Influences of light-dark cycle and water temperature on in vitro egg laying, hatching and survival rate of the monopisthocotyla Thaparocleidus vistulensis (Siwak, 1932)

doi:10.21203/rs.3.rs-4958124/v1

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Influences of light-dark cycle and water temperature on in vitro egg laying, hatching and survival rate of the monopisthocotyla Thaparocleidus vistulensis (Siwak, 1932)

https://doi.org/10.21203/rs.3.rs-4958124/v1

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The European catfish (Silurus glanis) is an important species with high economic value, and its growing demand has led to intensive farming practices for it. However, this species is increasingly challenged by parasitic infections, particularly from a specific gill monopisthocotylan parasite called Thaparocleidus vistulensis. To establish effective management strategies, it is crucial to comprehend the fundamental environmental variables that could influence the reproductive and survival behaviour of T. vistulensis. The present study conducted controlled in vitro experiments to observe the fecundity, hatching rate, and survival ability of T. vistulensis under varying light-dark conditions, as well as across a range of water temperatures from 5°C to 35°C. Interestingly, light exposure led to a threefold increase in egg production by adult T. vistulensis compared to constant darkness. While light or dark conditions did not significantly affect the hatching and survival rate of adults, they did significantly affect the survival rate of oncomiracidia. The parasite’s fecundity was optimal at 15°C. Eggs hatched fastest at 30°C, whereas no hatching occurred at 5°C and 35°C. The survival rate for both oncomiracidia and adults showed a negative correlation with increasing water temperatures. These findings provide fundamental insights into how varying environmental variables impact the life cycle of the parasite. The improved understanding of these findings provides a valuable basis for the management of T. vistulensis in cultured European catfish stocks in fish farms.

European catfish

Thaparocleidus vistulensis

Environmental factors

Fecundity

Hatching rate

Survival rate

Monopisthocotylan parasite

Global aquaculture production reached a record high of 130.9 million tonnes in 2022, achieving a 6.6% increase from 2020, highlighting its importance and growing role in the global food industry (FAO 2024). However, the rising demand for food has forced the rapid transformation of aquaculture practices, which has led to increasing incidences of diseases. Among them, parasitic infections are a major threat, inducing substantial economic losses for the aquaculture industry (Thoney and Hargis 1991; Shinn et al. 2015). Fish parasites can affect the cultivation process, reduce the nutritional value of the fish, and sometimes even deform the fish’s body, reducing its potential marketability (Hoffman 1998). Consequential losses due to these parasite infections are manifested in fish mortality, treatment costs, and low growth performance during and after the outbreaks, which hinder the expansion of aquaculture (Omeji et al. 2011).

As a large number of parasites is detrimental to the hosts’ fitness and resilience, it is important to understand the parasites’ reproductive and survival dynamics under different environmental conditions (Caminade et al. 2019). For most monogeneans (monopisthocotylans and polyopisthocotylans), development rates are influenced by natural factors like water temperature, light intensity, salinity, and the host species (Bauer et al. 1973; Gannicott and Tinsley 1997; Buchmann and Bresciani 2006).

The water temperature (Kashkovskii 1982; Buchmann 1988, 1990; Cecchini 1994; Cecchini et al. 1998, 2001; Gannicott and Tinsley 1998; Lackenby et al. 2007; Hirazawa et al. 2010; Maciel et al. 2017; Zhang et al. 2015, 2022) and alternating light and dark cycles (Kearn, 1963, 1973, 1982; Mooney et al. 2008; Hoai and Hutson 2014) are key elements that have been widely investigated as regulators in the reproduction of monopisthocotylans. Understanding the interaction between biotic and abiotic factors and parasitic infections is crucial to identifying vulnerable stages in the parasite’s life cycle, which can be targeted for effective and efficient management strategies (Dent 2000; Clausen et al. 2015).

European catfish (Silurus glanis) has been cultivated extensively in Central and Eastern Europe for more than 100 years (Linhart and Proteau 1993). The species possesses a high economic value because of its rapid growth and high-quality flesh (Tinkir et al. 2021). The increasing demand for European catfish has led to the introduction of intensive farming practices, where a high stock density increases susceptibility to infection by a specific gill monopisthocotylan parasite called Thaparocleidus vistulensis. This parasite is one of the most prevalent and serious threats in intensive farming of European catfish, causing a fatal disease in European catfish fry stocks (Molnár 1980; Molnár et al. 2019).

To date, studies done on this parasite have focused on its morphology, (Siwak 1932; Bychowsky and Nagibina 1957; Paladini et al. 2008; Wan Sajiri et al. 2024) reproductive strategies, infection dynamics, and life cycle parameters (oviposition, egg development, hatching, and survival rates) in optimal water temperatures (Molnár 1968; Wan Sajiri et al. 2023), as well as the gill histopathological alterations during the infection (Molnár 1980).

As there have been no previous studies about the effects of changing different environmental factors on various life stages of T. vistulensis, studies of these processes are crucial for assessing potential consequences on the host species. In this study, the effects of light-dark cycles and water temperature on the fecundity, hatching rate, and survivability of T. vistulensis were investigated through controlled laboratory experiments. The insights from these findings provide a valuable foundation for managing T. vistulensis in cultured European catfish stocks in fish farms.

Collection of Thaparocleidus vistulensis

Silurus glanis naturally infected by Thaparocleidus vistulensis were obtained from a commercial fish farm. The fish were transported to the Veterinary Medical Research Institute, Budapest, Hungary (HUN-REN VMRI), and settled for acclimatization in flow-through system tanks (60L) at 23 ± 1°C. To ensure the parasite was present, a gill biopsy from the first gill arch was conducted using a surgical scissors and forceps (Gussev 1983). The parasite population was then sustained in the laboratory by co-habitation, as described by Hutson et al. (2018). The adults, oncomiracidia, and eggs of T. vistulensis were collected following Wan Sajiri et al. (2023).

Study of the influence of light and darkness

Fecundity and survival rates of adult parasites

To investigate the impact of light and darkness on fecundity and survival rate, adult T. vistulensis specimens (n = 20; 2×[10wells×1individuals/well]) were collected and divided into two groups. One group was subjected to continuous light exposure with a 16:8 (L:D; light:dark) h photoperiod, while the other group was kept in constant darkness with 0:24 (L:D) h at 23°C. The parasites were then distributed individually in wells of the 24-well microtiter plates containing 2 ml of filtered (22 µm pore size) water from the fish tank. The number of eggs laid was recorded at intervals of 1, 2, 3, 4, 5, 6, and 24 h under a stereo microscope (Olympus SZX16, Olympus Optical, Tokyo, Japan). The survival ability of the adult T. vistulensis was observed at 24 h intervals until all the individuals died. Individuals that remained motionless, occasionally appeared swollen, and consistently failed to respond to gentle nudges with a needle were considered dead (Grano-Maldonado et al. 2011).

Egg hatching rates

To determine whether light and darkness affect the hatching rates of T. vistulensis eggs, the eggs (n = 300) were collected and evenly distributed into wells of 96-well microtiter plates containing 200 µl of filtered water from the fish tank. Two experimental groups (2×[5wells×10individuals/well, in triplicate]) were set up for hatching observation; one was exposed to a 16:8 (L:D) h photoperiod cycle, and the other was kept in constant darkness (0:24 (L:D) h). Both groups were maintained at a constant temperature of 23°C, with no water exchange or agitation applied (Whittington and Kearn 1988). The eggs were observed under a stereo microscope at 24 h intervals. The hatching rates were recorded daily until no further hatch events occurred within 24 h of the last observed hatching. Hatching rates were determined by counting the number of empty eggshells with an open operculum.

Survival rates of oncomiracidia

To evaluate the influence of light and darkness on the survival rate of oncomiracidia, T. vistulensis larvae (n = 90; 2×[5wells×3individuals/well, in triplicate]) were harvested and allocated into wells of 96-well microtiter plates containing 100 µl of filtered water from the fish tank. The experimental conditions were the same as described in the ‘Egg hatching rates’ section. The observations were done under a stereo microscope at 24 h intervals until all individuals had died. The criteria for determining the death of oncomiracidium were the same as described for the adult parasites.

Study of the influence of water temperature

Fecundity and survival rates of adult parasites

To assess the effect of water temperature on egg production, adult T. vistulensis (n = 70; 7×[10wells×1individuals/well]) were collected and divided into seven groups, with each group being exposed to a different water temperature (5, 10, 15, 20, 25, 30, 35°C) for the experiment. The parasites were then distributed into wells of 24-well microtiter plates containing 2 ml of filtered (22 µm pore size) water from the fish tank. The plates were preconditioned with a Peltier-cooled incubator (IPP 30, Memmert, Germany) set to the appropriate water temperatures. The number of eggs laid was recorded at intervals of 1, 2, 3, 4, 5, 6, and 24 h under a stereo microscope. The survival rate of the adult T. vistulensis was observed and recorded at 24 h intervals until all the individuals in the group had died.

Egg hatching rates

To evaluate the egg hatching rate of T. vistulensis at different water temperatures (5, 10, 15, 20, 25, 30, 35°C), eggs (n = 525; 7×[5wells×5individuals/well, in triplicate]) were collected and allocated into wells of 96-well microtiter plates containing 200 µl of filtered water from the fish tank. The egg hatching rate was monitored and recorded at 24 h intervals under a stereo microscope, and the observation was finished 24 hours after the last eggs hatched.

Survival rate of oncomiracidia

To investigate the influence of water temperature on the survival rate of T. vistulensis oncomiracidia, free swimming larvae (n = 210; 7×[5wells×2individuals/well, in triplicate]) were harvested and evenly distributed into wells of 96-well microtiter plates containing 100 µl of filtered water from the fish tank. The plates were kept at specified water temperatures (5, 10, 15, 20, 25, 30, 35°C), and were observed at 24 h intervals under a stereo microscope until all individuals had perished.

Statistical analysis

Under varying light conditions and water temperatures, biological parameters such as egg production, hatching rates, and survival rates were analysed. The data are presented as the mean ± standard deviation. Given the non-normal distribution of the experimental data groups, the Mann–Whitney test was conducted to compare egg production and hatching rates under light and dark conditions. The Kruskal-Wallis test, another nonparametric approach, was used to discern significant differences in egg production and hatching rates across various water temperatures. The Kaplan-Meier survival analysis was used to estimate the cumulative survival probability of T. vistulensis under different light and water temperature conditions, providing estimations of survival rates at various time intervals. The Log-rank (Mantel-Cox) test was used to compare the survival curve obtained from the Kaplan-Meier survival analyses for different water temperatures and light conditions to identify significant differences in survival between groups. The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software, version 29.0 (IBM Corp., Armonk, NY, United States), with a p-value of < 0.05 considered statistically significant.

Fecundity of T. vistulensis in relation to light and darkness

The impact of light and darkness on T. vistulensis egg production was assessed by observing it in 16:8 and 0:24 (L:D) h photoperiod conditions (Fig. 1). The number of eggs laid by the adults were three times more in the light (96 ± 3.5 eggs), compared to those in darkness (30 ± 1.15 eggs), which was a statistically significant difference (P < 0.001).

Hatching rates of T. vistulensis in relation to light and darkness

The hatching rates of T. vistulensis kept at a 16:8 (L:D) h photoperiod and constant darkness (0:24 (L:D)) h showed a similar tendency of hatching (Fig. 2). The eggs from both groups started to hatch on day 3 post-oviposition (POP), and ended on day 5 POP, with a hatching success of 88.0% and 88.7% in light and dark conditions, respectively. There was no statistically significant difference in hatching rates between the groups (P < 0.926).

Survival rates of adults and oncomiracidia of T. vistulensis in relation to light and darkness

The survival rates of T. vistulensis for adult and oncomiracidium groups were very similar in both light and dark conditions (Fig. 3, 4). For the adult parasites, the group exposed to darkness had its first mortality on Day 1 of post-isolation (PI), while the 16:8 (L:D) h exposed group recorded mortality on Day 2 PI. Individuals survived for less than three days in both groups (Fig. 3A). The survival curves of adult T. vistulensis for both conditions showed no significant differences (Fig. 3B), which was confirmed by a log-rank test (P < 0.142).

For oncomiracidia, individuals in both groups survived up to 5 days (Fig. 4A). The first death in the dark-exposed group was recorded on Day 3 PI, while the light-exposed group had its first death on Day 4 PI. All individuals in both groups perished by Day 6 PI. The survival curves of oncomiracidia T. vistulensis differed significantly in the two conditions (Fig. 4B), as confirmed by a log-rank test (P < 0.001).

Fecundity of T. vistulensis at different water temperatures

The fecundity of T. vistulensis was evaluated at different water temperatures (Fig. 5). Maximum egg production was observed at 15°C (52 ± 1.40 eggs), which showed a statistically significant difference compared to the data recorded at the other water temperatures (P < 0.001). At 20°C, 25°C, and 30°C, the number of eggs produced was 39 ± 1.10, 30 ± 1.15, and 26 ± 0.97, respectively. The number of eggs produced at 20°C was also statistically significant compared to the other water temperatures (P < 0.001). However, no statistical difference was observed between the egg production at 25°C and 30°C (P < 0.583). The egg production at 35°C (6 ± 0.70) and 10°C (11 ± 0.88) was low, but there was no statistically significant difference between them (P < 0.051). No eggs were produced at 5°C, which was statistically significant compared to the other water temperatures (P < 0.001).

Hatching rates of T. vistulensis at different water temperatures

The hatching rates of T. vistulensis were monitored across various water temperatures (Fig. 6). The total hatching success at 10°C, 15°C, 20°C, 25°C, and 30°C was 80%, 81.3%, 89.3%, 89.3%, and 88%, respectively. Notably, no hatching was observed at 0°C and 35°C, and no statistical difference was recorded between the two water temperatures (P < 1.000). The observations were terminated on Day 30 POP and Day 10 POP, respectively, due to the deterioration of the experimental medium. The shortest hatching times for the eggs were recorded at 30°C, followed by 25°C. The hatching started as early as Day 2 POP, and the last eggs were hatched on Day 3 POP and Day 4 POP, respectively. However, data at these two water temperatures showed no statistically significant difference (P < 0.528). At 20°C, the first eggs hatched on Day 3 POP and continued until Day 5 POP. Egg hatching was initiated on Day 5 POP and ended on Day 9 POP at 15°C. The longest hatching period occurred at 10°C, spanning between Day 12 POP and Day 19 POP. Egg hatching at 5°C and 35°C significantly differed (P < 0.001) compared to egg hatching at 10°C, 15°C, 20°C, 25°C, and 30°C.

Survival rates of adults and oncomiracidia of T. vistulensis at different water temperatures

To assess the survival rates of T. vistulensis, adults and oncomiracidia were studied at several water temperatures (Fig. 7, 8). The trend was found to be similar for adults and oncomiracidia, and the survival period shortened when the water temperature decreased.

For adult T. vistulensis (Fig. 7A), the group exposed to 35°C died quickly, with all of them perishing within 24 hours. Death began after Day 1 PI in the groups at 30°C and 25°C, and lasted until Day 2 PI and Day 3 PI, respectively. Mortality commenced on Day 3 PI for the 20°C group, and continued until Day 4 PI, while for the 15°C group, it started on Day 4 PI and persisted until Day 6 PI. A higher survivability was observed in the lower water temperature groups, 10°C and 5°C, where the adult parasites survived up to Day 8 PI and Day 12 PI, respectively. The survival curves of adult T. vistulensis differed significantly at all water temperatures (Fig. 7B), with a log-rank test confirming their statistical significance (P < 0.001).

For oncomiracidia of T. vistulensis, the larvae could only survive for less than one day at extreme water temperatures (35°C and 5°C) (Fig. 8A). At 30°C and 25°C, oncomiracidia stayed alive for three and four days, respectively. The survival trends for 20°C and 15°C were similar, starting on Day 3 PI and Day 4 PI, with the last oncomiracidia surviving until Day 7 PI. The survival period of the parasite larvae was the longest in the 10°C group, where the first death happened on Day 4 PI, and the last oncomiracidium stayed alive until Day 9 PI. A log-rank test confirmed the statistical significance of all survival curves (P < 0.001).

In the aquaculture industry, viruses, bacteria, fungi, and parasites can all be causative agents of various diseases (Woo 2006; Whittington and Chisholm 2008; Woo and Gregory 2014). Among them, parasites pose considerable challenges, demanding effective management strategies (Shinn et al. 2015) for the future of aquaculture. Metazoan ectoparasites, particularly monopisthocotylans, frequently cause significant economic and stock losses in finfish aquaculture (Ernst et al. 2002; Shinn et al. 2015). In order to create the right control strategies, it is crucial to know the ecology and infection dynamics of these parasites, especially the impacts of environmental factors on their life cycles (Villar-Torres et al. 2018; Huston et al. 2020). Most research on monopisthocotylans focus on reproductive strategies (i.e., egg production, hatching, and survival rates), which are often studied in vitro, such as the present study. However, the separation of parasites from their host reduces their viability due to starvation and other factors (Whittington 1997; Mooney et al. 2008). Although the present study may not reflect proper natural conditions, it serves as preliminary knowledge for the recognition of the biological and ecological features of the studied parasite, which are essential for further investigations to improve management and treatment in the field of aquaculture.

Several studies reported that the monopisthocotylans’ response to the daily light-darkness cycles (photoperiod) is one of the factors influencing their life cycles and reproductive strategies (Kearn 1963, 1973, 1982; Mooney et al. 2008; Hoai and Hutson 2014). The present study exposed T. vistulensis at distinct life stages (oncomiracidia and adults) to light and dark conditions of different durations to examine changes in biological parameters such as fecundity, egg hatching, and survival rates. Our observations showed that continuous exposure to darkness resulted in a lower egg production by adult T. vistulensis compared to the 16:8 (D:L) h conditions. A previous study revealed that parasitic activity is highly dependent on the host’s lifestyle, and is specific to each host (Shirakashi et al. 2021). Therefore, we believe that the reduced oviposition of adult T. vistulensis might be related to the behaviour of its host, the European catfish, which is mainly active and feeds at night (Boujard 1995; Slavík et al. 2007). This increased nocturnal activity creates a challenging environment for the parasite’s eggs, reducing their chances of attachment and further development due to the increased movement and water flow. In contrast, previous studies have found that the majority of eggs are released in the dark (Macdonald and Jones 1978; Mooney et al. 2008; Hoai and Hutson 2014; Woo et al. 2024). Macdonald and Jones (1978) suggest that the behaviour of Barbus meridionalis in its natural habitat, where it swims actively during the day, results in Paradiplozoon homoion producing fewer eggs during the day than at night. Some monogeneans keep their eggs in utero until light conditions are optimal for release (Mooney et al. 2006, 2008; Poddubnaya et al. 2017; Tinsley 2017). For example, to increase the likelihood of its hatching larvae finding a suitable new host, Zeuxapta seriolae accumulates eggs in utero and releases them at dusk or in darkness due to the behaviour of its specific host Seriola lalandi, which is active diurnally and congregates around submerged structures at dusk (Mooney et al. 2006). This strategy demonstrates that these parasites can adapt to their hosts’ light-regulated daily behaviour, thereby enhancing and maximizing the transmission of their larvae to a new host.

Our results showed that the trends of egg hatching were similar in both light and dark conditions. The eggs of T. vistulensis are able to hatch through the regulation of their internal developmental cues, regardless of the influence of environmental factors such as changes in light conditions. An optimal egg hatching rate is essential for infection success, as it ensures the release of the maximum number of viable larvae. However, host behaviour and physiology related to environmental conditions, especially light-dark cycles, may influence the hatching strategies of monopisthocotylan eggs (Ernst and Whittington 1996; Whittington and Ernst 2002). The results of light and dark conditions on hatching rates in the present study underscore the importance of further studying their rhythmic patterns such as the tendency of egg hatching (e.g., increased hatching in darkness or in light) under varying light conditions, as shown in several previous studies (Kearn 1963, 1973, 1982; Macdonald 1975; Whittington 1987; Gannicott and Tinsley 1997; Mooney et al. 2008; Hoai and Hutson 2014).

Similar to egg hatching, the survival rates of adult T. vistulensis were also found to be independent of variations in light and dark conditions. The recorded maximum lifespan of adult T. vistulensis was less than three days under both light and dark conditions, although adult T. vistulensis began to die earlier in darkness. Interestingly, statistical analysis of the survival rates of oncomiracidia from Days 3 to 5 POP revealed a significant difference between the two experimental settings, despite their maximum lifespan being the same in both conditions (less than six days). Nearly half of the oncomiracidia in the 16:8 (L:D) h photoperiod group survived up to five days. This suggests that this species might have a higher chance of successfully infecting its hosts during the day when the host is less active, as previously mentioned. However, thorough studies should be done to investigate this. It is interesting to note that the life span of T. vistulensis oncomiracidia is relatively long, allowing them more time to find a new host, whereas for most studied oncomiracidia, it usually does not exceed two days (Whittington et al. 2000; Whittington and Chisholm 2008; Whittington and Kearn 2011; Militz et al. 2014). This period is also longer than that of other monopisthocotylan species at similar water temperatures (20–25°C) (Prost 1963; Golovin and Shukhgalter 1979; Militz et al. 2014).

Environmental factors, particularly water temperature, are crucial in modifying parasite reproduction patterns (Tinsley 2004; Whittington and Chisholm 2008). Studying the effect of water temperature on the reproductive strategies of parasites is extremely important, considering the current use of antihelminths, which detach parasites from the gills by inducing paralysis (Burka et al. 1997; Watson 2009) without necessarily killing them (Woo et al. 2024). These alive, isolated parasites could continue egg production using the nutrients they have taken up, and understanding the effect of water temperature on this process assists in determining the optimal time for treatment (Whittington 1997). The present study found that the optimal water temperature for adult T. vistulensis to lay eggs is 15°C, like other monogenea such as Microcotyle sebastis (Woo et al. 2024). European catfish typically breed at water temperatures of 25–28°C (Linhart et al. 2002), and the present study demonstrated that T. vistulensis is capable of laying a considerable number of eggs per individual even within this water temperature range. The optimal water temperature for egg production for some of the other monopisthocotylan parasites is higher, such as Pseudodactylogyrus anguillae at 25°C (Buchmann 1990), Neobenedenia girellae at 30°C (Hirazawa et al. 2010), and Dactylogyrus vastator at 35°C (Zhang et al. 2015). Our results showed that T. vistulensis was able to lay a minimal number of eggs at 10°C and 35°C, indicating their wide water temperature tolerance that should be carefully considered in efforts to eradicate them within aquaculture systems. The large drop in egg production at these extreme water temperatures is possibly due to a reduction in metabolic activity exploited for egg production as a consequence of adaptation, as reported by Woo et al. (2024). Nevertheless, no eggs could be observed at 5°C, which mirrors the reported egg production behaviour of other parasites such as Urocleidus adspectus (Cone and Burt 1981), Pseudodactylogyrus bini (Chan and Wu 1984; Buchmann 1988), Diplectanum aequans (Cecchini 1994; Cecchini et al. 1998), and Dactylogyrus vastator (Zhang et al. 2022). It points to the impact of extreme water temperatures on the life cycle of T. vistulensis, which requires further in vivo investigations.

Water temperature also influences the hatching success of monopisthocotylan species (Tubbs et al. 2005). The observation of hatching rates of T. vistulensis in the present study revealed a high hatching success (> 80%) across a wide range of water temperatures (10–30°C). Our findings, which indicate that a decreased water temperature initiates a prolonged hatching process, align with a statement by Kearn (1986) that higher water temperatures shorten the developmental period of most monogenean eggs. The longer hatching period of T. vistulensis caused by decreased water temperature is consistent with previous findings for Dactylogyrus vastator (Zhang et al. 2015), D. extensus (Turgut 2012), Benedenia seriolae (Tubbs et al. 2005), Neobenedenia girellae (Bondad-Reantaso et al. 1995)d hirame (Yoshinaga et al. 2000). According to Molnár (1968), the hatching period for T. vistulensis eggs varies with water temperature, taking 6 to 6.5 days at 15–17°C, 3 days at 20–21°C, and 2.5 days at 24–25°C.

The results of the present study align with previously defined time ranges, showing hatching periods of 5 to 9 days at 15°C, 3 to 5 days at 20°C, and 2 to 4 days at 25°C. The developmental and egg hatching intervals can vary widely among species (Kearn 1986; Tubbs et al. 2005; Chen et al., 2010; Marchiori et al., 2015), demonstrating the adaptability of these parasites. The present study also showed that the eggs of T. vistulensis hatched with an average success rate of 80%, with the fastest hatching occurring on Day 2 after oviposition (POP) at 30°C. A similarly fast egg development and short hatching period at the same water temperature was also recorded for several other dactylogyrid monopisthocotylans, including Pseudodactylogyrus bini (Buchmann 1988) and P. anguillae (Buchmann 1990), which infect the European eel. The prolonged hatching and embryonic development period of T. vistulensis at 10°C demonstrates its adaptability to extreme environments, allowing it to maintain reproduction and parasitism, as stated in literature (Perry 1989; Thompson 2020; Marcus et al. 2023). Our results show that extreme water temperatures (5°C and 35°C) halt the development of T. vistulensis eggs, eventually causing their deterioration. The differences in hatching rates at various water temperatures emphasize the species-specific responses to temperature alterations, highlighting the importance of temperature control during fish rearing.

The survival rates of T. vistulensis (adults and oncomiracidia) negatively correlated with water temperature, just like the hatching rate. The present study confirmed that both adults and oncomiracidia exhibited considerably increased longevity as the water temperature decreased, consistent with other monopisthocotylans species from previous studies (Brazenor and Hutson 2015; Valles-Vega et al. 2019). Although all T. vistulensis adults and oncomiracidia died within 24 hours at 35°C in our experiments, Zhang et al. (2015) found that Dactylogyrus vastator oncomiracidia could survive 42 hours at this water temperature. Interestingly, some in vivo studies reported that at 34°C, the oncomiracidia of P. bini (Buchmann 1988) and P. anguillae (Buchmann 1990) could survive up to 14 days and 17 days, respectively. The present study also showed that the adult T. vistulensis could survive up to 12 days without a host at a lower water temperature of 5°C. However, despite their extended survivability, the parasites could not produce eggs at this extreme water temperature, as previously mentioned. Additionally, oncomiracidia died rapidly at 5°C, within less than a day. Careful adjustments of the water temperature can greatly enhance the effectiveness of treatments against T. vistulensis by disrupting their life cycle. As this study was conducted in vitro, further in vivo studies to validate these findings are recommended.

Our findings elucidate the significant impact of photoperiodicity and water temperature on the life cycle and reproductive strategies of T. vistulensis, providing insight into the parasite’s ecology and adaptability to environmental variables. The reproductive process of T. vistulensis is hardly affected by light-dark cycles, except with egg production, but is highly dependent on temperature conditions. Understanding the general biological features and behaviour of T. vistulensis, which infects the European catfish, is particularly relevant given the importance of this species in European fish farming. These findings are also useful to design prophylactic and therapeutic measures against the parasite in European catfish cultures. However, it is important to note that this study was conducted under controlled laboratory conditions and focused solely on an in vitro experimental setup, which may not fully represent the natural behaviour of the parasite. In planning future research, this should be kept in mind, and upcoming studies should be directed towards in vivo investigations to gain a better understanding of the reproductive strategies of T. vistulensis in aquaculture systems.

Financial interest

The authors have no relevant financial or non-financial interests to disclose.

Consent to participate

Not applicable.

Consent to publish
Not applicable.

Ethics approval
The experimental protocols, together with fish handling and sampling, were approved by the Institutional Animal Care and Use Committee of the Veterinary Medical Research Institute, Budapest, Hungary. All research involving experiments on fish (European catfish) was reviewed and approved by the Hungarian National Scientific Ethical Committee on Animal Experimentation under reference number: PE/EA/00081 − 4/2023. The authors complied with the ARRIVE guidelines (https://arriveguidelines.org).

Conflict of interest
The authors have declared that no competing interests exist.

Funding

This project was funded by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 956481.

Author Contribution

All authors contributed to this study. Conceptualization: W.S.W.M.H., B.S., S.C.; Methodology: W.S.W.M.H., B.S.; Formal analysis and investigation: W.S.W.M.H., B.S.; Writing - original draft preparation: W.S.W.M.H.; Writing - review and editing: W.S.W.M.H., B.S., SC; Funding acquisition: S.C.; Resources: S.C.; Supervision: B.S., S.C. The authors read and approved the published version of the manuscript.

Acknowledgement

The authors acknowledge Mr. Muhamad Faizal Mohd for their valuable assistance with the statistical analysis. The authors express their gratitude to Ms. Virág Marek for maintaining the fish in the laboratory.

Data availability

The data are available from the corresponding author upon reasonable request.

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Influences of light-dark cycle and water temperature on in vitro egg laying, hatching and survival rate of the monopisthocotyla Thaparocleidus vistulensis (Siwak, 1932)

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Abstract

Figures

Introduction

Materials and methods

Study of the influence of light and darkness

Fecundity and survival rates of adult parasites

Egg hatching rates

Survival rates of oncomiracidia

Study of the influence of water temperature

Fecundity and survival rates of adult parasites

Egg hatching rates

Survival rate of oncomiracidia

Statistical analysis

Results

Discussion

Conclusion

Declarations

Financial interest

Consent to participate

Consent to publish
Not applicable.

Conflict of interest
The authors have declared that no competing interests exist.

Funding

Author Contribution

Acknowledgement

Data availability

References

Additional Declarations

Status:

Version 1

Influences of light-dark cycle and water temperature on in vitro egg laying, hatching and survival rate of the monopisthocotyla Thaparocleidus vistulensis (Siwak, 1932)

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Study of the influence of light and darkness

Fecundity and survival rates of adult parasites

Egg hatching rates

Survival rates of oncomiracidia

Study of the influence of water temperature

Fecundity and survival rates of adult parasites

Egg hatching rates

Survival rate of oncomiracidia

Statistical analysis

Results

Discussion

Conclusion

Declarations

Financial interest

Consent to participate

Consent to publish Not applicable.

Conflict of interest The authors have declared that no competing interests exist.

Funding

Author Contribution

Acknowledgement

Data availability

References

Additional Declarations

Status:

Version 1

Consent to publish
Not applicable.

Conflict of interest
The authors have declared that no competing interests exist.