more than 120 years ago
[3±5]. To date, the extensive work of Bovet [
] and Khotenovsky [
] still represents the most
comprehensive morphological and taxonomical studies of diplozoid monogeneans. More
recent reviews provide useful information on general and functional morphology of
]. Numerous studies have already targeted their life cycle and pairing process [10±
17], while the other focused on molecular biological [18±22] and karyological [
of representatives from the family Diplozoidae. On the top of that, few immunomicroscopical
observations of the diplozoid nervous system were published [
]. Recent biochemical
analyses deal with the blood digestion in diplozoids [
Paradiplozoon homoion is a generalist diplozoid species parasitising a number of cyprinid
fish and, as such, represents a suitable model parasite for a range of studies. To date, most
studies have concentrated on Paradiplozoon spp. genetic characterisation and identification, its life
cycle under experimental conditions [
], abnormalities in the attachment apparatus and
fluctuating asymmetry [30±32], morphology of the digestive tract [
] and excretory system [
ultrastructure of the tegument and attachment structures [
]. However, only few fluorescent
or methodical studies focusing on Paradiplozoon spp. were published to date [36±38]. A recent
study visualised the trace element accumulation sites in Paradiplozoon adults [
Though molecular and biochemical studies are becoming increasingly prevalent, routine
microscopic methods, such as electron microscopy and confocal laser scanning microscopy, in
combination with immunohistochemistry, still provide a strong tool for investigating different
aspects of a parasite's biology, including its functional morphology and any adaptive
mechanisms. A number of structures and systems have repeatedly been analysed through
microscopy, including the parasite's surface and tegumental structures, the attachment organs with a
significant role in host-parasite interactions, its nervous and sensory system, the body's
musculature and mobility, along with its reproductive, excretory and alimentary systems [
majority of these studies, however, were based on a single microscopic approach or were
narrowly focused on a particular structure or system. Apparently, the investigation of
morphological adaptations to parasitism in metazoan organisms requires a more complex approach using
a combination of microscopy methods (e.g. [
]). Hence, the aim of this study was to provide
a complex analysis of P. homoion adult-stage body architecture in relation to adaptation to an
ectoparasitic life-style. Here, we describe those structures involved in parasite host-attachment,
movement, host blood-sucking and excretion.
Material and methods
Samples of Paradiplozoon homoion (Bychowsky et Nagibina, 1959) were collected from the
gills of roach Rutilus rutilus (L.), bleak Alburnus alburnus (L.) and gudgeon Gobio gobio (L.).
The fish were caught by electrofishing or using gillnets in MuÏsov lowland reservoir (southern
Moravia, Czech Republic) during the year 2013. The fish collection was carried out by external
collaborators from Institute of Vertebrate Biology, Academy of Science, Czech Republic (wild
fish collection of Institute of Vertebrate Biology is approved by certificate issued by Ministry of
Agriculture No. 3OZ31162/2011-17214). Fish were transported in aerated original water to the
laboratory facilities of Faculty of Science, Masaryk University, Brno, Czech Republic (Permit
No. 16256/2015-MZE-17214). Fish were sacrificed by stunning and cutting the spine, and all
efforts were made to minimize suffering (in accordance with the Act No. 246/1992 Coll., on
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Prevention of Cruelty to Animals). Gills were removed according to the standard protocol [
and examined. This study was carried out in strict accordance with Act No.207/2004 of the
Collections of Laws of the Czech Republic on the Protection, Breeding and Use of
Experimental Animals. The study was approved by the Animal Care and Use Committee at the Faculty of
Science, Masaryk University, Czech Republic and followed to Ministry of Education, Youth
and Sports (Permit No. 13715/2011-30).
Confocal laser scanning microscopy (CLSM)
Diplozoid worms were flat-fixed between microscopic slides in freshly prepared 4%
paraformaldehyde in 0.1 phosphate buffered saline (PBS) for 4 h at 4ÊC and then transferred into
fresh fixative. For the labelling of filamentous actin (F-actin), specimens were washed for 24 h
in antibody diluent (AbD) containing 0.1 M PBS, 0.5% Triton X-100, 0.1% bovine serum
albumin and 0.1% NaN3 at pH 7.4. The samples were subsequently incubated in
phalloidin±tetramethylrhodamine B isothiocyanate (phalloidin-TRITC; Sigma-Aldrich, Czech Republic) and
AbD (10μl/1ml) for 48 h at room temperature and then washed again in AbD for 24 h at 4ÊC.
For double fluorescent labelling, specimens were fixed, washed and permeabilised for 48 h in
0.5% Triton X-100 (Sigma-Aldrich, Czech Republic). The samples were then incubated with
mouse monoclonal anti-α-tubulin antibody (Clone B-5-1-2, Sigma-Aldrich, Czech Republic)
at 4ÊC for six days, washed for 24 h in AbD and finally incubated with mouse polyvalent
immunoglobulins (1:125) in PBS with 1% BSA at 37ÊC for four days. The specimens were then
washed and incubated in TRITC-phalloidin as described above. Controls were labelled with
FITC-conjugated secondary antibody only without the primary antibody. For localisation of
cell nuclei, preparations were counterstained with either DAPI and mounted in 9:1 glycerol/
PBS containing 2.5% 1,4-diazabicyclo [2.2.2] octane (DABCO, Sigma-Aldrich) or with
Hoechst and mounted in VECTASHIELD1 (Vector Laboratories, USA). Gomori trichrome
staining was used for 3D visualisation of sclerotised structures [
]. The hydrochloric carmine
staining of whole-mount preparations follows published protocols [
All slides were examined and documented using an Olympus IX81 microscope equipped
with a laser-scanning FluoView 500 confocal unit (Olympus FluoView 4.3 software) or an
Olympus BX60 microscope with FluoView 1.26 (Fluoview 2.0 software). Some confocal
micrographs were processed using Fiji software (an image-processing package based on ImageJ,
developed at the National Institute of Health).
For scanning electron microscopy (SEM), the specimens were first washed several times in tap
water to remove any fish mucus, fixed in either hot 4% formaldehyde or 4% glutaraldehyde at
4ÊC for 24 h and postfixed for 1 h in 1% OsO4. The samples were subsequently dehydrated
through a graded ethanol series and dried in a Pelco CPD II critical point drying apparatus
(Bal-Tec) using liquid CO2. The dried samples were finally mounted on aluminium stubs with
double-sided adhesive tape or disks, coated with gold in a Polaron E5100 sputter coating unit
(Balzers) and examined in a MIRA 3 TESCAN SEM operating at 15 kV.
This study focuses on individuals that have already paired and formed the juvenile/adult
stages. As in other members of the family Diplozoidae, the body of the P. homoion in the adult
stage typically resembles a letter X. This X-shaped body is comprised of two forebodies and
two hindbodies along with the haptors of the two fused individuals (Fig 1A).
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Fig 1. Surface topology of forebody tegumentary structures in Paradiplozoon homoion adults. A) Overall view of
an adult. The white circles indicate the area of the mouths and the white rectangles the area of the haptors. SEM. B)
Ventral view of the forebody, with a subterminal mouth and two apically located, round projections. SEM. C)
Micrograph of an opened mouth revealing two buccal suckers and uniciliated sensory structures. SEM. D) The mouth
area covered by numerous uniciliated sensory structures and pits. SEM. E) Detail of the mouth border (the area
marked by the white rectangle in D). Note the tegument with numerous pits and sensory structures. SEM. F) Detail of
the buccal cavity surface enlarged by numerous foliate and tubular digitations. SEM. asterisks±round projections, black
arrowheads±pits, black arrows±tubular digitations, bs±buccal sucker, fb 1± forebody 1, fb 2 –forebody 2, hb 1 –
hindbody 1, hb 2 –hindbody 2, mo±mouth, white arrowheads±uniciliated sensory structures, white arrows±foliate
The mouth is situated subterminally on the ventral side of the forebody, with two rounded
projections protruding above it (Fig 1B). The surface of the tegument around the mouth bears
numerous uniciliated sensory structures and pits (Fig 1C±1E). The buccal cavity is equipped
with two well-developed buccal suckers (Fig 1C). The mouth is limited by a brush border (Fig
1C). The surface of the buccal cavity is substantially enlarged by abundant foliate and tubular
digitations (Fig 1F).
Fluorescent labelling of F-actin with phalloidin allowed visualisation of the forebody
muscular layer arrangement (Fig 2A±2D), revealing muscular organs such as buccal suckers and
pharynx (Figs 2B and 3A±3H). The forebody wall musculature is arranged in three layers;
an external circular muscle layer, a deeply situated longitudinal layer and several diagonal
layers (Figs 2A±2C and 3A). In addition, numerous perpendicular, long and thin muscles
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Fig 2. Forebody wall musculature of Paradiplozoon homoion adults. A-B) General views of the body wall
musculature arrangement. CLSM, phalloidin-TRITC. C-D) Detail of the body wall musculature. CLSM,
phalloidinTRITC (C) and phalloidin-TRITC/DAPI (D). A-B are composite views created by flattening a series of optical sections,
while C-D represent single optical sections. black arrowheads±flame cells, bs±buccal suckers, cm±circular muscles, dm±
diagonal muscles, lm±longitudinal muscles, mo±mouth opening, pm±perpendicular muscles.
interconnect the tegument and parenchyma (Fig 2D). The apex of the forebody is segmented
by muscular trabeculae and filled with numerous prominent nuclei (Fig 3B and 3C).
The most prominent structures in the apical part of the forebody were the two oval buccal
suckers (Fig 3A±3C). The border of the bowl-like buccal sucker is clearly demarcated by
densely arranged radial muscle fibres, while a transverse U-shaped muscle bundle is visible at
its centre (Fig 3B). The suckers are controlled by muscles connected to the body wall
musculature and individual muscle fascicles oriented towards the forebody apex or the pharynx, with
some localised obliquely between the two suckers (Fig 3B). The pharynx is a conspicuous
elongated muscular organ situated in the medial plane near to the posterior margins of the buccal
suckers. The pharynx is composed of lumen passing through its central part and a muscular
wall, with massive muscle bundles enabling movement during blood sucking (Fig 3A±3H).
The circular musculature of the pharyngeal lumen is fixed to the pharynx wall by numerous
trabeculae (Fig 3E and 3F). Numerous cell nuclei located between the radial muscular
trabeculae appear to be those of glandular pharyngeal cells (Fig 3C and 3F). The pharynx wall
musculature is arranged into layers of radial, longitudinal and circular fibres (Fig 3A±3G). The
anterior part of the pharynx is lined with an obvious muscular collar bearing four F-actin-rich
oval structures (Fig 3H).
Phalloidin staining enabled detection of two groups of circular structures (of unknown
origin and function) symmetrically arranged in the middle of the forebody apical end (Figs 4A,
4B and 5A), sensory structures (Figs 4A, 4B and 5B) and flame cells, these representing the
basic elements of an excretory system (Fig 5B and 5C).
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Fig 3. The main muscular organs in the forebody of Paradiplozoon homoion adults. A) General view of the
forebody. CLSM, phalloidin-TRITC. B-C) The forebody in median plane optical sectioning. Note the muscles forming
thin trabeculae in the forebody apical part and the numerous cells with prominent nuclei located between the
trabeculae (C). CLSM, phalloidin-TRITC (B) and phalloidin-TRITC/DAPI (C). D) Detail showing the pharynx muscle
wall arrangement and the muscles controlling pharynx movement. CLSM, phalloidin-TRITC/DAPI. E-F) Various
optical sections of the pharynx showing the transverse trabeculae and pharyngeal cell nuclei. G-H) Apical view of the
pharynx. CLSM, Phalloidin-TRITC (E, H) and phalloidin-TRITC/DAPI (F, G). A-H are composite views created by
flattening a series of optical sections. black arrowheads±muscles controlling the pharynx, black arrows±muscles
controlling the buccal suckers, bs±buccal suckers, dm±diagonal muscles of the body wall, lm±longitudinal muscles of
the body wall, ph±pharynx, pm±muscle fibres fixed perpendicularly to the tegument, um±U-shaped muscle bundle of a
sucker, white arrowheads±two sensory structures localised in the area of the round projections, white arrows in E, F±
trabeculae, white arrows in H±four round structures.
Immunofluorescent labelling of α-tubulin localised the forebody nerve cords, and especially
the longitudinal dorsal and ventral cords and transverse connective cords (Fig 5D and 5E).
The α-tubulin antibody also enabled visualisation of the excretory system, comprising a
number of flame cells distributed between the transverse nerve connectives in the apical part (Fig
5D and 5E). The apical part of the forebody, including the U-shaped middle part of buccal
suckers (Fig 6A), appears to be tubulin-rich (Figs 6A, 6B, 6D, 6E, 6G, 7B and 7C). Several
preparations revealed an ability of parasite to retract the pharynx and buccal suckers into the body,
resulting in a half-closed appearance of the mouth (Figs 6B, 6D±6G and 7B). Concentration of
α-tubulin around the mouth border confirms rich innervation of this region (Fig 6D, 6E and
6G), with regularly arranged muscle fibres anchored to the peripheral rim (Fig 6F). Numerous,
regularly distributed uniciliated sensory structures with a raised circular rim and one long
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Fig 4. Forebody of Paradiplozoon homoion adults, with emphasis on the apical sensory and circular structures. A)
The apical part of the forebody, revealing the distribution of sensory structures and a group of circular structures
(encircled). The white rectangle indicates the area of the apical part of the pharynx containing four round structures.
CLSM, phalloidin-TRIC. The micrograph is a composite view created by flattening a series of optical sections. B) Single
medial plane optical section of the forebody. CLSM, phalloidin-TRIC/DAPI. black arrows±muscles controlling the
pharynx, bs–buccal sucker, ph±pharynx with four circular openings, white arrowheads±sensory structures, white arrows±
muscles controlling the buccal suckers.
cilium were detected in the mouth area (Fig 6C). The circular rim is formed of F-actin (Fig 6B
and 6D), while the cilium is strongly labelled for α-tubulin (Fig 6D, 6E and 6G).
The forebody surface is obviously folded, creating transverse ridges with shallow pits
(Fig 7A). The tegument covering the forebody bears numerous uniciliated sensory structures
distributed along the transverse ridges at regular intervals (Fig 7A, 7B and 7E±7H).
Immunofluorescent labelling of α-tubulin indicated that innervation of the forebody comprises
longitudinal nerve cords interconnected with transverse cords (Fig 7C and 7D). Peripheral
innervation of the forebody forms a dense mesh of fine nerve fibres surrounding the main
nerve cords (Fig 7B±7D). The peripheral nerve fibres are associated with tegumentary ridges
with individual uniciliated sensory structures (Fig 7B, 7F and 7G). The arrangement of the
single uniciliated receptor is similar to the sensory structures localised around the mouth. The
circular rim is formed of F-actin (Fig 7G and 7H) and the tubulin-rich cilium is anchored by
radially organised septa embedded in the tegument (Fig 7E).
In Platyhelminthes, the protonephridial excretory system generally consists of terminal
organs, i.e. flame cells consisting of terminal cells and adjacent canal cells, and a system of
interconnected collecting ducts opening to the body surface [
immunofluorescent labelling of α-tubulin was only able to reliably visualise abundant flame cells, which are
regularly distributed along the entire forebody (Fig 8A, 8B, 8D and 8E). The prominent flame
cell nucleus was easily detected through Hoechst counterstaining (Fig 8B and 8E). The ciliated
tuft (i.e. flame) and roots of the cilia were conspicuously labelled by the α-tubulin antibody
(Fig 8B, 8D and 8E). Labelling of F-actin revealed the non-ciliated, barrel-shaped part of the
flame cell, consisting of both terminal and adjacent canal cells (Fig 8C and 8E).
The major part of the hindbody surface is covered with transverse, discontinuous tegumentary
folds (Fig 9A, 9B and 9D), while papilla-like structures prevail in the middle part of the ventral
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Fig 5. Forebody of Paradiplozoon homoion adults, with emphasis on the muscular, excretory and nervous systems.
A) Total view of the musculature and the main muscular organs of the forebody. CLSM, phalloidin-TRITC/DAPI. B)
Detail of two flame cells located above the buccal suckers and the area with apical circular structures (encircled). CLSM,
phalloidin-TRITC (the output image is uncoloured). C) Distribution of flame cells in the area around the pharynx and
buccal suckers. CLSM, phalloidin-TRITC. D-E) Forebody nerve cords and excretory system. CLSM, IFA-FITC/
Hoechst (D) and IFA-FITC (E). A, D and E are composite views created by flattening a series of optical sections, while
B and C represent single median optical sections. bc±buccal sucker, black arrowheads±flame cells, black arrows±
longitudinal (dorsal and ventral) nerve cords, ph±pharynx, white arrowheads±uniciliated sensory structures, white
arrows±transverse connective cords.
side above the haptor (Fig 9B±9D). The haptor bears four pairs of clamps organised in two
rows on its ventral side (Fig 9D). These clamps are covered with a thin layer of tegument (Figs
9D and 10A). The base of the clamps is comprised of both musculature and sclerotised parts.
The clamp sclerites form a pincer-like mechanism allowing for opening and closing
movements (Fig 9F and 9G). The clamp musculature, controlled by well-developed muscle bundles,
enables the sclerites to move and attach the clamps to host tissue (Figs 9E, 10B and 10D).
Gomori staining enabled 3D visualisation of both the clamp sclerites and the pair of marginal
hooks between the rows of clamps (Fig 9F and 9H). The dorsal side of the haptor is divided
into three (one central and two lateral) lobed structures (Fig 10A). The haptor's musculature
appears massive (especially in the central lobe) and is arranged as longitudinal, circular and
diagonal muscle fibres (Fig 10B and 10C). The flame cells are more abundant in the central
lobe and surrounding the clamps (Figs 10C, 10E, 10F and 11B). The sensory structures,
distinguishable due to an F-actin-rich rim, are also frequent in the central lobe (Fig 10C). Situated
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Fig 6. Mouth border of Paradiplozoon homoion adults. A) Median plane optical sectioning of the forebody. Note the
accumulation of α-tubulin associated with the forebody apical part and buccal suckers. CLSM,
IFA-FITC/phalloidinTRITC/Hoechst. B) View of the forebody showing the tubulin-rich apical end. CLSM, IFA-FITC/phalloidin-TRITC/
Hoechst. C) Detail of a uniciliated sensory structure with a raised circular rim and one long cilium. SEM. D-E) A
different optical section of the specimen in B) revealing the tubulin-rich border of the mouth opening and the
distribution of uniciliated sensory structures. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst (D) and IFA-FITC/
Hoechst (E). F) Arrangement of the muscle fibres around the border of the mouth opening. CLSM, phalloidin-TRITC.
G) Detail of uniciliated sensory structures in the forebody apical end. CLSM, IFA-FITC/DAPI. A-B, D-E and G are
composite views created by flattening a series of optical sections, while F represents a single optical section. black
arrow±raised circular rim, black arrowheads±flame cells, bs±buccal suckers, ph±pharynx, white arrow±cilium, white
arrowheads±uniciliated sensory structure.
near the haptor, the nervous system (strongly labelled for α-tubulin) is represented by
peripheral nerve fibres with a mesh-like arrangement in the central area that most likely innerve the
abundant sensory structures on the tegument surface (Fig 11A and 11C). The clamps are well
innervated and strong bundles of nerve fibres surround and copy the sclerites of the clamp
jaws (Figs 11B and 10C). The middle part, above the haptor, is packed with numerous cells
and is densely innervated (Fig 11C).
Cellular morphology of adults stained with hydrochloric carmine
This staining revealed the presence of large gland-like cells with prominent nuclei. Majority of
these cells are randomly scattered over the body, with increased occurrence in the forebody
and haptor regions (Fig 12A and 12B). Of special interest is the accumulation of putative gland
cells located in the area of apical circular structures (described above), and around the
pharynx. Furthermore, we detected a pair of club-shaped sacs lying laterally to the pharynx and
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Fig 7. Forebody innervation in Paradiplozoon homoion adults. A) Tegumentary ridges and uniciliated sensory
structures. SEM. B) Micrograph revealing the distribution of uniciliated sensory structures. CLSM, IFA-FITC. C-D)
Longitudinal and transverse connective nerve cords. CLSM, IFA-FITC. E) Detail of the sensory structure, with visible
cilium anchoring. SEM. F-G) Arrangement of uniciliated sensory structures and peripheral nerve fibres within the
tegumentary ridges. CLSM, IFA-FITC (F) and IFA-FITC/phalloidin-TRITC (G). H) Superficial distribution of
uniciliated structures, a view comparable with B). The inset to the right shows a detail of two sensory structures from
H) (encircled), while the left inset shows the circular rim rich in F-actin only; both views are magnified five times.
CLSM, IFA-FITC/phalloidin-TRITC. A, C, D, F and G are composite views created by flattening a series of optical
sections, while H represents a single median optical section. asterisk±cilium, black arrowhead±circular rim, black
arrowsÐlongitudinal nerve cords, mo–mouth opening, white arrowheads±uniciliated sensory structures, white arrows±
transverse connective cords.
opening towards the prepharyngeal/pharyngeal region. These so far undetected structures
exhibit no staining affinity to carmine and appear dark without fluorescence signal, indicating
the absence of cell structures.
Ectoparasitic diplozoid monogeneans exhibit a range of unique biological characteristics and
sophisticated functional adaptations to their bloodsucking life style. The most significant of
these structures are those related to host searching, attachment, feeding/metabolism, pairing
and protection against host responses [
]. As previous microscopy studies have shown the
benefits of CLSM analysis with fluorescent labelling for detection of specific structures, we
used phalloidin labelling of F-actin for visualisation of muscle structures and tubulin staining
for detection of the nervous or excretory systems [
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Fig 8. Visualisation of the excretory system of Paradiplozoon homoion adults using α-tubulin immunolabeling. A)
Micrograph showing the distribution of flame cells and peripheral nerve fibres in the forebody region. CLSM,
IFA-FITC. B) Flame cells (encircled) counterstained with Hoechst to indicate the nuclei of terminal cells. Note the
green stained ciliated tufts and rootlets. CLSM, IFA-FITC/Hoechst. C) Detail of the flame cells. The barrel non-ciliated
part involves both the terminal and adjacent canal cell. CLSM, phalloidin-TRITC. D) Detail of one flame cell with the
ciliated tuft of the terminal cell. CLSM, IFA-FITC. E) Detail of two flame cells. CLSM, IFA-FITC/phalloidin-TRITC/
Hoechst. A and B are single median optical sections, while C and D are composite views created by flattening a series
of optical sections. black arrowheads±flame cells, black arrows±roots of tuft cilia, white arrows±transverse connective
The tegument is the primary surface for host-parasite interaction during the search for a
suitable niche on the host's gills and also plays an essential role during contact with other
individuals during pairing and reproduction. With its shallow pits, the tegument of P. homoion
resembles that of some other monogenean species, e.g. Allodiscocotyla diacanthi [
Empleurosoma pyriforme [
] and Eudiplozoon nipponicum [
]. Similar to E. nipponicum [
unlike Paranaella luquei , Marcorgyrodactylus congolensis [
] or Diclidophora merlangi
], we observed no microvilli or microvilli-like projections on the tegument surface of P.
Diplozoids have a number of superficially located sensory structures responsible for the
reception and evaluation of information from the external environment (e.g. water flow),
facilitating selection of suitable attachment sites on the surface of the host [
]. We showed that the
well-innervated sensory structures are distributed over the entire body surface, being more
concentrated in the forebody and hindbody areas. Abundant uniciliated sensory structures
surrounding the mouth opening of P. homoion are likely related to surface perception of host
tissue during attachment and food intake (blood sucking). They could also function as
tangoreceptors with a tactile function or rheoreceptors for perception of water current during the
parasite's orientation [
]. The ultrastructure of the P. homoion sensory cilia has been described
]. A comparison of the P. homoion cilium anchoring (shown in Fig 7E) with
tangoreceptor (sensory structure with a single cilium) reconstructions for Gyrodactylus sp. and
Entobdella soleae [
] indicates a resemblance with the striated transitional fibres arising from
the basal body of the cilium that extend to the dense collar of the nerve bulb. Double
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Fig 9. Hindbody of Paradiplozoon homoion adults. A) Lateral view of both hindbodies with prominent haptors.
SEM. B) Detail of tegumentary papillae and the folds covering the middle part of the hindbody. SEM. C) Detail of the
tegument with papillae. The micrograph shows the area marked by a white rectangle in B). SEM. D) The haptor, with
eight clamps organised in two rows. SEM. E) Musculature of four clamps with operating muscle bundles. Note the
distribution of flame cells. CLSM, phalloidin-TRITC. F) 3D visualisation of the clamp sclerites. CLSM, Gomori
staining. G) Clamp sclerites. LM, bright field. H) Pair of marginal hooks localised between the two rows of clamps.
CLSM, Gomori staining. D, E, F and H represent composite views created by flattening a series of optical sections.
black arrowheads±flame cells, black arrows±tegumentary folds, cl±clamps, double white arrows±marginal hooks, h±
haptor, white arrowheads±tegumentary papillae, white arrows–extrinsic muscle bundles.
fluorescent labelling confirmed that the rim of the sensory structures, clearly visible under
SEM (Fig 7E), is rich in actin microfilaments (Fig 7H). This area corresponds to the septate
] enveloping the nerve bulb and attached to the syncytium, which is likely to be
F-actin rich in the same way as the septate junction previously described for invertebrates [
The fixed, long and movable cilium arising from the centre of the rim contained a high
concertation of α-tubulin, a basic protein forming the microtubules [
]. We assume that the
Factin-rich peripheral rim may help position the cilium through constriction and dilation. This
type of uniciliated sensory structures is similar to those observed in other monogeneans
[46,51,54±56]. Moreover, the type of sensory structure in P. homoion corresponds to those
surrounding the mouth area, though the rim appears more massive.
In addition to the uniciliated sensory structures, the apical part of the forebody has two
round projections (Fig 1B) similar to those observed in E. nipponicum [
]. Using CLSM, we
observed two circular, F-actin-rich structures (Fig 4A and 4B) similar to those observed in
12 / 21
Fig 10. Hindbody with haptor in Paradiplozoon homoion adults, with emphasis on musculature. A) Lateral view of
the haptor, with one row of four clamps and three lobed structures (one central and two lateral lobes). SEM. B)
Muscular haptor equipped with four pairs of clamps with extrinsic muscle bundles and a central lobe. CLSM,
phalloidin-TRITC. C) Central lobe of the haptor, with numerous flame cells. Note the muscle arrangement
(longitudinal, circular and diagonal). CLSM, phalloidin-TRITC. D) Detail of the massive muscle bundles controlling
the clamp. CLSM, phalloidin-TRITC. E) Detail of the clamp musculature surrounded by flame cells. Note the strong
Factin labelling localised in the barrel part of the flame cells. CLSM, phalloidin-TRITC. F) α-tubulin labelling of flame
cell ciliated tufts located near the clamps. CLSM, IFA-FITC/DAPI. B-F represent composite views created by flattening
a series of optical sections. black arrows±extrinsic muscle bundles, black arrowheads±flame cells, ce–central lobe, cl±
clamps, cm±circular muscles, dmÐdiagonal muscles, la–lateral lobes, lm±longitudinal muscles, white arrowheads±
diporpa and other stages of E. nipponicum [
]. In E. nipponicum, two nerve cords terminated
in this area [
], while our study showed dense innervation of the apex, likely related to the
round projections. Hence, we speculate that these two projections could function as
non-ciliated papillae [
]. Phalloidin labelling revealed the presence of thin trabeculae oriented
longitudinally towards the apex, the space in between being packed with nuclei, most likely
belonging to the secretory cells. This suggests that some of the circular structures could be
involved in secretion of substances involved in host-parasite interaction. This hypothesis is
supported by a staining with hydrochloric carmine for CLSM revealing the presence of the
putative gland cells accumulating in the region of apical circular structures. Similar gland cells
were described in other monogeneans as adhesive glands . Goto in his original description
of E. nipponicum termed these as gigantic cells [
]. Another accumulation of gland-like cells in
13 / 21
Fig 11. Hindbody with haptor in Paradiplozoon homoion adults, with emphasis on innervation. A) Detail of the
haptor central lobe. Note the distribution of uniciliated sensory structures (F-actin) and peripheral nerve fibre endings
(α-tubulin). The micrograph shows the area marked by a white rectangle in C). CLSM, IFA-FITC/phalloidin-TRITC.
B) Double F-actin and α-tubulin labelling of the region surrounding the two clamps. The micrograph shows the area
marked by a red rectangle in C). The dense red structures represent autofluorescence of the clamp sclerites. CLSM,
IFA-FITC/phalloidin-TRITC/Hoechst. C) General view of the hindbody labelled for α-tubulin and counterstained
with Hoechst. CLSM, IFA-FITC/Hoechst. A-C represent composite views created by flattening a series of optical
sections. black arrowheads±flame cells, black arrows±innervation of clamps, cl±clamps, white arrowheads±uniciliated
sensory structures, white arrows±peripheral nerve fibres.
the middle region of P. homoion haptor may be also associated with adhesive function as
proposed earlier [
The paired bowl-shaped buccal suckers in the mouth cavity appear to be the main
attachment organs of the forebody. These suckers also help in sucking the host's blood. As also
observed in E. nipponicum [
], the U-shaped septum is located in the middle of the suckers.
This structure is rich in F-actin and α-tubulin, and appears to be related to sucker innervation.
Sucker functioning is controlled by prominent muscles that are directed around the pharynx
posteriorly, and muscles that cross each other and are directed towards the apical part of the
forebody. The muscles controlling the suckers are similar to those of E. nipponicum [
except for the previously unobserved muscles fascicles oriented obliquely between the suckers.
The buccal cavity surrounding the suckers is covered with numerous foliate and tubular
digitations up to the rim of the mouth, thereby expanding the parasite's surface in contact with
the host tissue. These tubular digitations have previously been described for P. homoion as
short, irregular lamellae [
]. Similar projections were also recorded in the buccal cavity of E.
]. These digitations may enable insertion of biochemically active compounds
14 / 21
Fig 12. Cellular morphology of Paradiplozoon homoion adults stained with hydrochloric carmine. A) Micrograph
showing the forebody region. The white circles indicate some of the putative unicellular glands. The white rectangle
demarks the putative glands in the area of apical circular structures. CLSM, output image not coloured. B) Micrograph
showing the haptor. The accumulation of putative gland cells is demarcated by white circle. CLSM, output image not
coloured. A-B are composite views created by flattening a series of optical sections. asterisks±paired club-shaped sacs,
bs±buccal suckers, ce–central lobe, cl±clamps, g±foregut, ph±pharynx.
for modifying host tissue during sucking, or could interact with the host's blood before
digestion. We expect the prominent gland-like cells surrounding the retractable pharynx in
wholemount preparations stained with hydrochloric carmine to be responsible for enzymatic
secretion, with the paired club-shaped sacs opening towards the (pre)pharyngeal region and serving
as secretory reservoirs.
The well-developed pharynx musculature and muscles controlling eversion and protruding
of the pharynx correspond with those previously described in E. nipponicum [
stretched muscles with deeply retracted pharynx (Fig 4C) and relaxed muscles with clearly
shortened pharynx (Fig 4D) confirm the retractile function of these muscles. In general, the
ultrastructure of the pharynx  corresponds with our CLSM observations. The four
F-actinrich circular openings detected on the apical end of the pharynx (corresponding with the pits
in E. nipponicum visible under SEM ([
], Fig 6D), could represent ducts for releasing
proteolytic enzymes during extraintestinal digestion, as proposed by ValigurovaÂ et al. [
described in previous ultrastructural studies, these secretions could be produced by gland cells
opening into the pharynx lumen [
As in other Platyhelminthes [
], the body wall musculature is organised into three
main layers, an outer circular layer, an inner longitudinal layer and diagonal muscles. In P.
homoion, additional muscles of unknown function run perpendicularly to the tegument,
apparently corresponding to the dorso-ventral fibres reported in other studies [
stages of P. homoion exhibit no obvious differences when compared to E. nipponicum; except
for globular glandulo-muscular organs that appear to be species-specific for both the juvenile
and adult stages of E. nipponicum [
Basic diagram showing the nervous system in E. nipponicum was published by Goto [
Later immunomicroscopical study focused on changes and fusion of central nerve elements in
paired individuals of E. nipponicum, using a range of staining techniques to demonstrate
cholinergic elements and FaRPergic innervation, along with serotonin immunostaining and gold
labelling highlighting neuropeptide immunoreactivity [
]. Another study visualised the
innervation of diporpa of P. ichtyoxanthon [
]. In our study, in addition to visualising the
longitudinal nerve cords and transverse connectives of the central nervous system,
immunofluorescent labelling of α-tubulin proved helpful in detecting a network of peripheral nerves
reaching up to the tegumentary folds. Furthermore, this relatively simple method also helped
visualise the fine innervation of sensory structures and their cilia. Nevertheless, the staining
techniques used by Zurawski [
] do provide more specific results when identifying individual
elements of the diplozoid nervous system.
Increased staining in the P. homoion apex indicates a higher accumulation of sensory
structures and higher sensitivity of the entire mouth area. A similar combination of F-actin and
tubulin staining was used to visualise musculature and innervation in a previous work on
Schistosoma mansoni cercariae [
], though the antibody used detected β-tubulin. While the
cephalic ganglia and main nerve cords were clearly stained for synapsin, β-tubulin was
restricted to fine muscle innervation, especially in the caudal cercaria area, which was not
labelled with synapsin. The antibody specific for acetylated α-tubulin was used in the above
16 / 21
mentioned study to show the distribution of sensory papillae and the periphery of the
Anti-α-tubulin antibodies were also used to localise P. homoion flame cells. Flame cells
form part of the protonephridial system, which comprises a terminal (flame cell), an adjacent
canal cell and a system of associated collecting ducts serving for osmoregulation, as previously
described for other flatworms [
]. In selected monogenean species, previous studies focused
on general organisation [
] and ultrastructure of the excretory system (e.g. [
isoforms of tubulin were used for investigation of the protonephridial system of S. mansoni
. Unfortunately, this method also visualises part of the nervous system. In order to avoid
this cross-reaction, the authors used proteinase K to suppress nerve staining. The flame cell
tuft roots were subsequently labelled with anti-phospho S/T antibody and DAPI, while the
basket-like structure of the barrel (the non-ciliated part of the flame cell consisting of both the
terminal and the adjacent canal cells) were stained with anti-phospho tyrosine and phalloidin. In
our study on P. homoion, we achieved good results by using double fluorescent labelling,
which stained the ciliated tuft (i.e. the `flame') and cilia root for α-tubulin and the barrel for
Factin. The nucleus of the flame cell was easily detected using Hoechst counterstaining. While it
is likely that the collecting ducts were also visualised using α-tubulin labelling (similar to a
study on S. mansoni [
]), it was impossible to distinguish them reliably from the stained parts
of the peripheral nervous system. Labelling of flame cells for β-tubulin in S. mansoni [
corresponded to our results using the anti-α-tubulin antibody.
The hindbody of P. homoion plays an important role in attachment to host tissue and
moving on the host's gills. Compared to E. nipponicum, where prominent folds and species-typical
lobular extensions play an important role in attaching this robust parasite within the gill
], P. homoion has less prominent tegumentary folds and three distinct lobes on the
haptor rather than lobular extensions. These lobes are highly mobile and equipped with a
conspicuous three-layer musculature innervated with abundant uniciliated sensory structures on
the surface. Besides the clamps and putative adhesive glands, these lobes serve presumably for
attaching the parasite to the host's gills. In contrast to E. nipponicum, in which the lateral
hindbody is equipped with non-ciliated papillae (most likely involved in reception of
environmental stimuli) , P. homoion had similar non-ciliated papillae clustered above the clamps. Such
non-ciliated papillae have also been reported in Entobdella soleae [
]. It is generally
assumed that these function as mechanoreceptors in direct contact with the host, with
information from host-parasite interaction being utilised during attachment/detachment when
relocating on the host's surface . Alternatively, they could serve as proprioreceptors for
sensing the relative position of the haptor during movement.
As the primary structures fixing the parasite to the host surface, the clamps are organised in
a similar manner to those in other Diplozoidae; i.e. four pairs of clamps in two parallel rows.
While the sclerotised parts of the clamps exhibit autofluorescence and also are easily
recognised under a light microscope equipped with Nomarski differential interference-contrast
(NDIC; Fig 9G), the staining with Gomori trichrome appears to be the most suitable method
for 3D visualisation of the sclerites and the marginal hooks [
]. Similar results were achieved
in Paradiplozoon sp. using the HoÈrens trichrome [
]. Phalloidin staining confirmed that the
clamp musculature, which is well developed and robust, is controlled by muscle bundles,
which corresponds with previous observations on E. nipponicum [
] and Diplozoon
paradoxum . This musculature system controls individual clamps and facilitates parallel
movement of an entire row of four clamps when translocating on host gill lamellae. In contrast to
the visualisation of peptidergic and serotoninergic parts innervating the main muscles
controlling the clamps in E. nipponicum [
], α-tubulin labelling in our study not only revealed
innervation of the clamps but also the nerve fibres lining individual sclerites.
17 / 21
In conclusion, this study demonstrated the major structures important for the ectoparasitic
life style of P. homoion. Overall, P. homoion exhibits a number of sophisticated functional
adaptations to its ectoparasitic life-style, similar to those previously described for other
helminth parasites (e.g. [
]). The original combined fluorescent labelling and SEM used in this
study, however, revealed much more details in organisation and morphology of individual
structures. The well-developed musculature and innervation of buccal suckers and haptor
equipped with sclerotised clamps indicate their significant role in attachment and movement
on the host. The hydrochloric carmine staining confirmed the increased accumulation of
gland cells with proposed adhesive function in these regions . The parasite is well equipped
for blood sucking thanks to its heavily innervated mouth opening with abundant sensory
structures, buccal cavity covered with numerous digitations, muscular buccal suckers and
retractable pharynx with F-actin-rich circular openings on its apical end (likely representing
the ducts for releasing proteolytic enzymes during extraintestinal digestion). On the top of
that, we showed the presence of putative unicellular glands surrounding the pharynx along
with so far not reported pair of club-shaped sacs that might function as secretory reservoirs.
Using the fluorescent labelling, we were able to visualise the body wall musculature along with
its peripheral innervation reaching up to the tegumentary folds, the distribution and
innervation of uniciliated sensory structures and flame cells involved in parasite's excretion.
The study was financially supported by ECIP (European Centre of
Ichthyoparasitology)±Centre of Excellence, Czech Science Foundation Project No. P505/12/G112. All authors
acknowledge support from the Department of Botany and Zoology of the Faculty of Science at
Masaryk University towards the preparation of this manuscript.
Conceptualization: Iveta HodovaÂ, Andrea ValigurovaÂ.
Formal analysis: Iveta HodovaÂ, Radim Sonnek, Andrea ValigurovaÂ.
Funding acquisition: Milan Gelnar.
Investigation: Iveta HodovaÂ, Andrea ValigurovaÂ.
Methodology: Iveta HodovaÂ, Radim Sonnek, Andrea ValigurovaÂ.
Project administration: Milan Gelnar.
Resources: Iveta HodovaÂ, Milan Gelnar, Andrea ValigurovaÂ.
Validation: Iveta HodovaÂ, Andrea ValigurovaÂ.
Visualization: Iveta HodovaÂ, Radim Sonnek, Andrea ValigurovaÂ.
Writing ± original draft: Iveta HodovaÂ, Andrea ValigurovaÂ.
Writing ± review & editing: Iveta HodovaÂ, Milan Gelnar, Andrea ValigurovaÂ.
18 / 21
19 / 21
89(1):198±200. https://doi.org/10.1645/0022-3395(2003)089[0198:MEONCB]2.0.CO;2 PMID:
20 / 21
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