Introduction
In the complex life cycle of trematodes, an important stage of transmission is the endotrophic cercaria larva, which infects the second intermediate host (SIH) or the definitive host (DH). Cercariae have a complex set of behavioural reactions promoting dispersal and infection of a wide range of animal hosts, both invertebrate and vertebrate (reviewed in: Haas Reference Haas2003). Many factors, exogenous as well as endogenous, influence the transmission of cercariae (reviewed in: Combes et al. Reference Combes, Fournier, Mone and Theron1994; Marcogliese Reference Marcogliese2001; Galaktionov and Dobrovolskij Reference Galaktionov and Dobrovolskij2003; Pietrock and Marcogliese Reference Pietrock and Marcogliese2003; Lafferty and Kuris Reference Lafferty, Kuris, Thomas, Renaud and Guégan2005). Their lifespan is limited, which was attributed to the depletion of glycogen reserves accumulated during the development in the molluscan first intermediate host (FIH) (reviewed in: Ginetsinskaya Reference Ginetsinskaya1968; Morley Reference Morley2011). The time during which cercariae remain infective is even more limited, ranging in the larvae of different species from a few hours (e.g. Miller and McCoy Reference Miller and McCoy1930; Anderson and Whitfield Reference Anderson and Whitfield1975; Lowenberger and Rau Reference Lowenberger and Rau1994; McCarthy Reference McCarthy1999; Karvonen et al. Reference Karvonen, Paukku, Valtonen and Hudson2003; de Montaudouin et al. Reference de Montaudouin, Blanchet, Desclaux-Marchand, Lavesque and Bachelet2016; Born-Torrijos et al. Reference Born-Torrijos, van Beest, Vyhlidalova, Knudsen, Kristoffersen, Amundsen, Thieltges and Soldanova2022) to several days (Pekkarinen Reference Pekkarinen1987) or even weeks (Wetzel and Esch Reference Wetzel and Esch1995). Age-related dynamics of infectivity differ significantly cercariae of all species studied in this respect.
Cercariae of some species reach maximal infectivity only after ca. 1–3 hr after leaving the molluscan host. An analysis of the literature on the age dynamics of cercarial infectivity (Table 1) showed that the methodological aspect, that is, the minimum age of cercariae used in the experiment, was often the key to the discovery of the time delay before the attainment of the maximum infectivity (TDMI). In most cases, the larvae were tested starting from the age of 1 hr after leaving the FIH (Table 1), which means that the period of their decreased infectivity might have already ended. At the same time, the minimum and the most frequently observed TDMI made up about 1 hr (McCarthy Reference McCarthy1999; Toledo et al. Reference Toledo, Munoz-Antoli, Perez and Esteban1999; Munoz-Antoli et al. Reference Muñoz-Antoli, Trelis, Espert, Toledo and Esteban2002; Whitfield et al. Reference Whitfield, Bartlett, Khammo and Clothier2003; Paller et al. Reference Paller, Kimura and Uga2007), and so could not be revealed in experiments with older cercariae (Stirewalt and Fregeau Reference Stirewalt and Fregeau1968; Malek Reference Malek1977; Karvonen et al. Reference Karvonen, Paukku, Valtonen and Hudson2003) (Table 1). Experiments with cercariae of Schistosoma mansoni are a case in point. Stirewalt and Fregeau (Reference Stirewalt and Fregeau1968) tested them starting from the age of 1 hr and did not find any TDMI. However, as shown later by Whitfield et al. (Reference Whitfield, Bartlett, Khammo and Clothier2003), cercariae of S. mansoni do exhibit a decreased infectivity at an age of less than 1 hr, while at an age of 1 hr, the infectivity reaches the maximum (Table 1). Therefore, it cannot be ruled out that cercariae of other trematode species also have a decreased infectivity in the first minutes (before the age of 1 hr) of their life in the environment.
Table 1. Conditions and results of experiments on detection of time delay before the attainment of the maximum infectivity (TDMI) by cercariae of trematodes based on literature data and the data from this study
TDMI may be due to the need for some morpho-physiological changes during the transition of the cercariae from the existence in the molluscan host to the life in the external environment (Whitfield et al. Reference Whitfield, Bartlett, Khammo and Clothier2003). The adaptive significance of the delay is usually attributed mainly to the necessity of spatial dispersion of the larvae and the avoidance of superinfection of the SIH in the vicinity of the cercariae-shedding molluscan FIH (Evans and Gordon Reference Evans and Gordon1983; McCarthy Reference McCarthy1999; Toledo et al. Reference Toledo, Munoz-Antoli, Perez and Esteban1999; Lowenberger and Rau Reference Lowenberger and Rau1994; Whitfield et al. Reference Whitfield, Bartlett, Khammo and Clothier2003). This is particularly important under conditions of low hydrodynamics (e.g. in lentic or slow-flowing freshwater bodies), if the SIH is sedentary and/or if the role of the SIH can be also played by the FIH. All these factors are characteristics of transmission of echinostomatid cercariae, which were found to exhibit TDMI of 1–3 hr (Lo and Cross Reference Lo and Cross1975; Evans and Gordon Reference Evans and Gordon1983; McCarthy Reference McCarthy1999; Toledo et al. Reference Toledo, Munoz-Antoli, Perez and Esteban1999) (Table 1). To note, echinostomatid larvae are highly pathogenic for their molluscan SIH when the infection intensity is high (Kuris and Warren Reference Kuris and Warren1980; Fried et al. Reference Fried, Idris and Ohsawa1995; Ataev Reference Ataev2010). Cercariae of Echinostoma trivolvis may also have TDMI, but only at the first stages of their life in the environment, which is why it was not recorded in the experiments of Pechenik and Fried (Reference Pechenik and Fried1995), who tested cercariae starting from the age of 45 min (Table 1).
Superinfection of an agile downstream host by cercariae shed by the mollusc is unlikely, which means that TDMI found in larvae of S. mansoni (Whitfield et al. Reference Whitfield, Bartlett, Khammo and Clothier2003) and Centrocestus armatus (Paller et al. Reference Paller, Kimura and Uga2007) (Table 1) must be due to some other factors. The primary factor in this case is probably the need for a period of physiological maturation of the cercariae in the environment. To note, TDMI was not found in cercariae of Transversotrema patialense infecting agile fish hosts (Anderson and Whitfield Reference Anderson and Whitfield1975; Anderson et al. Reference Anderson, Whitfield and Mills1977), though these larvae were involved in the experiment as early as 15 min after emergence from the molluscan host. Information about the absence of TDMI in cercariae of Diplostomum spathaceum infecting fish requires verification since the minimum age of cercariae in the experiment was 3 hr (Karvonen et al. Reference Karvonen, Paukku, Valtonen and Hudson2003).
The need for a period of maturation in the external environment after emergence from the molluscan host cannot be ruled out for cercariae of other trematodes. All available data were obtained on freshwater species whose cercariae are usually transmitted under conditions of low hydrodynamics. There is no information on the age dynamics of infectivity of cercariae infecting SIHs in marine biotopes with an increased hydrodynamics such as the intertidal.
This lack of this information prompted our study of cercariae of two trematode species transmitted in the coastal ecosystems of the White Sea, Himasthla elongata (Mehlis, 1831) (Himasthlidae) and Renicola parvicaudatus (Stunkard & Shaw, Reference Stunkard and Shaw1931) (Renicolidae), which use mussels (Mytilus edulis) as the SIH. The FIH of these trematode species are snails Littorina littorea, while DH are seabirds (Stunkard and Shaw Reference Stunkard and Shaw1931; Werding Reference Werding1969; Galaktionov et al. Reference Galaktionov, Solovyeva and Miroliubov2021, Reference Galaktionov, Solovyeva, Blakeslee and Skirnisson2023).
It should be noted that the family Himasthlidae and the family Echinostomatidae are very closely related. Previously, the himasthlids were even considered as a subfamily of the Echinostomatidae (Kostadinova Reference Kostadinova, Jones, Bray and Gibson2005; Tkach et al. Reference Tkach, Kudlai and Kostadinova2016). This means that our results obtained on H. elongata can be compared with those on freshwater echinostomatids.
Materials and methods
Sample collection
Periwinkles Littorina littorea (Linnaeus, 1758) were collected in the intertidal zone of the Chupa Inlet of the Kandalaksha Bay (the White Sea) in July 2023. Individuals of L. littorea shedding cercariae of Himasthla elongata (or Renicola parvicaudatus) were considered as infected and used as a source of cercariae. Mussels (Mytilus edulis) were taken from artificial substrates of the sea farm. The sea farms in the White Sea are situated at a distance of at least 50 m from the nearest shore, and mussels cultivated there are uninfected with renicolid and himasthlid larvae (Kulachkova Reference Kulatchkova and Lukanin1985; Levakin et al. Reference Levakin, Losev, Nikolaev and Galaktionov2013).
Experiment
Experiments on the infection of mussels with cercariae of H. elongata and R. parvicaudatus were carried out in August 2023 at the White Sea Biological Station of the Zoological Institute of the Russian Academy of Science (Cape Kartesh, Kandalaksha Bay of the White Sea). In each experiment, a pool of cercariae was taken from 15 individuals of L. littorea infected with H. elongata and the same number of periwinkles was infected with R. parvicaudatus. In this way, possible interclonal differences in the infectivity of cercariae were levelled (Levakin et al. Reference Levakin, Losev, Nikolaev and Galaktionov2013). After that, the molluscs were exposed to diffused sunlight in 1 l jars with seawater. Cercariae were sampled for the experiment after 10 min of exposure of the snails to light (ca. 500 Lx). Groups of cercariae for the infection of one mussel (20 cercariae of H. elongata or 50 cercariae of R. parvicaudatus) were placed into separate Petri dishes with a volume of 5 ml filled with fresh seawater with a temperature of 20°C and a salinity of 24 ppt. The cercariae were kept in these Petri dishes at 20°C for 0.5; 1; 1.5; 2; 2.5; 3; 4; 5; and 6 hr and then used for mussel infection.
Uninfected mussels with a shell length of 18–20 mm were acclimated to 20°C for 2 days in aerated tanks with seawater. After that, they were placed in separate (100 ml) jars with fresh seawater with a salinity of 24 ppt. Then, either 20 cercariae of H. elongata or 50 cercariae of R. parvicaudatus aged 0.5; 1; 1.5; 2; 2.5; 3; 4; 5; and 6 hr were added to each jar with mussels. Taking into account the dependence of the pumping activity of the White Sea mussels (E, l/hr) on the mean geometric length (l), height (h), and width (w) of their shell [L = (l*h*w)1/3, mm], E=0.04L 1.6 (Lezin et al. Reference Lezin, Agat’eva and Khalaman2006), the volume of water pumped by a mussel of the size used in our experiments (L = 9.2±0.2 mm) during the time of incubation with the cercariae (0.25 hr) makes up 348 ± 12 ml. This volume is more than 3 times greater than that of the experimental jar (100 ml), which guarantees the host-parasite encounter and removes the possible influence of the behavioural differences between the larvae of the two trematode species involved in our experiments (Prokofiev Reference Prokofiev2002; Nikolaev et al. Reference Nikolaev, Prokofiev, Levakin and Galaktionov2017) on the success of mussel infection.
A total of 180 mussels were infected (10 molluscs with each age gradation of cercariae). The molluscs were exposed to cercariae for 15 min at 20°C with regular room light (about 500 Lx), after which they were rinsed with fresh water to remove cercariae that had not penetrated during the exposure time and transferred to jars with fresh seawater (100 ml), where they were kept for 24 hr and then dissected. To detect the resulting infection, the mussels’ soft tissues were squeezed and screened under a stereomicroscope, and the metacercariae were counted.
Statistics
Statistical treatment of the data followed standard recommendations (Sokal and Rohlf Reference Sokal and Rohlf1995; Underwood Reference Underwood1997). Infection success was estimated individually for each mussel as a ratio of the number of metacercariae found in a mussel to the number of cercariae used to infect this mussel (20 cercariae of H. elongata or 50 cercariae of R. parvicaudatus). The mean success of mussel infection with cercariae of the same age was considered as the infectivity of the larvae of this age. Statistical significance of the influence of cercarial age on their infectivity (mean infection success) was assessed with the help of one-way of Analysis of Variance (ANOVA). The data were arcsin-transformed before performing ANOVA. Means and confidence limits of means were back-transformed. Confidence intervals are given for a 95% significance level. The computations were carried out in R (https://www.r-project.org/).
Results and Discussion
Changes in the infectivity of cercariae of H. elongata and cercariae of R. parvicaudatus from 0.5 hr to 6 hr were insignificant (ANOVA; H. elongata: F (8.81) = 1.053, P>0.05, η 2 = 9.4%; R. parvicaudatus: F (8,81) = 2.047, P>0.05, η 2 = 16.8%) (Figure 1). This means that TDMI was absent in cercariae of H. elongata and R. parvicaudatus aged 0.5 hr and more.
Figure 1. Age-dependent dynamics of infectivity of cercariae of Himasthla elongata and Renicola parvicaudatus.
At first sight, this finding seems strange. These larvae, especially those of H. elongata, can be highly pathogenic for SIHs, which are represented by sedentary bivalves such as mussels (Lauckner Reference Lauckner and Kinne1983). Besides, both H. elongata and R. parvicaudatus can use Littorina spp. (FIH) as SIHs (Lauckner Reference Lauckner and Kinne1983). Cercariae of echinostomatids, which are closely related to Himasthla, have precisely this combination of features and exhibit a pronounced TDMI (see the Introduction) (Table 1). It is not entirely impossible that the cercariae of the two species in our study do have TDMI in the first minutes of their life (up to 0.5 hr) and that they need it to reach full maturity, as, for example, larvae of S. mansoni (Whitfield et al. Reference Whitfield, Bartlett, Khammo and Clothier2003). However, considering the biological features of cercariae of H. elongata and R. parvicaudatus and the fact that they are transmitted in marine intertidal biotopes, the adaptive character of TDMI appears less important for them than for cercariae of freshwater digeneans in lentic and/or slow-flowing water bodies.
Under littoral conditions, passive transport by currents and microcurrents contributes significantly to cercarial dispersion (de Montaudouin et al. Reference de Montaudouin, Wegeberg, Jensen and Sauriau1998; Fingerut et al. Reference Fingerut, Zimmer and Zimmer2003; Zimmer et al. Reference Zimmer, Fingerut and Zimmer2009). In addition, cercariae can be rapidly transported outside the host contact zone by wave action and tidal currents. These features considerably decrease the probability of superinfection of mussels in the vicinity of the mollusc that has emitted the larvae. The same is true of their FIH, periwinkles, which, not being specific SIH for cercariae of H. elongata and R. parvicaudatus, are infected with them with a very low efficiency. In the area of material (FIH) collection, where 100% prevalence and a high abundance of metacercariae of H. elongata and R. parvicaudatus in mussels is observed, periwinkles infected with these larvae are extremely rare (our observations).
Besides environmental factors, superinfection of SIHs may also be prevented by the features of cercarial emergence and biology. Cercariae of H. elongata have a relatively small average daily output, 707.9 ± 89.2 (438–1,225) (Prokofiev et al. Reference Prokofiev, Galaktionov and Levakin2016). Therefore, the absence of TDMI can increase the chances of an individual larva finding itself in the vicinity of a potential SIH and infecting it. On the other hand, a high hydrodynamics at the intertidal combined with the mobility of the FIH, L. littorea, decrease the risk of superinfection of sedentary SIHs (mussels). Renicolid metacercariae are less pathogenic for SIHs (Thieltges Reference Thieltges2006), but the daily output of their cercariae is much higher than in H. elongata, 2,276.2 ± 342.2 (854–3,728) (Prokofiev et al. Reference Prokofiev, Galaktionov and Levakin2016). Having left the FIH, cercariae of R. parvicaudatus, which are already infective for SIHs, rise to the upper water layers, thus leaving the ‘host space’, the habitat of mussels on the sediment. They concentrate near the bottom, thus returning to the ‘host space’, only after 2–4 hr (Prokofiev Reference Prokofiev2002; Nikolaev et al. Reference Nikolaev, Prokofiev, Levakin and Galaktionov2017). This ensures a more or less uniform distribution of the larvae within the littoral site, increasing the probability that they would find mussels in sparse settlements. This behavioural feature of cercariae of R. parvicaudatus can also be considered as a mechanism of preventing the superinfection of SIHs in the absence of TDMI.
The average encystment rate of cercariae of H. elongata in our experiments was 40.6 ± 4%, while that of cercariae of R. parvicaudatus did not exceed 12.7 ± 1.7%. These differences are probably associated with the fact that the mussel is not the preferred host for renicolid larvae. The results of experimental infections (Thieltges and Rick Reference Thieltges and Rick2006) confirmed that the preferred host for metacercariae of R. parvicaudatus is the mollusc Cerastoderma edule (Lauckner Reference Lauckner and Kinne1983). Therefore, an initial retreat of renicolid cercariae from the ‘host space’ is especially relevant in littoral biotopes of temperate seas inhabited by cockles Cerastoderma edule.
Conclusion
We would like to note that the presence or absence of TDMI in cercariae does not seem to depend on their taxonomic position. It appears to be determined by the transmission conditions for a particular species. In this regard, it would be interesting to study the age dynamics of the invasive capacity of echinostomatid cercariae that infect highly agile fish hosts and that are transmitted in a lentic freshwater biotope, such as Isthmiophora melis (Beaver Reference Beaver1941; Radev et al. Reference Radev, Kanev, Hrusanov and Fried2009) and Drepanocephalus spathans (Alberson et al. Reference Alberson, Rosser, King, Woodyard, Khoo, Baumgartner, Wise, Pote, Cunningham and Griffin2022).
Acknowledgments
We are grateful to the staff of the White Sea Biological Station of the Zoological Institute of the Russian Academy of Sciences for their help during the work. We are grateful to Natalia Lentsman for her help with the translation into English.
Financial support
This study was funded by the Russian Science Foundation (grant number 23-14-00329). The fieldwork at the White Sea Biological Station was supported by the research programme of the Zoological Institute of the Russian Academy of Sciences (project numbers 122031100260-0 and 122031100283-9).
Competing interest
None.
Ethical standard
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional institutional guides on the care and use of laboratory animals.
