Parasitology International 56 (2007) 179 – 183

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Investigation of Paradiplozoon homoion (Monogenea, Diplozoidae) life

cycle under experimental conditions
Martina Pečínková a,⁎, Iveta Matějusová b , Božena Koubková a , Milan Gelnar a
a
Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
b
FRS Marine Laboratory, P. O. Box 101, Victoria Road, Aberdeen AB11 9DB, United Kingdom
Received 17 May 2006; received in revised form 16 January 2007; accepted 30 January 2007
Available online 6 February 2007

Abstract

Diplozoids (Diplozoidae, Monogenea) are fish ectoparasites with a direct life cycle without intermediate hosts. Their free swimming larva, the
oncomiracidium, hatches from eggs, invades a fish host and metamorphoses into a post-oncomiracidial larval stage, the diporpa. Later, two
diporpae fuse and live as a pair in cross-copulation during their adult life. An experimental study was designed to investigate the life cycle of
Paradiplozoon homoion (Monogenea, Diplozoidae) parasitizing their common fish hosts, gudgeon (Gobio gobio). A total of 35 gudgeon
parasitized by diplozoids were collected from their natural environment of the Vlára River, Czech Republic, and kept together in tanks with 41
non-parasitized gudgeons reared in a laboratory environment. In total, 100 adult specimens of P. homoion were collected from the Vlára River
gudgeon and a new parasite generation was expected to be observed on fish reared in the laboratory environment. Eight days after the first
diplozoid eggs appeared on fish gills, the presence of diporpae with one or two pairs of clamps was noted. The appearance of the first juveniles
was recorded at the same time as diporpae. Development of P. homoion from egg to sexually mature adult stage took 33 days at a constant
temperature of 20 °C. The development of eggs in adults of the second generation was observed 2 days after the first observation of these adults.
The behavior of oncomiracidia was also studied and this free swimming stage of diplozoids survived for 22 h in the absence of a host. When
host fish were experimentally infected by oncomiracidia, diporpae were found attached to the fish gill apparatus within 2 h of infection.
© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Paradiplozoon homoion; Ectoparasite; Gudgeon; Infection; Experiment; Life cycle

1. Introduction juvenile stage undergo development of attachment apparatus

The Diplozoidae, Palombi, 1949, are oviparous monoge-
neans and live on the gills of mainly cyprinid fish. Their eggs
have a long polar filament, which helps them to attach to a
substrate in the water or less commonly to the gills of host fish
[1]. Free swimming oncomiracidia hatch from eggs and within a
short time have to find a host fish for further development. After
attachment to fish gills, the oncomiracidium changes morphol-
ogy, loses the eyespot and surface cilia and develops a branched
intestine. This post-oncomiracidium stage is called the diporpa.
Diporpae, like all other following developmental stages of
diplozoids, are blood-feeders. Later, two diporpae fuse and as a
⁎ Corresponding author. Tel.: +420 549497363; fax: +420 541211214.
E-mail address: pecinkova@sci.muni.cz (M. Pečínková).
1383-5769/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.parint.2007.01.010
and reproductive organs.
The fully formed attachment apparatus of adult diplozoids
consists of four pairs of clamps and one pair of central hooks on
each specimen of a pair. First pair of clamps and a pair of central
hooks are presented already in the oncomiracidium to establish
their attachment on host gills. After invading a host, the central
hooks lose their function and in addition to the first pair of
clamps, additional pairs of clamps are developed towards the
anterior part of diplozoid body [2]. Two diporpae with two or
three pairs of attachment clamps usually fuse together. If a
diporpa does not find another specimen to pair with,
development of a full attachment apparatus is possible;
however, these unpaired specimens fail to mature.
Diplozoids stay as a pair for all their adult life. Each
specimen of a pair is hermaphroditic and nervous, muscle,
180 M. Pečínková et al. / Parasitology International 56 (2007) 179–183

Table 1
Overview of studies on life cycle of the diplozoids
Parasite species Host fish Study type Temperature of Observed stage Reference
experiment
Paradiplozoon homoion Barbus meridionalis Experiment 8, 24 °C and fluctuating Egg (production and [18] a
room temperature hatching)
Eudiplozoon nipponicum Cyprinus carpio Field Whole life cycle [10]
P. homoion Rutilus rutilus Field Whole life cycle [7]
E. nipponicum C. carpio Experiment 25 °C Diporpa (development and [15]
pairing)
Diplozoon paradoxum, Abramis bjoerkna, Abramis brama, Experiment 15 to 20 °C Egg and development of [16]
E. nipponicum, P. bliccae, P. homoion, C. carpio, R. rutilus, Vimba vimba embryo in the egg,
P. rutili oncomiracidium
Field Whole life cycle
D. paradoxum A. brama Experiment 24 °C Egg, oncomiracidium [1]
D. paradoxum, Diplozoon sp. A. brama, R. rutilus Experiment 20 °C (4 °C) All stages [14]
(oncomiracidium)
Field Whole life cycle
a
Studied relationship between egg hatching and host behaviour.

alimentary and also genital systems are fused reciprocally [3].
Gelnar et al. [4] also described a sub-adult stage, which
represents specimens with fully formed attachment apparatus
but not sexually mature.
The life cycle of monogenean (especially diplozoid) para-
sites has been studied several times in the past from different
aspects and available information are summarized in Table 1.
The present study investigated the development of Paradiplo-
zoon homoion (Bychowsky et Nagibina, 1959) on gudgeon
Gobio gobio (Linnaeus, 1758). P. homoion is a common
parasite of gudgeon and has been considered as generalist as its
presence is recorded from eighteen species of cyprinids in the
Czech and Slovak Republics [5,6]. Together with other
diplozoids, P. homoion exhibits a low pathogenicity to its
hosts [7]. The development of P. homoion was studied under
experimental conditions in order to obtain precise time scales
for development of each parasite stage, from egg to sexually
matured diplozoid. Data on longevity of diplozoid stages is
important when planning experimental studies, e. g. the effect of
parasites on host immuno-competence or parasite developmen-
tal instability.
2. Materials and methods
2.1. Collection of fish hosts
Two different populations of gudgeon (G. gobio) were used
in the experiment (Table 2). Thirty-five fish were collected from
their natural environment, Bohuslavice nad Vláří at Vlára River
basin, Czech Republic (49°5′ N, 17°55′ E) in November 2003.
From long-term research at this locality, it is known that the
occurrence of P. homoion on gudgeon is common here, the
parasite population is well established. In the autumn period of
2001 and 2002 respectively, adult parasites were found on the
gill apparatus with prevalence 73.33% and 90.00% respectively
and mean intensity of infection 3.64 and 4.83 respectively [8].
The second population of gudgeon was reared in laboratory
conditions and has never been parasitized by P. homoion.
Standard length and weight of fish from the natural population
were larger than in fish reared in the laboratory (ANOVA,
P b 0.001) (Table 2).
2.2. Design of the experiment
Gudgeon from the Vlára River, collected by electrofishing,
were transported alive to the laboratory in the aerated tanks of
river water. At the time of fish collection, the water temperature
in the Vlára River was 5 °C so fish were acclimatized to the
experimental conditions by increasing water temperature by
1 °C/day. Acclimatized fish from the Vlára River were then
transferred into tanks and cohabited with gudgeon reared under
the laboratory condition. In total, 35 separate tanks with a pair
of fish from both populations were positioned in a large tank
with stood tap water to ensure consistent environmental
conditions for all the experimental tanks (the water temperature
stabilized at 20 °C and natural light dawn to dusk). Laboratory
reared fish were never exposed to any parasite infection prior to
experiment. To distinguish gudgeon specimens of a pair in the
separate tanks, a caudal fin was clipped on the laboratory reared
gudgeon (see Guy et al. [9]). Temperature 20 °C is suggested as
the optimum temperature for fast development of diplozoids
[10] and corresponds to the highest temperature measured
during study at the Vlára River. It is easily maintained in
experimental conditions. As no suitable substrate for attachment
of parasite eggs was provided in the experimental tanks, the
diplozoid eggs were observed attached on the gudgeon gills.
Table 2
Number, length and weight of gudgeon (Gobio gobio) used in the present
experiment
Gudgeon from Vlára River Laboratory reared
gudgeon
Number of fish 35 41
Standard length (cm) 7.72 ± 0.51 5.55 ± 0.55
Mean ± SD
Weight (g) Mean ± SD 6.23 ± 1.32 2.98 ± 0.94
 M. Pečínková et al. / Parasitology International 56 (2007) 179–183
Table 3
Comparison of width and length of P. homoion eggs (mean ± SD, range)
Our results (n = 17) Khotenovsky (1985) (range)
Egg length (μm) 250.17 ± 17.85, 233–257
207.48–281.59
Egg width (μm) 88.32 ± 6.42, 90–103
81.20–105.62
Eggs that sank to the bottom of tanks were not considered.
Presence of eggs on randomly chosen, dissected gudgeon from
the population reared in the laboratory environment was
recorded after 20 days from the capture of gudgeon in the
Vlára River; this day was noted as day 1 of experiment. After
that, every 24 to 48 h one gudgeon from the population reared
under the laboratory conditions (always from another separate
tank) was dissected and examined for presence of P. homoion
parasitic stages using a stereomicroscope. All diplozoid speci-
mens found were mounted on a microscope slide and fixed with
a mixture of glycerine-ammonium picrate [11,12]. Diplozoid
developmental stages, such as diporpae, juvenile diplozoids and
adult diplozoids were identified using a light microscope
equipped with differential interference contrast (Nomarski
DIC). In the present experiment, the sub-adult stage was not
determined and all specimens with fully developed attachment
apparatus were classified as adults. The experiment was
finished when adult matured diplozoids, able to produce eggs,
appeared on the laboratory reared gudgeon. Prevalence, mean
and range of intensity of infection were calculated according to
Bush et al. [13].
Heavy infection of P. homoion was established on both
populations of gudgeon under experimental conditions. Due to
heavy infection by both P. homoion and protozoans, six
individual laboratory reared gudgeon died during the experiment
and were replaced by individuals from the same population.
The Spearman rank correlation test was applied to analyze a
relationship between number of adult P. homoion and weight or
standard length of the laboratory reared gudgeon. Only fish,
dissected after first mature P. homoion adults were observed,
were included in this analysis.
2.3. Study of oncomiracidium transmission and survival
A large quantity of P. homoion eggs was obtained from gills
of dissected gudgeon during the experiment. Eggs were
collected and incubated in a Petri dishes and standing tap
water under conditions of natural daylight and room temper-
ature. The condition of eggs was monitored using a stereomi-
croscope. Hatching occurred after 1–2 days.
Most of hatched oncomiracidia were used for the transmis-
sion study to estimate a percentage of oncomiracidia success-
fully attached to its host. Ten to fifteen oncomiracidia specimens
were transferred into the aerated and stood tap water in a 1.5 l
aquarium with one non-infected laboratory reared gudgeon.
Single fish were examined for the presence of diporpae attached
to the gills 30 min after the transfer of oncomiracidia into the
tank, using a stereomicroscope. Identical experiments with a
181
number of attached diporpae recorded on a fish were performed
1, 1.5, 2, 6, 12 and 24 h after the transfer of oncomiracidia into
the tank.
Five collected eggs were used to study survival time of
oncomiracidium and were kept in a Petri dish in the absence of a
suitable host. The time of hatching was recorded and oncomir-
acidia were observed every half an hour by a stereomicroscope.
3. Results
3.1. Life cycle of P. homoion
A total of 100 adult P. homoion were recovered from the gills
of gudgeon from the Vlára River during our experiment with
prevalence of 82.86% and mean intensity of infection of 3.45.
More than six hundreds eggs were found attached to the gills of
fish from both populations. The length and width of 17 eggs
were determined (Table 3). Production of eggs was highest on
the 6th day of the experiment. After the 16th day, eggs were
very rarely recorded in the experiment.
The first diporpae appeared 8 days after the presence of eggs
and mainly diporpae with one or two pairs of clamps were
recorded. During our experiment, only one diporpa with three
pairs of clamps and none with four pairs of clamps were found.
The first juveniles were recorded at the same time as diporpae,
confirming that diplozoids attempt to fuse quickly to continue
their development. Diporpae and juveniles were found
abundant for the remaining period of the experiment. In total,
771 diporpae with maximum intensity of infection 248 diporpae
on the gills of one laboratory reared fish were found during the
duration of experiment. 228 juveniles were collected from gills
of the laboratory reared fish with maximum 62 juveniles on fish
gills. On day 15 of the experiment, juveniles with a developed
third pair of clamps were recorded, the fourth pair of clamps
started to develop on day 18. However, another 15 days were
necessary for juveniles to reach maturity. Juveniles with
different numbers of pairs of clamps on each haptor were
identified (juveniles with one and two, two and three and three
and four pairs of clamps on each haptor, respectively).
The first mature adult of P. homoion was found on gills of
the fish reared in the laboratory environment 33 days after
detection of eggs; 2 days later, newly formed eggs were found.
Adult P. homoion of the second generation recovered from gills
Fig. 1. Occurrence of Paradiplozoon homoion developmental stages during
experiment (in days). +Note: The experiment was finished 36 days after
detection of eggs on gudgeon gills.
182 M. Pečínková et al. / Parasitology International 56 (2007) 179–183
Table 4
The percentage of successful oncomiracidia in the invasion of host fish
(experiment of oncomiracidium transmission and survival)
Time of observation after the No. of Percentage of
transfer oncomiracidia into the tank oncomiracidia used successful
with host for experiment oncomiracidia (%)
0.5 15 0.0
1h 10 0.0
1.5 h 10 0.0
2h 13 23.1
6h 15 20.0
12 h 15 26.7
24 h 15 40.0
of the laboratory reared gudgeon (dissected after first mature
P. homoion adults were observed) were found with a prevalence
of 47.83% and mean intensity of infection of 3.91. Fig. 1 shows
that the occurrence of certain developmental stages overlaps.
There was no relationship between weight of the laboratory
reared gudgeon and the number of adult matured P. homoion
(Spearman rank correlation, r = 0.243, P = 0.264). Also no
correlation between standard length of the laboratory reared
gudgeon and the number of adult P. homoion was found
(Spearman rank correlation, r = 0.356, P = 0.095).
3.2. Study of oncomiracidium transmission and survival
No diporpae were observed on fish dissected in the time
period from 0.5 to 1.5 h after infection. After 2, 6 and 12 h, more
than 20% of oncomiracidia were found attached and developed
to diporpae. After 24 h from the transfer oncomiracidia into the
tank with parasite-free gudgeon, six diporpae were recorded
attached on the gills of the host fish (Table 4).
When no suitable host was present, survival time of five
oncomiracidia was observed using a stereomicroscopy. The first
oncomiracidium was found dead after 9 h of observation. Two
of them died after 20 h and the maximum longevity of two
oncomiracidia was 22 h.
4. Discussion
In the natural environment, the life cycle of diplozoids
usually takes less than 1 year, with sub-adult specimens
wintering on fish gills and rapidly sexually maturing in the
spring months [2]. In the present study, the duration of
P. homoion transformation from egg into mature, adult speci-
mens was observed under laboratory conditions with water
temperature stabilized at 20 °C. Although positive correlations
between host size and size of some attachment clamp sclerites
of P. homoion or the intensity of infection have been reported in
literature [6,7], no effect of host size on the intensity of infection
of P. homoion was observed in the present study. The whole
process of development of P. homoion took 33 days from egg to
adult stage. Mature diplozoids of the new generation, able to
produce eggs, where found on the 35th day after detection of
eggs on gills of fish. Whereas, Bovet [14] recorded that in room
temperature oncomiracidia developed into sexually matured
specimens in 54 to 60 days after infection of bream and roach.
The results of Bovet [14], and also the findings of present
experimental study, confirm results of Gelnar and Koubková
[10] who stated that diplozoids are able to reach maturity during
the same vegetative period in the natural environmental with
average water temperature higher than 20 °C.
Diplozoid larvae and their longevity have been studied in
several experiments [14,15] and the life cycle of diplozoids was
also observed in the natural environment [10,14,16] (Table 1).
In our study, the unparasitized gudgeon were infected by
P. homoion and the second generation of parasites was
successfully bred under experimental conditions. During the
experiment, many eggs were found attached by a filament on
the gills of host fish. Seventeen eggs collected from the
experiment were measured and the data obtained were
compared to results of Khotenovsky [2] (see Table 3).
Measurements of eggs obtained during our experiment exhibit
a larger range for both width and length of eggs than previously
published by Khotenovsky [2], however there was no
information on how many eggs have been measured.
During the experiment, only diporpae with one or two pairs
of clamps were recorded and already at this stage, they were
able to pair and form juvenile diplozoids. In contrast, Hirose
et al. [15] only recorded juvenile specimens formed from
diporpae with the third or (less often) fourth clamps developed
and referred that the diporpa with three pairs of clamps are ready
for pairing. Bovet [14] reported that the diporpae are ready to
pair and develop into a juvenile specimen stage 4 days after
attachment to the host gills, when the second pair of clamps is
developed. In agreement with the previous study of Bovet [14],
we found juveniles with different numbers of clamp pairs on
each haptor. However, juveniles with one pair of clamps were
not usually found in the natural environment [14]. Juveniles
with third and later with fourth pairs of clamps were observed 7
and 10 days after presence of the first juveniles was observed. In
addition, Bovet [14] studied the development of diplozoid pairs
of clamps and recorded that Diplozoon had the fully formed
attachment apparatus 30 to 35 days after pairing of diporpae.
In the present experiment, oncomiracidium survival was
observed in conditions of presence and absence of potential fish
hosts. Longevity of the oncomiracidium stage is the shortest in
the life cycle of diplozoids. Within few hours, the oncomir-
acidium requires to find a suitable host to ensure successful
development into juvenile diplozoid. It was confirmed that
monogenean larvae respond to chemical hatching factors of host
origin (e.g. substances in their skin and gill secretions) (for
review see Kearn [17]). In the present study, more than 20% of
oncomiracidia were successful in the search for the host fish and
were found attached to the fish gills and transformed into
diporpae after 2 to 12 h. After 24 h, 40% of oncomiracidia were
successfully attached on a gudgeon and transformed into
diporpae. Success of oncomiracidia in finding a host was higher
in the present experiment compared to Bovet [14], who found
only 10% of oncomiracidia able to successfully attach to the
host and transform to diporpae.
In the study by Hirose et al. [15], diporpae of E. nipponicum
were confirmed on carp gills as soon as one hour after post-
infection at temperature of 25 °C, however, in our experiment no
 M. Pečínková et al. / Parasitology International 56 (2007) 179–183
diporpae were found in the period 0.5 to 1.5 h post-infection.
Higher experimental temperature in Hirose et al. [15] study might
explain faster development of particular developmental stages of
diplozoids. However, the possibility of differences in duration of
life cycle for different species of the family Diplozoidae should
also be considered. Khotenovsky [16] recorded a difference in
duration of development of eggs of five diplozoid species at water
temperature of 15 to 20 °C. Eggs of P. homoion needed 8 to
10 days to hatch, whereas the D. paradoxum eggs hatched in 10 to
11 days.
In the present study, the maximum survival of P. homoion
oncomiracidia in absence of a potential host was 22 h.
According to Bovet [14], the average longevity of oncomir-
acidium is 6 h in water of 18 to 20 °C. The maximum survival of
oncomiracidia was extended to 92 h when water temperature
was lowered to 4 °C, oncomiracidia were kept in darkness and
in Ringer's medium without glucose.
To conclude, the life cycle of P. homoion was successfully
completed in experimental conditions and data on the
occurrence of each developmental stage of this diplozoid was
obtained and will be used for future experimental studies
focused on host/parasite systems. The ability to rear the
diplozoids under laboratory conditions and precise information
on their development will provide data valuable for the study of
host–parasite relationship studies, cytochemical studies, or
studies focused on immune response of fish host.
Acknowledgements
The authors would like to thank M. Palíková from the
University of Veterinary and Pharmaceutical Sciences Brno,
Czech Republic, for providing laboratory reared gudgeon, P.
Jurajda and J. Huml from Institute of Vertebrate Biology
(Academy of Sciences of the Czech Republic) for help with
collection of wild population of fish. This study was supported
by the Grant Agency of the Czech Republic (Project No. 524/
04/1115). BK was funded by the Research Project of Masaryk
University (MSM0021622416) and MP was partly supported
by the Grant Agency of the Czech Republic (No. 524/05/H536)
and by Ministry of Education, Youth and Sports of the Czech
Republic (Ichtyoparasitology Research Centre – Project No.
LC522).
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