Ligula intestinalis (Linnaeus, 1758)

Occurrence of Ligula intestinalis View in CoL and rate of infection

We found infection with L. intestinalis in freshwater bream from Lakes Onego, Ladoga, Svyatozero and Konchezero. This is the first record of this cestode in bream from these water bodies. Infection indices could not be calculated because the material from these lakes was limited and collected in different years.

In Lake Syamozero infection rates of bream with L. intestinalis were low: prevalence 8.9% with ratio 1.52, intensity of infection from 1 to 3 tapeworms per fish, abundance 0.12 ( Fig. 2 View Figure 2 ). These values are similar to those recorded in 1975, when prevalence was 6.7% and mean abundance was 0.07 ( Novokhatskaya et al., 2008). In June 2024, we also examined several individuals of roach R. rutilus and white bream Blicca bjoerkna but did not find any plerocercoids of L. intestinalis in them.

Phylogenetic analysis

We obtained partial sequences of two mtDNA regions, COI (396 bp) and Cyt b 405 bp), from 28 individuals of L. intestinalis from Karelia. They were used for phylogenetic reconstruction together with the previously published sequences of Ligula spp. from different hosts and localities ( Table 1, Fig. 3).

Ecologica Montenegrina , 80, 2024, 21-37

Table 1

Ab738 PQ356679 PQ329020 Abramis brama Russia: Karelia,

Ab739

PQ356680 PQ329021

Abramis brama Russia: Karelia, Ab740

PQ356681 PQ329022

Abramis brama Russia: Karelia, 126a

OP933968 OP908173

Abramis brama Russia: 126b

OP933988 OP908193

Abramis brama Russia 126c

OP933969 OP908174

Abramis brama Russia 126g

OP933970 OP908175

Abramis brama Russia: 126h

OP933971 OP908176

Abramis brama Russia:

brama_Lip20 OP408033 OP390380

Abramis brama Czech Republic:

brama_Lip1 OP408034 OP390381

Abramis brama Czech Republic:

brama_Lip2 OP408035 OP390382

Abramis brama Czech Republic:

brama_Lip14 OP408036 OP390383

Abramis brama Czech Republic:

brama_Rimov1 OP408037 OP390384

Abramis brama Czech Republic:

EE1Ab

JQ279121 JQ279085

Abramis brama Estonia

EE2Ab

EU241192 EU241275

Abramis brama Estonia:

EE3ab

EU241160 EU241276

Abramis brama Estonia:

EE4Ab

EU241195 EU241294

Abramis brama Estonia:

EE5Ab

JQ279122 JQ279086

Abramis brama Estonia:

FR30Ab EU241201 EU241259

Abramis brama France:

RU3Ab

EU241212 EU241252

Abramis brama Russia:

RU4Ab

EU241158 EU241253

Abramis brama Russia:

RU5Ab

EU241210 EU241309

Abramis brama Russia:

RU8Ab

EU241211 EU241310

Abramis brama Russia:

CZ14Ab EU241182 EU241263

Abramis brama Czech Republic: Nove

CZ16Ab EU241179 EU241283

Abramis brama Czech Republic: Nove

CZ17Ab EU241183 EU241284

Abramis brama Czech Republic: Nove

CZ18Ab EU241184 EU241285 Abramis brama Czech Republic: Nove

Table 1 CZ24Ab EU241180 EU241267 Abramis brama Czech Republic: Nove

OK

OP933980 OP908185

Rutilus rutilus Czech Republic

UA3 OP933986 OP908191

Rutilus rutilus Ukraine:

CZ90Rr EU241178 EU241282

Rutilus rutilus Czech Republic:

S4

OP933972 OP908177

Rutilus rutilus Iran: Alborz

CZ7Rr EU241159 EU241278

Rutilus rutilus Czech Republic:

CR22 OP933981 OP908186

Rutilus rutilus France:

FR67Aa JQ279124 JQ279088 Alburnus alburnus

CZ106Pc EU241167 EU241244 Podiceps cristatus eryth_Most1 OP408040 OP390387 Scardinius erythrophthalmus

France:

Czech Republic:

Czech Republic:

blicc_Lip OP408038 OP390385

Blicca bjoerkna Czech Republic: MCf-FB04919371 MW602520 Pusa hispida saimensis Finland: Lake

IE2Rr

EU241206 EU241250

Rutilus rutilus Ireland: Lough

GB2Pp

EU241175 EU241304 Phoxinus phoxinus

United Kingdom: Ru

OP933995 OP908201 Hemiculter lucidus

Russia: Lake

CN1 Hb

EU241153 EU241229 Hemiculter bleekeri

China: lac

AU1Gt

EU241146 EU241222 Galaxias truttaceus

Australia Au2

OP933951 OP908156 Galaxias maculatus

Australia: Goodga Au3

OP933952 OP908157 Galaxias maculatus

Australia:

TN63Ps

JQ279139 JQ279102 Pseudophoxinus callensis

Tunisia: Remel,

IE3 Gg

EU241188 EU241305

Gobio gobio Ireland: Lough

ALG1Bc

JQ279109 JQ279074

Barbus sp. Algeria

IE4Gg

EU241208 EU241290

Gobio Gobio Ireland: Lough

H11 OP933994 OP908199

Neogobious Hungury:

ALG2Bc

EU241143 EU241219

Barbus sp. Algeria: Hamiz

TN62Ps

JQ279138 JQ279101 Pseudophoxinus callensis

Tunisia: Joumine

CN4Nt EU241157 EU241237 Neosalanx taihuensis China: Zhanghe Ecologica Montenegrina , 80, 2024, 21-37

Table 1 CN8 OP933997 OP908203 Neosalanx taihuensis China

CA19Sa EU241150 EU241224 Semotilus atromaculatus

CA5Sa EU241149 EU241226 Semotilus atromaculatus

Oregon_C4 OP934005 OP908211 Rhinichthys osculus

CA1Cp EU241152 EU241228 Couesius plumbeus

Canada

Canada

Canada: Mckenzie

Canada

ET2 OP934000 OP908206

Barbus sp. Ethiopia

ET5 OP934001 OP908207

Barbus sp. Ethiopia

K1CO OP934009 OP908215

Barbus sp Kenya 1c

OP934010 OP908216 Rastrineobola argentea

Kenya

C2

OP934011 OP908217 Rastrineobola argentea

Kenya

C3

OP934012 OP908218 Rastrineobola argentea

Kenya 4c

OP934013 OP908219 Rastrineobola argentea

Kenya 5c

OP934014 OP908220 Rastrineobola argentea

Kenya 6c

OP934015 OP908221 Rastrineobola argentea

Kenya 7c

OP934016 OP908222 Rastrineobola argentea

Kenya 8c

OP934017 OP908223 Rastrineobola argentea

Kenya 9c

OP934018 OP908224 Rastrineobola argentea

Kenya

10c OP934019 OP908225 Rastrineobola argentea

Kenya

K3CO OP934020 OP908226 Rastrineobola argentea

Kenya

K4CO OP934021 OP908227 Rastrineobola argentea

Kenya Tanz2 OP934022 OP908228 Engraulicypris sardella Tanzania Tanz3 OP934023 OP908229 Engraulicypris sardella Tanzania Tanz8a OP934024 OP908230 Engraulicypris sardella Tanzania

Bn151 OP934025 OP908231

Barbus anoplus South

SA_Mpum OP934026 OP908232 Barbus anoplus South DRC OP934027 OP908233 Barbus sp Democratic Republic

Table 1 Namibia 2 OP934028 OP908234 Barbus paludinosus Namibia

SA_Limpop OP934029 OP908235

Barbus anoplus South Namibia 1 OP934030 OP908236 Barbus paludinosus Namibia

SA_Buff OP934031 OP908237

Barbus anoplus South

Bn159 OP934032 OP908238

Barbus anoplus South

NZ 2 OP934033 OP908239 Gobiomorphus breviceps

New

NZS OP934034 OP908240 Gobiomorphus breviceps New

Ecologica Montenegrina , 80, 2024, 21-37

The phylogenetic analysis (BI) and maximum Likelihood (ML) analysis of concatenated partial genes showed that there were six geographically distinct lineages within the monophyletic L. intestinalis complex ( Fig. 3). This result is consistent with the previous studies (Bouzid et al. 2008; Štefka et al. 2009; Nazarizadeh et al., 2022, 2023). On the phylogenetic tree, all samples from Karelia were placed in the clade “ L. intestinalis Lineage A ”, which included parasites of various cyprinid fish from around the world. The clade position was supported by high posterior probability (1.0) and bootstrap support (100) ( Fig. 3).

Haplotype analysis

Haplotype analysis was performed for the sequences of L. intestinalis from freshwater bream obtained in this study and those available in GenBank. The indices of genetic diversity in L. intestinalis datasets of concatenated sequences of cox1 and cytb and separate datasets of the same genes are presented in Table 2.

Haplotype analysis of the 53 concatenated sequences revealed 40 haplotypes. Most of the individual plerocercoids had their own unique haplotype ( Fig. 4). Only six haplotypes were shared by two or more tapeworms. The most numerous haplotype was noted in four individuals of the parasite in Lake Syamozero as well as in a cestode from Lake Řimov in Czech Republic. One haplotype was identified in three tapeworms from different geographical localities ( Estonia, Czech Republic, Rybinsk). Another haplotype was detected in three cestodes from Rybinsk. Two other haplotypes were found in two plerocercoids from Lake Syamozero each (Ab643/Ab649 and Ab665/Ab666). One haplotype was noted in two tapeworms: one from Lake Syamozero and the other from Lake Konchezero (Ab657 and Ab660).

Out of the 40 haplotypes revealed in our study, 22 haplotypes were found in specimens from Karelian lakes, and 21 of them were unique. Each plerocercoid sampled from bream in Lake Ladoga and Lake Onego had its own unique haplotype ( Fig. 4). Fifteen haplotypes, each corresponding to an individual tapeworm, were found in cestodes from Lake Syamozero ( Fig. 4). Within Lake Syamozero, three haplotypes represented by the greatest number of tapeworms were sampled in the Kurmoila Bay ( Fig. 1 View Figure 1 ), which might be associated with the larger amount of material sampled in this locality.

P-distances of concatenated cytb+cox1 of samples from Karelian lakes varied from 0.1 to 2.3 %. P-distances of the same markers between the Karelian samples and the tapeworms from Rybinsk, Estonia, Czech Republic and France were 0.45%, 0.75%, 0.7% and 0.5%, respectively.

Examination of partial sequences separately for cox1 and Cyt b revealed different patterns ( Table 2). All Cyt b sequences were divided into 26 haplotypes. Karelian tapeworms were represented by 13 haplotypes, including 10 unique ones. At the same time, one of the most common haplotypes was noted only in Karelian specimens from lakes Syamozero, Konchezero, Onego, Ladoga (Ab650, Ab656, Ab657, Ab660, Ab661, Ab663, Ab667, Ab740). One haplotype was shared by five cestodes from Syamozero (Ab641 Ab646 Ab736 Ab738 Ab739) and worms from Czech lakes Rimov and Lipno. One haplotype of tapeworms from Syamozero and Onego (Ab640, Ab643, Ab649) was shared with those from Rybinsk reservoir (RU3Ab) and Lake Pepsi (EE2Ab) in Estonia. Another haplotype was common for tapeworms from Lake Syamozero and Lake Pepsi (EE4Ab) in Estonia.

The least variable site was cox1: 17 haplotypes in total and 12 haplotypes in Karelia ( Table 2). Five haplotypes were unique for L. intestinalis from Karelian bream, while all the others were shared with tapeworms from other geographical locations. The best-represented haplotype was found in 11 tapeworms from Karelia (Syamozero, Konchezero) and 11 cestodes from Rybinsk, Estonia, Czech Republic and France. Two less common haplotypes, each in a different group of worms, were identified in two cestodes from Syamozero and four tapeworms from Rybinsk. Similarly, two haplotypes were found in two different groups including tapeworms from Syamozero and Estonia. Haplotypes of cestodes from Ladoga and Svyatozero coincided with those from Czech reservoirs.

Discussion

Our results indicate an expanding dispersal of the cestodes Ligula intestinalis parasitizing freshwater bream in Karelia. One of the reasons is the dispersal of the bream itself, which has been noted in ichthyological studies ( Sterligova et al. 2016). In Lakes Onego, Ladoga, and Svyatozero, where we recorded L. intestinalis in bream for the first time, these parasites had been previously recorded in other fish: roach Rutilus rutilus and crucian carp Carassius carassius L., 1758 ( Rumyantsev 2007) in Lakes Onego and Svyatozero and roach R. rutilus , vimba bream Vimba vimba L., 1758, blue bream Ballerus ballerus L., 1758 and bleak Alburnus alburnus L., 1758 ( Rumyantsev & Mamontova 2008) in Lake Ladoga. The infection rates in all these water bodies were low (prevalence less than 7%, mean abundance 0.1). In Lake Konchezero, freshwater bream was introduced in the 1960s ( Sterligova et al. 2016) and has acclimatised. Our results show that its parasitic fauna now includes L. intestinalis .

Lake Syamozero was the only Karelian lake where plerocercoids of L. intestinalis have been recorded in bream before the present study. The only other host in which L. intestinalis has been noted in Lake Syamozero is bleak, A. alburnus , and infection indices are low (prevalence 6%, mean abundance 0.06) (Novokhatskaya 2008).

No infection of freshwater bream with L. intestinalis had been noted in Syamozero in the 1950s ( Shulman 1962) ( Fig. 2 View Figure 2 ), the first record dating back to 1973 ( Malakhova & Ieshko 1977). Since that time, the abundance of this parasite has varied, the fluctuations being possibly associated with the state of the bream population. In 1970s-1990s, it was mainly represented by immature individuals (about 70%); the maturation rates were slow, and the fecundity was low. This long-term depression was probably due to fishing restrictions and eutrophication of the lake. The numbers of bream increased as a result of long-term ban on bream fishing, and there was not enough benthos, which is an energy-rich resource, for all the bream in the lake. Moreover, eutrophication, caused by the use of fertilizers, resulted in a depression of the benthic communities, while the abundance and biomass of plankton increased. Under these conditions, bream mostly fed on zooplankton, which is a low-energy resource. The role of copepods in bream diet became more significant, and the infection rates of bream with L. intestinalis plerocercoids increased correspondingly. The parasite probably depressed the growth rate of the host even further ( Ieshko & Malakhova 1982; Novokhatskaya et al. 2008; Sterligova et al. 2016). Current infection rates of bream with L. intestinalis are similar to those from 1975, which indicates that the share of plankton in the bream diet is fairly high.

We provided new gene sequences of L. intestinalis from A. brama and identified new haplotypes. Haplotype diversity was high both for the parasites from different countries and for Karelia (0.98–0.99), but the nucleotide diversity was low (0.000.006–0.007). Tajimaʼs D values were negative both in Europe and in Karelia, with statistically insignificant values, suggesting that L. intestinalis population in freshwater bream in the European part of Palearctic is genetically diverse and rapidly expanding. Our data support the hypothesis, based on historical demography modeling, that isolation with continuous gene flow is the most likely scenario of the divergence of L. intestinalis ( Nazarizadeh et al. 2024) .

All the samples of plerocercoids involved in our study were placed into Lineage A of L. intestinalis ( Nazarizadeh et al. 2023) . The authors have suggested that L. intestinalis from freshwater bream have certain haplotypes that are almost never found in other cyprinids ( Nazarizadeh et al. 2022). Having examined water bodies situated at a distance of 50-150 km from each other, they concluded that the differences in prevalence between fish host species in different lakes might be influenced not only by the parasite’s ecology but also by its genetic diversity ( Nazarizadeh et al. 2022). We arrived at the same conclusion in this study. Different haplotypes of L. intestinalis from bream could be found in the same location in lake (e.g. Kurmoila Bay of Lake Syamozero), while the same haplotypes could be found in locations separated by a distance of 5-20 km within the lake.

Nazarizadeh et al. (2022) note that the heterogeneity of the helminth population in the sample of bream is due to the fact that the material is collected in different seasons. We caught bream individuals in Kurmoila Bay for 12 days in June 2024 but our sample was also rather heterogeneous (22 haplotypes). Large numbers of bream spawn and migrate in the lake in the study period, and their populations mix, which affects the diversity of the parasites ( Sterligova et al. 2016).

In summer, the mixing and dispersal of the plerocercoids is facilitated by the feeding of young bream from different populations in shallow and well-warmed littoral areas and numerous bays of Lake Syamozero. Conditions are favourable there for copepods, which are the first intermediate hosts of L. intestinalis . Accumulations of young fish in such places attracts the final hosts, fish-eating birds. Large numbers of gulls (Black-headed Gull, Chroicocephalus ridibundus and Herring Gull, Larus argentatus ), which are probably the main hosts of L. intestinalis , have been observed at Lake Syamozero ( Sazonov 2004). In this way, the transmission of the parasite in the ecosystem is promoted. Similar results have been obtained in a study of Ligula circulation in some aquatic ecosystem in south-western Spain ( Capasso et al. 2024).

Variation in the occurrence of haplotypes of L. intestinalis in different locations may be due to the different species of fish-eating birds, their definitive hosts, as well as their migration pathways. Among the definitive hosts of Ligula intestinalis indicated by Dubinina (1980), different species of gulls, goosanders, grebes, cormorants, making both short- and long-distance migrations for wintering or nesting, are found in Northwestern Russia ( Noskov et al. 2016). Nazarizadeh et al. (2022) suggest that fish-eating birds such as Great Cormorant P. carbo , grebes Podiceps auritus (Linnaeus) , P. cristatus and P. nigricollis Brehm, Goosander Mergus merganser , and Common Pochard Aythya ferin a (Linnaeus) may be potential final hosts of L. intestinalis in the Czech Republic.

So far, L. intestinalis has been noted in Karelia only in Great Crested Grebe Podiceps cristatus and Great Cormorant Phalacrocorax carbo ( Dubinina 1980; Yakovleva et al. 2020) but this may be due to the limited scope of parasitological research. Though P. carbo has been shown to expand into the water area of Lake Ladoga, this bird has not yet been noted at Syamozero, Onego, Svyatozero and Konchezero ( Lapshin & Mikhaleva 2021).

Our molecular data on L. intestinalis indirectly support the connections between water bodies on the migratory routes of fish-eating birds, particularly, gulls, discovered by Noskov et al. (2016). The numbers of Herring Gull and Black-headed Gull in the Karelia has been increasing in recent decades ( Zimin et al. 1993; Noskov et al. 2016). Numerous colonies of these birds are observed along the shores of many Karelian water bodies, and local residents report that gulls often feed on discarded fish. These species of fish-eating birds may transmit L. intestinalis both between lakes within Karelia and between Karelia and Europe. Black-headed Gull and Herring Gull, wintering on the southern coast of the North Sea and the Baltic Sea ( Noskov et al. 2016), are likely to maintain a more homogeneous population of the parasite within the northern part of their range. This hypothesis is supported by the occurrence of the same haplotypes in bream in the Karelian water bodies examined in our study and in water bodies in other regions ( Fig. 4).

The study of the first intermediate hosts of L. intestinalis , i.e. crustaceans, would be interesting for identification of factors influencing the dispersal rate, the survival and the host specificity of this parasite. The life products of bird colonies strongly influence the zooplankton in the littoral zone of freshwater bodies ( Krylov et al. 2012). The birds do not only release infective agents into water but also change the structure and abundance of zooplankton. These changes are likely to affect the implementation of the life cycle of L. intestinalis and the survival of its lineages/subspecies.

Conclusion

In this study, we obtained data on the occurrence of the cestode L. intestinalis in freshwater bream inhabiting several lakes in Northwestern Russia and examined the genetic structure of its plerocercoids using two mitochondrial genes (Cyt b and COI). Our results highlight the need to study this parasite in other fish of the region in order to understand its specificity to the second intermediate host. It is also important to obtain the data on the bird species that serve as the main infection vectors of L. intestinalis . These data would contribute to epidemiology, control and treatment options of Ligula infection.

Acknowledgements

We are grateful to our colleagues, particularly to Drs. Olga Sterligova, Eugeny Ieshko, Sergey Bugmyrin, and Fedor Fariseev (IB KarRC RAS), for their help with material collection. We extend our sincere thanks to the two anonymous reviewers for their valuable comments on the first version of the manuscript.

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

Ethics Approval

The paper does not contain any studies involving animal experiments. The wild animal study protocol was approved by the Institute of Biology of Karelian Research Centre, the Russian Academy of Sciences (protocol no. 7 of 8 July 2023). Research Fishing was under Permit of North-West Territorial Administration of the Federal Agency for Fishery (7820240317689) of 14 May 2024.

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