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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
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 The Diplozoidae, Palombi, 1949, are oviparous monogeneans 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 . 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 morphology, 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: firstname.lastname@example.org (M. Pečínková). 1383-5769/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2007.01.010
juvenile stage undergo development of attachment apparatus 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 . 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,
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Table 1 Overview of studies on life cycle of the diplozoids Parasite species Paradiplozoon homoion Eudiplozoon nipponicum P. homoion E. nipponicum Diplozoon paradoxum, E. nipponicum, P. bliccae, P. homoion, P. rutili D. paradoxum D. paradoxum, Diplozoon sp. Host fish Barbus meridionalis Cyprinus carpio Rutilus rutilus C. carpio Abramis bjoerkna, Abramis brama, C. carpio, R. rutilus, Vimba vimba Study type Temperature of experiment Experiment 8, 24 °C and fluctuating room temperature Field Field Experiment 25 °C Experiment 15 to 20 °C Observed stage Egg (production and hatching) Whole life cycle Whole life cycle Diporpa (development and pairing) Egg and development of embryo in the egg, oncomiracidium Whole life cycle Egg, oncomiracidium All stages (oncomiracidium) Whole life cycle Reference  a    
A. brama A. brama, R. rutilus
Field Experiment 24 °C Experiment 20 °C (4 °C) Field
Studied relationship between egg hatching and host behaviour.
alimentary and also genital systems are fused reciprocally . Gelnar et al.  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) parasites 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 Paradiplozoon 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 . 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 developmental 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 . 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. ). Temperature 20 °C is suggested as the optimum temperature for fast development of diplozoids  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 Number of fish Standard length (cm) Mean ± SD Weight (g) Mean ± SD 35 7.72 ± 0.51 6.23 ± 1.32 Laboratory reared gudgeon 41 5.55 ± 0.55 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) Egg length (μm) Egg width (μm) 250.17 ± 17.85, 207.48–281.59 88.32 ± 6.42, 81.20–105.62 Khotenovsky (1985) (range) 233–257 90–103
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 oncomiracidia were observed every half an hour by a stereomicroscope. 3. Results
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 specimens 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. . 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 temperature. The condition of eggs was monitored using a stereomicroscope. Hatching occurred after 1–2 days. Most of hatched oncomiracidia were used for the transmission study to estimate a percentage of oncomiracidia successfully 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
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.
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 transfer oncomiracidia into the tank oncomiracidia used with host for experiment 0.5 1h 1.5 h 2h 6h 12 h 24 h 15 10 10 13 15 15 15 Percentage of successful oncomiracidia (%) 0.0 0.0 0.0 23.1 20.0 26.7 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 . In the present study, the duration of P. homoion transformation from egg into mature, adult specimens 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  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 , and also the findings of present experimental study, confirm results of Gelnar and Koubková  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  (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 , 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.  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  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 , 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 . 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  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 oncomiracidium 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 ). 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 , who found only 10% of oncomiracidia able to successfully attach to the host and transform to diporpae. In the study by Hirose et al. , diporpae of E. nipponicum were confirmed on carp gills as soon as one hour after postinfection 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.  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  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 , the average longevity of oncomiracidium 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). References
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