Молекулярная и морфологическая характеристика метацеркарий Diplostomum spathaceum из Abramis brama l. Озера Сямозеро (Северо-Запад России)

Полный текст

Lake Syamozero (Baltic Sea drainage basin), located in South Karelia, has long been a unique water body for studying the dynamics of ecosystem eutrophication. Lake Syamozero is the third largest water body in South Karelia (Lake Onego drainage basin, Shuya River catchment). Its area including islands is 270.3 km². The lake is mesotrophic. It is a relatively shallow reservoir with a slight decrease in oxygen concentration in the bottom layers. The lake has abundant zooplankton, and the benthic fauna is relatively rich, with a predominance of chironomids. The fauna of molluscs and insect larvae is also diverse. The ichthyofauna of the lake includes 24 species of fish, among which Cyprinids prevail (Novokhatskaya, 2008).

Various aspects of the lake ecosystem functioning, such as the fish fauna, hydrological parameters, fish parasites, have been studied by staff of the Karelian Research Centre RAS since the 1950s. Long-term multidisciplinary studies have shown that increased anthropogenic pressure in the 1970s–1980s has significantly altered the Syamozero ecosystem (Reshetnikov et al., 1982). In the 1990s, the lake has partly recovered ecologically as the anthropogenic pressure decreased (Sterligova et al., 2016).

Research on fish parasites in Syamozero has been underway for more than 50 years (Shulman, 1962; Shulman et al., 1974; Malakhova, Ieshko, 1977; Ieshko, Malakhova, 1982; Novokhatskaya, 2008; Novokhatskaya et al., 2008; etc.). The variations observed in the diversity and prevalence of parasites indicate the extreme lability of parasitic systems, which are responsive to any changes in the hydrological and hydrobiological conditions (Rumyantsev, 1996).

However, for an accurate overview of the changes in the parasite fauna of fish it is necessary to accurately identify these parasites, which is not always easy. For example, larvae of the genus Diplostomum von Nordmann, 1832 are difficult to identify morphologically. This is a large genus of widely distributed trematodes. Their life cycles involve freshwater snails as the first intermediate hosts, various fish species as the second intermediate hosts, and fish-eating birds as definitive hosts (Shigin, 1986).

Since the advance of the molecular methods, the systematics of many parasite species, including Diplostomum spp., has been revisited, and molecular analysis is now essential for the identification of metacercariae (Astrin et al., 2013; Faltýnková et al., 2014, 2022; Lebedeva et al., 2022; Sokolov et al., 2023; etc.).

The objective of our study is to provide a morphological and molecular description of Diplostomum metacercariae from the eyes of the freshwater bream Abramis brama L., 1758 in Lake Syamozero as an integral part of investigating the parasite composition in the bream and long-term monitoring of changes in its parasite fauna.

MATERIALS AND METHODS

Sample collection

The work is based on the parasitological studies of bream from the south-western part of Lake Syamozero (Syargilahta Bay). Syargilahta Bay (Fig. 1) is a sheltered bay with small sparse aquatic vegetation and shares all of the abovementioned features with the lake.

 

Figure 1. Sampling location: Lake Syamozero. Syargilahta Bay is marked with a black circle.

 

Breams Abramis brama (20 specimens) were examined by complete parasitological dissection according to the method of Bykhovskaya-Pavlovskaya (1985). Fish aged from 4+ to 9+ years were caught with 30-60 mm mesh nets in June 2024. Diplostomum metacercariae were found in the eyes, removed under a preparation stereomicroscope, washed in distilled water and counted. Some larvae were immediately fixed in 96% ethanol and some stained with acetic acid carmine. We used definitions for vouchers in a molecular context proposed by Astrin et al. (2013). Metacercaria paragenophores (morphological voucher) stained with acetic acid carmine were mounted in Canadian balsam for the morphological investigation (Sudarikov, Shigin, 1965). Ten hologenophores (molecular voucher), each from an individual host, fixed in ethanol were used for the molecular studies. They are metacercariae with different body shapes corresponding to those of paragenophores.

The ecological metrics of fish infection, prevalence (Е) and mean abundance (M), were calculated according to Bush et al. (1997).

Morphological examination

Photomicrographs of paragenophores of metacercariae were made with a Levenhuk C1400 NG digital camera attached to Olympus BX-53 microscope using LevenhukToupView image analysis software (V 3.5) at the Core Facility of the Karelian Research Centre of the Russian Academy of Sciences, Petrozavodsk, Russia. The morphology of 40 metacercariae from the eye lens of A. brama was investigated. Thirteen morphological characteristics according to Shigin (1986) were scored (in μm): body length (BL), body width (BW), oral sucker length (OSL), oral sucker width (OSW), ventral sucker length (VSL), ventral sucker width (VSW), holdfast organ length (HL), holdfast organ width (HW), pseudosucker length (PSL), pharynx length (PHL), pharynx width (PHW), distance from center of ventral sucker to anterior end of body (O), and number of excretory bodies. Six indices of the relative values of these parameters were used: BW × BL/HW × HL, BW × BL/VSW × VSL, OSW × OSL/VSW × VSL, HW × HL/ VSW × VSL, BW/BL (%), O/BL (%).

Morphological characteristics of the parasites were assessed by means of discriminant analysis in PAST v. 4.05 (Hammer et al., 2001). Values of ten morphometric parameters of metacercariae of different Diplostomum species were chosen for the canonical discriminant analysis (BL, BW, OSL, OSW, VSL, VSW, HOL, HOW). The choice was based on the availability of published data and their relevance for our study. We used average measurements of D. spathaceum metacercariae from Abramis brama (Shigin, 1986), Gasterosteus aculeatus Linnaeus, 1758, Salvelinus alpinus Linnaeus, 1758 (Faltýnková et al., 2014), Cyprinus carpio Linnaeus, 1758 (Niewiadomska, 2010; Pérez-del-Olmo et al., 2014), multiple hosts: Acipenser ruthenus Linnaeus, 1758; Abramis brama Linnaeus, 1758; Blicca bjoerkna Linnaeus, 1758; Chondrostoma nasus Linnaeus, 1758; Leuciscus aspius Linnaeus, 1758; Rutilus pigus (Lacépède, 1804); Rutilus rutilus Linnaeus, 1758; Vimba vimba Linnaeus, 1758; Silurus glanis Linnaeus, 1758 (Kudlai et al., 2017); and average measurements of D. rutili Razmashkin, 1969 (Shigin, 1986) metacercaria from the lens of Hypophthalmichthys molitrix (Valenciennes, 1844) to determine their ordination in the canonical axes.

The taxonomy and nomenclature of Diplostomum spp. followed the latest studies by Schwelm et al. (2021), Achatz et al. (2022), Faltýnková et al. (2022) and Sokolov et al. (2023).

Slides of paragenophore specimens (DspAb1-DspAb6) of the parasites are deposited in the Helminthological Collections of the Karelian Research Centre, Russian Academy of Sciences (Petrozavodsk, Karelia, Russia).

Molecular and phylogenetic analysis

Genomic DNA was isolated from 10 ethanol-fixed specimens using the DNA-Extran kits (Synthol, Moscow, Russia).

For each of these larvae, we amplified a fragment of the mtDNA cox1 gene using the primers Cox1_schist_5’ (5’-TCTTTR GAT CAT AAG CG-3’) and Cox1_schist_3’ (5’-TAA TGC ATM GGA AAA AAA CA-3’) of Lockyer et al. (2003). The PCR assay was carried in 20 μl of reaction mixture containing 5XScreenMix (Evrogen, Moscow Russia), 1.5 pmol of each primer, an 2 µl of DNA; an annealing temperature of 50⁰C was used for the amplification. PCR products were purified using the Cleanup Standard Extraction Kit (Evrogen, Moscow, Russia) following the manufacturer’s instructions and then sequenced directly with the automatic sequencing system ABI PRISM 3100-Avant (Applied Biosystems Inc., Foster City, CA, USA). Consensus sequences were assembled within MEGA v. 10 (Kumar et al., 2018) to the 1170 bp length and deposited in GenBank under the accession numbers PQ461127 – PQ461136.

The identity of the newly generated sequences was checked with the Basic Local Alignment Search Tool (BLASTn) (www.ncbi.nih.gov/BLAST/, accessed on 10 October 2024). In total, the novel sequences were aligned with those of 32 representatives of the genus Diplostomum in MEGA v. 10 and trimmed to the shortest length, 384 nt. Blasting of the sequences showed a more than 99% match with a large number of sequences of Diplostomum spathaceum. However, we used only 15 of them for the phylogenetic analysis. In this case, we used specimens of the species D. spathaceum, for which both molecular and morphological data are available, especially from bream, as well as specimens of this species in different development phases from different geographical locations (Georgieva et al., 2013; Blasco-Costa et al., 2014; Faltýnková et al., 2014; Pérez-del-Olmo et al., 2014; Locke et al., 2015; Kudlai et al., 2017; Achatz et al., 2022; Lebedeva et al., 2023; Diaz-Suarez et al., 2024). We also applied some sequences of Diplostomum spp. of “non-lens” localization (Achatz et al., 2022).

The cox1 sequence JX986909 of Tylodelphys clavata (Nordmann, 1832) was used as an outgroup.

To assess the phylogenetic relationships of the newly found Diplostomum metacercariae, we applied Bayesian inference analysis (BI) and Maximum Likelihood (ML). ML analyses of HKY+G+I parameter distances were carried out using MEGA v10; nodal support was estimated using 1000 bootstrap resamplings. The best-fitting model for BI was identified as TVM + G + I with jModelTest v2.1.2 (Darriba et al., 2012). Bayesian inference analyses were conducted using MrBayes (v3.2.3) (Ronquist et al., 2012). Markov chain Monte Carlo (MCMC) simulations were run for 3,000,000 generations, log-likelihood scores were plotted, and only the final 75% of trees were used to produce the consensus tree. Posterior probability was calculated to estimate nodal support. FigTree v1.4 (Rambaut, 2012) was used to visualize the tree.

Distance matrices (p-distance) were calculated with MEGA v. 10 (Kumar et al., 2018). The cox1 haplotypes of Diplostomum spp. from the lens of the freshwater bream, Abramis brama, collected in the present study and previous study in Slovakia (Kudlai et al., 2017) countries were identified with DnaSP v. 6 (Rozas et al., 2017). The haplotype network was reconstructed using the Median-Joining method in PopART software v 1.7 (Population Analysis with Reticulate Trees, http://popart.otago.ac.nz).

RESULTS

We investigated different eye tissues of bream, and Diplostomum metacercariae were found only in the lens. Prevalence was 100%, mean abundance 25.3 specimens per fish, intensity from 1 to 51 specimens.

Phylogenetic analysis included ten metacercariae taken from the eyes of different bream specimens fish and indicated that their partial sequences of the cox 1 gene were identical to the published ones of the species D. spathaceum (Rudolphi, 1819) Olsson, 1876 (Fig. 2).

 

 

Figure 2. Bayesian inference (BI) and maximum likelihood (ML) analyses tree for Diplostomum spp. based on the partial cox1 mtDNA sequences (384 bp). Nodal supports from both analyses are indicated as BI/ML. The scale bar indicates the expected number of substitutions per site. Only significant values of the posterior probabilities (≥ 0.5) are indicated. Newly obtained sequences are in bold. Abbreviations: ad – adult, mtc – metacercariae, Ab – Abramis brama, Bb – Blicca bjoerkna, Cm – Cyprinion macrostomum, La – Larus argentatus, Pp – Pungitius pungitius, Rr – Rutilus rutilus, Sa – Salvelinus alpinus, Ch – China, E – Estonia, G – Germany, H – Hungary, I – Iraq, Ic – Iceland, R – Russia, S – Slovakia, Sp – Spain.

 

The metacercariae we studied joined in a clade with representatives of D. spathaceum at different developmental stages and from different countries, confirming a wide distribution and occurrence in different host species worldwide (Fig. 2). The p-distance between ten investigated parasites from Syamozero was 0.08 – 1%. And p-distances among samples of D. spathaceum were 0.03 – 1.5%.

There were seven haplotypes (Fig. 3) among the larvae from bream, with haplotype diversity at 0.93. Four of the sequences each had its own haplotype (PQ461127, PQ461129, PQ461130, PQ461134), and three more haplotypes were represented by two specimens each (PQ461132 and PQ461133; PQ461135 and PQ461136; PQ461128 and PQ461131, respectively).

 

Figure 3. Haplotype network for Diplostomum spathaceum based on novel and published partial cox1 mtDNA sequences of Kudlai et al. (2017). Unsampled intermediate haplotype is represented by a short intersecting line; each branch corresponds to a single mutational difference and connective lines represent one mutational step. Circle size is proportional to the number of isolates sharing a haplotype; black circles indicate transitive haplotypes that have not been found.

 

Comparison against the only available cox1 mtDNA sequences of D. spathaceum metacercariae from breams from Slovakia (Kudlai et al., 2017) showed that only one haplotype was shared between two Karelian metacercariae (PQ461128, PQ461131) and one Slovakian metacercaria (KY653964).

Along with the phylogenetic analysis, paragenophores (taken from the same host individuals as the larvae taken for the molecular studies) were examined morphologically (Fig. 4).

 

Figure 4. Metacercariae of Diplostomum spathaceum with different shape of body from the lens of Abramis brama in Lake Syamozero. Scales: 100 μm.

 

Thirteen morphological attributes of 40 newly found D. spathaceum metacercariae and six ratios of attributes were obtained.

Body elongate-oval, 296–499 × 194–313 (403 × 243), with maximum width just anterior to ventral sucker. Oral sucker elongate-oval, 33–70 × 36–61 (54 × 46). Pseudosuckers strongly muscular, mostly prominent, ear-like, 40 – 75 (58) long. Pharynx elongate-oval, 25–39 × 18–28 (34 × 20); oesophagus short, bifurcates closely posterior to pharynx; caeca long, narrow, reaching posterior to holdfast organ. Ventral sucker transversely oval, 32–59 × 40–75 (47 × 53), smaller or larger than oral sucker [sucker width ratio 1:0.92 0.96 (1:1.02)], slightly posterior to mid-body length. Distance from anterior extremity of body to ventral sucker 154 294 (242). Holdfast organ relatively small, transversely oval, bipartite, contiguous with ventral sucker, 57–119 × 61–146 (78 × 82). Excretory vesicle small, V-shaped; reserve excretory system of diplostomid type; excretory concretions small, 177–380 (259) in number. Ratios are BW × BL/HW × HL 9–16 (15); BW × BL/VSW ×VSL 35–44 (40); OSW × OSL/VSW ×VSL 0.92–0.96 (1); HW × HL/VSW ×VSL 2.6–3.9 (2.5); BW/BL (%) 60–66 (63); О/BL (%) 52–60 (59).

Metacercariae from different host specimens did not significantly differ in the number of excretory granules (p > 0.05). The parasites’ dimensions also were not significantly different (p > 0.05). The morphological attributes of metacercariae were also similar, except for the shape of body and pseudosucker (Fig. 4). The body shape of metacercariae from bream’s eye lens varied from rounded to elongated oval. A specific feature of the larvae was the morphology of pseudosuckers, which in a majority of metacercariae protruded forward beyond the anterior part of the body and oral suckers.

Discriminant analysis of the newly obtained and previously published data on the mean dimensions of Diplostomum spp. metacercariae parasitizing the lens was conducted (Fig. 5). The metacercariae of D. spathaceum from Lake Syamozero fell within a single cohort with a 95% confidence interval (large ellipse in Fig. 5).

 

Figure 5. Ordination of the traits of Diplostomum spathaceum and D. rutili metacercariae by discriminant analysis. The mean values of nine traits, including morphometry, from the present study and the literature were involved: 1 – green rhombus: D. spathaceum (Shigin, 1986); 2 – black asterisk: D. spathaceum (Niewiadomska, 2010); 3 – blue cross: D. spathaceum (Faltýnková et al., 2014); 4 – yellow oval: D. spathaceum (Pérez-del-Olmo et al., 2014); 5 – navy square: D. spathaceum (Kudlai et al., 2017); 6 – pink triangle: D. rutile (Shigin, 1986); 7 – black circle: means of dimensions of D. spathaceum from Lake Syamozero (the coloured points – red, light blue, dark blue, green, brown, purple – represent the specimens taken from different host individuals).

 

Only three individuals from different hosts did not fit into the size group. The most significant contribution to the divergence of the species included in the analysis was from the metacercariae body length and width (BL and BW) on both axes. The length and width of the holdfast organ (HL and HW) were the most significant on the first canonical axis, while OSL, VSL and VSW were the most significant on the second canonical axis. The confidence ellipse includes both data on the dimensions of D. spathaceum from the present study and from Cyprinus carpio (Pérez-del-Olmo et al., 2014) as well as on D. rutili from Hypophthalmichthys molitrix (Shigin, 1986). Metacercariae of D. spathaceum from Abramis brama of Shigin (1986) were the most similar in mean dimensions to the larvae found in Cyprinus carpio (Niewiadomska, 2010). While neither of these samples fit within the confidence zone of the Lake Syamozero sample, they proved to be very close to it. Average dimensions of D. spathaceum from G. aculeatus and S. alpinus (Faltýnková et al., 2014) and from multiple hosts (Kudlai et al., 2017) were far both from the confidence ellipse of the Lake Syamozero sample and from each other (Fig. 5).

DISCUSSION

The data we obtained demonstrate that the species D. spathaceum parasitizes the lens of freshwater breams in Lake Syamozero. Previously, this species was recorded by researchers either as Diplostomum sp. (Shulman, 1962; Ieshko, Malakhova, 1982) or as D. chromatophorum (Brown, 1931) Shigin, 1986 (Novokhatskaya et al., 2008). This identification is solely due to the alteration in the systematics of the genus, when different authors tried to distinguish species only on the basis of morphological data and host specificity (Sudarikov et al., 2002; Niewiadomska, 2010). However, owing to the use of molecular methods, the situation with identification and description of diplostomids tends to improve. The partial cox1 mtDNA sequences of the newly found parasites matched numerous previously published sequences for the species D. spathaceum, noted not only at the metacercariae stage in different fish species (Faltýnková et al., 2014; Kudlai et al., 2017), but also at the adult stage (Pérez-del-Olmo et al., 2014).

We identified seven haplotypes in ten metacercariae retrieved from bream from Lake Syamozero. Among the 14 cyprinid species in Danube River in the study by Kudlai et al. (2017), A. brama had the largest haplotype diversity (8 haplotypes). In total, our data and the above-mentioned report by Kudlai et al. (2017) revealed 14 haplotypes in Diplostomum spathaceum metacercariae from bream, with haplotype diversity (Hd) of 0.96.

Our integrated approach (molecular and morphological features) also enabled a detailed analysis of the morphological attributes of D. spathaceum larvae (Fig. 5).

The morphology of D. spathaceum metacercariae in our study (Fig. 5) is not in full agreement with the descriptions by other authors, i.e. Faltýnková et al. (2014) and Kudlai et al. (2017). The newly investigated specimens differ from those of Faltýnková et al. (2014) in having a shorter and wider body, smaller oral and ventral sucker, holdfast organ. Metacercariae from the host mix of Kudlai et al. (2017) had a smaller length of the body and oral sucker, but larger dimensions of the holdfast organ compared to the parasites from Syamozero. While situated in the very periphery of the confidence ellipse, the larvae reported by Pérez-del-Olmo et al. (2014) had dimension characteristic of the species. Such variability of parasites is probably due to different sample collection and fixation methods, as well as different host species.

The most interesting finding of our analysis is that the mean dimensions of the species D. rutili described from the lens of Hypophthalmichthys molitrix by Shigin (1986) fall within the confidence ellipse of the newly studied metacercariae of D. spathaceum. In addition, Figure 5 demonstrates that the mean dimension values of the species fall within the range between those of the newly described metacercariae and larvae of D. spathaceum from Shigin (1986) and Niewiadomska (2010). Furthermore, such а specific feature of the larvae as pseudosuckers protruding forward beyond the anterior part of the body and oral suckers was described as a species-specific trait only for the metacercariae of D. rutili Razmashkin, 1969 (Shigin, 1986). Thus, the matching sizes, the presence of the characteristic ear-shaped protruding pseudosuckers, and the similar range of Cyprinid hosts leads us to the assumption that the species D. rutili Razmashkin, 1969 may be conspecific to D. spathaceum (Rudolphi, 1819) Olsson, 1876.

The joint analysis of our own and published data shows that the rates of Diplostomum metacercariae infection in bream of Lake Syamozero have have increased significantly since 1980 (Fig. 6).

 

Figure 6. Long-term changes of Diplostomum sp. metacercariae infection parameters in eyes of the bream Abramis brama in Lake Syamozero.

 

A similar trend was previously noted in many other fish species in Syamozero (Ieshko, Novokhatskaya, 2008; Novokhatskaya et al., 2005; Novokhatskaya, 2008). It is likely that intensive vegetation growth in the littoral zone induced by eutrophication has created favourable conditions for gastropods, the intermediate hosts of diplostomids (Novokhatskaya, 2008). In addition, the concentration of juveniles of many fish species in shallow waters (Sterligova et al., 2016) and the growing number of gulls in the lake area (Sazonov, 2004) also promote the high abundance of diplostomids.

CONCLUSIONS

Thus, our study has established that the species D. spathaceum parasitizes the eye lens of breams in Lake Syamozero. The mtDNA sequences of the species match those obtained earlier, but their morphological features only partially correspond to those described in the literature. Furthermore, discriminant analysis of the size characteristics in combination with the presence of a well-defined three-lobed anterior end of the body suggests that D. rutili is a junior synonym of D. spathaceum. This study once again confirms the importance of using an integrated approach to diplostomid larvae identification. In addition, this approach opens great opportunities for investigating the species morphological variability within populations.

Long-term data demonstrate a considerable increase in bream infection with D. spathaceum and reflect changes occurring both in the host population and in the ecosystem as a whole.

ACKNOWLEDGEMENTS

The authors are very grateful to Dr. Olga P. Sterligova for assistance in collecting the material and to Dr. Sergey V. Bugmyrin for help with conducting discriminant analysis. Special thanks are due to Olga S. Kislova (Chief Translator of Department for International Cooperation, KRC RAS) for help with proofreading of the text.

FUNDING

The study was funded by the Russian Science Foundation, project no. 24-26-00251.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Research Fishing is under Permit of North-West Territorial Administration of the Federal Agency for Fishery (7820240317689) from 14 May 2024.

CONFLICT OF INTEREST

The authors of this work declare that they have no conflicts of interest.

Об авторах

Д. И. Лебедева

Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences

Автор, ответственный за переписку.
Email: daryal78@gmail.com
Россия, 11 Pushkinskaya St., Petrozavodsk, 185910

И. В. Суховская

Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences

Email: daryal78@gmail.com
Россия, 11 Pushkinskaya St., Petrozavodsk, 185910

А. А. Кочнева

Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences

Email: daryal78@gmail.com
Россия, 11 Pushkinskaya St., Petrozavodsk, 185910

Л. А. Лысенко

Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences

Email: daryal78@gmail.com
Россия, 11 Pushkinskaya St., Petrozavodsk, 185910

Н. П. Канцерова

Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences

Email: daryal78@gmail.com
Россия, 11 Pushkinskaya St., Petrozavodsk, 185910

Д. О. Зайцев

Petrozavodsk State University

Email: daryal78@gmail.com
Россия, 33 Lenin Ave., Petrozavodsk, 185910

Н. П. Милянчук

Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences

Email: daryal78@gmail.com
Россия, 11 Pushkinskaya St., Petrozavodsk, 185910

Е. П. Иешко

Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences

Email: daryal78@gmail.com
Россия, 11 Pushkinskaya St., Petrozavodsk, 185910

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