Introduction
In marine ecosystems, trophic interactions drive the transmission and dispersal of parasites with complex life cycles, such as marine tapeworms (Cestoda) (Marcogliese, Reference Marcogliese2002). These cycles require larval stages to move through one or more hosts via predator-prey interactions until reaching sexual maturity in their definitive hosts (Parker et al. Reference Parker, Chubb, Ball and Roberts2003). For elasmobranch tapeworms, mapping these pathways remains challenging due to several constraints: the inability to morphologically identify larval forms of some lineages at the species level, poor taxonomic resolution in earlier literature, the presence of cryptic species, and the fact that less than half of elasmobranchs have been studied for their cestode parasites (Caira and Jensen Reference Caira and Jensen2014; Franzese et al. Reference Franzese, Facal and Menoret2023). Consequently, the identification and molecular characterisation of cestode larvae from their intermediate/paratenic hosts are essential steps towards providing missing data to resolve these life cycles.
The common squid Loligo vulgaris is widely distributed in the northeast Atlantic Ocean, ranging from the Celtic Sea, North Sea, and western Baltic Sea to the northwestern coast of Africa (Angola). Its distribution also extends throughout the Mediterranean, Adriatic, Ionian, and Aegean Seas (Jereb et al. Reference Jereb, Allcock, Piatkowski, Hastie and Pierce2015). The bathymetric distribution of the squid ranges from 38 to 170 m, with the highest occurrence at depths of 50–100 m. It also exhibits an ontogenetic vertical migration related to its reproduction and life stages (Quetglas et al. Reference Quetglas, de Mesa, Guijarro, Massutí, Ordinas, Zaragoza and Valls2026).
The lifespan of the common squid extends from 9 to 15 months (Bettencourt et al. Reference Bettencourt, Coelho, Andrade and Guerra1996; Moreno et al. Reference Moreno, Azevedo, Pereira and Pierce2007; Vila et al. Reference Vila, Silva, Torres and Sobrino2010). This species is sexually dimorphic, and males reach greater maximum size and weight (640 mm; 2302 g) than females (362 mm; 1320 g) (Moreno et al. Reference Moreno, Pereira, Arvanitidis, Robin, Koutsoubas, Perales-Raya, Cunha, Balguerías and Denis2002; 2007), although growth parameters may vary geographically (Jereb et al. Reference Jereb, Allcock, Piatkowski, Hastie and Pierce2015). Reproduction occurs continuously throughout the year, with males attaining sexual maturity around one month prior to females (Bettencourt et al. Reference Bettencourt, Coelho, Andrade and Guerra1996; Quetglas et al. Reference Quetglas, de Mesa, Guijarro, Massutí, Ordinas, Zaragoza and Valls2026; Vila et al. Reference Vila, Silva, Torres and Sobrino2010). Furthermore, it is semelparous, dying after its only reproductive cycle (Rocha and Guerra Reference Rocha and Guerra1996).
At early life stages, L. vulgaris exhibits a selective diet composed of small pelagic crustaceans. As juveniles grow, they incorporate fish larvae into their diet, whereas adults become more opportunistic, with fish representing the dominant prey, along with lower proportions of cephalopods, crustaceans, and polychaetes (Jereb et al. Reference Jereb, Allcock, Piatkowski, Hastie and Pierce2015; Turk et al. Reference Turk, Hanlon, Bradford and Yang1986; Vila et al. Reference Vila, Silva, Torres and Sobrino2010). On the other hand, this species is an important component of the food web, serving as prey for a wide range of elasmobranch and osteichthyan fish as well as marine mammals (Jereb et al. Reference Jereb, Allcock, Piatkowski, Hastie and Pierce2015).
In marine ecosystems, squids constitute important intermediate or paratenic hosts for several heteroxenous helminths, particularly metacestodes (Hochberg Reference Hochberg1983; Jensen and Bullard Reference Jensen and Bullard2010), which infect multiple tissues including the digestive tract, mantle cavity, mantle muscle, and gills (Naidenova et al. Reference Naidenova, Nigmatullin and Gaevskaya1981; Reference Naidenova, Nigmatullin, Gaevskaya and Hargis1985; Nitta et al. Reference Nitta, Matsumoto and Urabe2023; Occhibove et al. Reference Occhibove, Lopez-Verdejo, Mazzella, Cusano, Palomba, Aco-Alburqueque and Santoro2025). Through trophic interactions, these infected cephalopods serve as a major pathway for parasite transmission to elasmobranchs (Hochberg Reference Hochberg1983). Predation on infected squids facilitates the transmission of cestode larvae to elasmobranchs, in which they develop into adult stages, thereby confirming the role of elasmobranchs. These helminths generally have complex life cycles involving trophic transmission and can therefore be used to better understand the host’s role within the food web and its interactions in the ecosystem (Tedesco et al. Reference Tedesco, Quadrone, Albonetti, Marcer, Caffara, Fioravanti and Gustinelli2025).
Several metacestode species have been reported in L. vulgaris, mainly from the Mediterranean Sea, including Nybelinia lingualis (Cuvier, 1817) Dollfus, Reference Dollfus1929 (Trypanorhyncha); Heteronybelinia estigmena (Dollfus, 1960) Palm, 1999 (Trypanorhyncha); Phyllobothrium sp. Van Beneden, 1850 (Phyllobothriidea); Scyphophyllidium pruvoti (Guiart, 1933) Joyeux and Baer, 1936 (Phyllobothriidea); and Spirometra gracilis (Baer, 1927) (Diphyllobothriidea) as well as species from the ‘Scolex’ group, such as ‘Scolex de Bavay’ Bavay, 1873 and ‘Scolex polymorphus Rudolphi, 1819’ (Dollfus Reference Dollfus1929, Reference Dollfus1931, Reference Dollfus1942, Reference Dollfus1958; González et al. Reference González, Pascual, Gestal, Abollo and Guerra2003; Joyeux and Dollfus Reference Joyeux and Dollfus1931; Pascual et al. Reference Pascual, Gestal, Estévez, Rodríguez, Soto, Abollo and Arias1996).
To our knowledge, no studies have specifically investigated the molecular characterisation of cestode larvae in the common squid L. vulgaris. However, fragmentary records of ‘Scolex larvae’, generally limited to schematic illustrations, have been reported over the past century (Dollfus Reference Dollfus1923, Reference Dollfus1929). The present study aimed to investigate the cestode larvae infecting L. vulgaris from Tunisian waters using an integrative approach combining morphological examination and molecular analysis of the partial 28S ribosomal DNA (rDNA) region.
Materials and methods
Sampling and morphological analysis
From February to June 2025, 58 specimens of Loligo vulgaris were examined from two study areas off Tunisia (Table 1): the Bay of Bizerte (37° 17’ N, 9° 58’ E) in the north and the Gulf of Tunis (36° 48’ N, 10° 10’ E) in the northeast. Freshly collected, the samples were immediately transported in a cooler filled with ice to the Laboratory of Biodiversity, Parasitology and Ecology of Aquatic Ecosystems at the Faculty of Sciences of Tunis for parasitological examination. After the dissection, the viscera were examined under a stereomicroscope (Leica EZ4). The still-living cestode larvae were identified under a binocular microscope (Zeiss Primo Star 1). Larvae were collected using a plastic pipette and then transferred to Petri dishes filled with filtered seawater. Prior to fixation, morphometric measurements were performed on five live larvae relaxed under minimum light intensity. Two larvae were preserved in 96% ethanol for molecular analyses, while the remaining larvae were preserved in 70% ethanol for morphological study. For the flattening process, cestode larvae were placed for 24 hours between a slide and a coverslip in a Petri dish containing 70% ethanol. Staining was performed with Mayer–Schuberg’s acetic carmine solution. The stained larvae were mounted in Canada balsam on glass slides. All specimens were photographed using a Zeiss Primo Star 1 microscope equipped with a digital camera.
Loligo vulgaris biometrics and parasitological indices of Anthobothrium sp. larvae collected from the study sites
Table 1. Long description
The table contains 10 columns: Collection site, N E H, T L, T W, N I H, N P, Site of infection, P (percent), m I, and m A.
Row 1: Gulf of Tunis. N E H: 34. T L: 27.85 plus or minus 4.77. T W: 173.46 plus or minus 109.22. N I H: 18. N P: 67. Site of infection: Caecum, stomach. P (percent): 52.94. m I: 3.72 plus or minus 4.21. m A: 1.97 plus or minus 3.56.
Row 2: Bay of Bizerte. N E H: 24. T L: 24.83 plus or minus 3.96. T W: 148.76 plus or minus 70.14. N I H: 6. N P: 20. Site of infection: Caecum, stomach. P (percent): 25.00. m I: 3.33 plus or minus 3.83. m A: 0.83 plus or minus 2.32.
Initialisms are defined as: N E H is Number of examined hosts; T L is total length (mantle length) in cm; T W is total weight in g; N I H is number of infected hosts; N P is number of collected parasites; P is prevalence; m I is mean intensity; m A is mean abundance.
NEH: Number of examined hosts; TL: total length (mantle length) (cm) mean ± standard deviation; TW: total weight (g) mean ± standard deviation; NIH: number of infected hosts; NP: number of collected parasites; P (%): prevalence; mI: mean intensity; mA: mean abundance.
The parasitological indices, prevalence (P%), mean intensity (mI), and mean abundance (mA) were calculated according to Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
DNA extraction, PCR amplification, and sequencing
Genomic DNA of two larvae was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s protocol. Digestion of biological material was performed in a GFL 1003 incubation bath at 56°C for 2 hours. The amplification of the 28S rDNA (region D1–D3) was performed with the universal primer pair ZX-1 (forward: 5′-ACCCGCTGAATTTAAGCATAT-3′; modified from Van der Auwera et al. Reference Van der Auwera, Chapelle and De Wächter1994) and 1500R (reverse: 5′-GCTATCCTGAGGGAAACTTCG-3′; Littlewood and Olson, Reference Littlewood, Olson, Littlewood and Bray2001). Polymerase chain reaction (PCR) was performed in a reaction volume of 25 μL containing 0.5 μL of each primer, 12.5 μL of Taq DNA polymerase (HotStarTaq-QIAGEN), 2 μL of extracted genomic DNA, and 9.5 μL of ultrapure water. PCR amplifications were run in a thermocycler (Bio-Rad T100) as follows: denaturation step at 94°C for 2 min, followed by 34 cycles of 50 s at 94°C, 50 s at 55°C, and 2 min at 72°C, and an extended elongation step at 72°C for 7 min. PCR products were analysed by electrophoresis in a 1% agarose gel with 1 × Tris-borate-EDTA buffer stained with a fluorescent dye (EZView Stain BIOMATIK). PCR products were enzymatically purified using the ExoSAP-IT PCR Product Cleanup Reagent. Bidirectional Sanger sequencing was then performed by Microsynth (France) on an ABI 3500 Genetic Analyzer (Applied Biosystems) using the amplification primers.
Phylogenetic analyses
The amplified forward and reverse fragments were visually trimmed and assembled using BioEdit Sequence Alignment Editor version 7 (Hall Reference Hall1999). Consensus sequences were compared against the GenBank database using BLASTn to verify target gene orthology amplification and assess sequence similarity with available reference data. For phylogenetic analysis, a total of 38 sequences were retrieved from GenBank (Table 2). The cestode sequence of Nybelinia sp. (PV156517.1) was used as an outgroup, as the Trypanorhyncha order represents a basal lineage diverging outside the ‘Tetraphyllidea’ clade (Caira et al. Reference Caira, Jensen, Waeschenbach, Olson and Littlewood2014).
GenBank accession numbers of Anthobothrium spp. 28S rDNA sequences used in this study
Table 2. Long description
A table with five columns: Parasite species, Host species, Accession number, Sequence length in b p, and Reference.
Key entries include:
* Anthobothrium sp. larva from Ariopsis felis, accession G Q 4 7 0 1 6 4, length 1252.
* Anthobothrium sp. adult from Carcharhinus cautus, accession P Q 1 4 6 1 9 6, length 1268.
* Anthobothrium sp. larva from Loligo vulgaris, accessions P Z 2 9 5 7 2 3 and P Z 2 9 5 7 2 4, lengths 867 and 1021 respectively, noted as from the present study.
* Anthobothrium caseyi adult from Prionace glauca, accessions K F 6 8 5 8 7 9, O Q 7 8 8 3 3 4, and P V 1 5 6 5 0 8.
* Anthobothrium sp. larva from Trichiurus lepturus, with five separate accessions ranging from G Q 4 7 0 1 5 8 to G Q 4 7 0 1 7 0.
* Nybelinia sp. outgroup from Raja asterias, accession P V 1 5 6 5 1 7, length 1778.
The table includes sequences from various shark hosts like Carcharhinus limbatus and Carcharhinus obscurus, as well as cephalopods like Octopus vulgaris and Sepioteuthis lessoniana.
Phylogenetic analysis was performed using MEGA 12 software (Kumar et al. Reference Kumar, Stecher, Suleski, Sanderford, Sharma and Tamura2024), and the sequences were aligned using the MUSCLE alignment tool. To determine the most appropriate evolutionary model for the dataset, the ‘Find Best DNA Model’ tool in MEGA 12 was used, and the model selection was based on the lowest Bayesian Information Criterion (BIC) score. Consequently, a maximum-likelihood (ML) tree was inferred with nodal support assessed by 1000 bootstrap replicates. The phylogram was constructed based on 41 sequences under the general time-reversible model with a discrete Gamma distribution (GTR+G).
Pairwise distance matrices were calculated for the 41 sequences across 1094 sites, including all pairwise comparisons, intergroup mean distances, and within-group mean distances, using the p-distance method and accounting for both transitions and transversions.
The sequences generated in this study were deposited in GenBank under the accession numbers PZ295723 and PZ295724.
Results
Morphological description
Order: Tetraphyllidea
Family: Tetraphyllidea incertae sedis
Genus: Anthobothrium Van Beneden, 1850
The larva of Anthobothrium sp. has an elongated, brownish body that gradually tapers towards the posterior end. The measurements taken on five still-living larvae indicated that the total length of the larvae ranges from approximately 689 to 1604 μm (1048.0 ± 390.7), while the width ranges from approximately 380 to 484 μm (425.0 ± 30.5). The scolex bears four subspherical acetabula, ensuring attachment to the host tissues. Their length ranges from 99 to 173 μm (133.2 ± 37.5), while their width ranges from 31 to 59 μm (93.6 ± 31.8). The presence of an apical sucker was also observed at the apex of the scolex, measuring from 93 to 115 μm (101.4 ± 10.1) in length and from 74 to 96 μm (85.3 ± 8.9) in width (Figure 1). A checklist and comparative overview of collected Anthobothrium sp. larvae reported in previous studies, including their morphological assignment categorised according to Jensen and Bullard (Reference Jensen and Bullard2010), are shown in Table 3.
Morphology of Anthobothrium sp. larvae. A: Two larvae attached to the caecum of Loligo vulgaris; B: Live larva (in vivo); C: Stained larva. White arrows: larvae; black arrows: acetabula; red arrows: apical sucker.
Figure 1. Long description
Panel A is a photo of two small white larvae attached to the translucent, folded tissue of a Loligo vulgaris caecum. White arrows point to the larvae. A scale bar in the bottom right indicates 1 mm.
Panel B is a micrograph of a live larva in vivo. The body is elongated and granular. At the anterior end, a red arrow points to a central apical sucker. Four black arrows point to the surrounding acetabula, which appear as translucent, rounded lobes. A scale bar at the bottom indicates 200 micrometers.
Panel C is a micrograph of a stained larva, appearing pink. The red arrow points to the apical sucker at the very top. Three black arrows point to the acetabula, which are more clearly defined as circular muscular structures in this stained preparation. A scale bar at the bottom indicates 100 micrometers.
A checklist and comparative overview of collected Anthobothrium sp. larvae reported in previous studies
Table 3. Long description
A data table with five columns and eight primary data rows.
Column headers: Fish and cephalopod species; Larval type; Site of infection; Geographical locality; References.
Row 1: Loligo vulgaris; G 1; Caecum, stomach; Mediterranean Sea (Gulf of Tunis and Bay of Bizerte, Tunisia); Present study.
Row 2: Sardinella aurita; G 2; Pyloric caeca; Mediterranean Sea (Gulf of Tunis); Saad et al. 2026.
Row 3: A list of 32 species including Siganus fuscescens and Chrysophrys auratus; G 1, G 2; Intestine; Pacific Ocean (Moreton Bay, Australia); Cutmore et al. 2024.
Row 4: Sepioteuthis lessoniana; G 1, G 2; Gills; Pacific Ocean (Wakayama, Japan); Nitta et al. 2023.
Row 5: Octopus vulgaris; G 1; Caecum, intestine; Mediterranean Sea (Sicily, Italy); Tedesco et al. 2020.
Row 6: Ariopsis felis; G 1; Digestive tract; Gulf of Mexico (Mississippi, U S A); Jensen and Bullard 2010.
Row 7: Trichiurus lepturus; G 1, G 2; Digestive tract; Gulf of Mexico (Mississippi, U S A); Jensen and Bullard 2010.
Row 8: Loligo vulgaris; G 1; Stomach, digestive tract; Northeast Atlantic (Gironde and Finistère, France); Dollfus 1929 and Dollfus 1923.
Footnotes indicate larval types G 1 and G 2 follow Jensen and Bullard 2010 classification, and some larvae were originally reported as Scolex de Bavay.
a The larval type category (G1, G2) is given according to the classification of Jensen and Bullard (Reference Jensen and Bullard2010).
b Larvae are reported as ‘Scolex de Bavay’ Bavay, 1873.
Molecular results
The sequencing of the 28S rDNA region of Anthobothrium sp. cestode larvae yielded a sequence of 867 bp (PZ295723) from a specimen collected in the Bay of Bizerte, and a sequence of 1021 bp (PZ295724) from a specimen collected in the Gulf of Tunis. BLAST analysis of both sequences revealed the highest identity values of 99.42% and 99.41%, respectively (with 100% coverage), with Anthobothrium sp. larvae collected in Sepioteuthis lessoniana Lesson, 1831 from Japan (LC771487). The alignment of the two sequences revealed that sequence PZ295723 contained one transition and one transversion.
The maximum-likelihood phylogenetic analysis (Figure 2), inferred by aligning our sequences with 38 Anthobothrium spp. sequences retrieved from GenBank, showed a tree with two main clades. The first, a major clade (GQ470167-PQ146221) with moderate bootstrap support (bootstrap value = 56%), included eight adults and twenty-one larval sequences, within which both sequences obtained in the present study (PZ295723 and PZ295724) were clustered. Within this clade, the sequences were clustered into four well-supported sub-clades (Sub-clades 1.1–1.4). Our sequences, included in Sub-clade 1.2, were most closely related to PQ146197 and PQ146218 from Australia (Cutmore et al. Reference Cutmore, Bennett and Cribb2024), generated from an adult specimen infecting Carcharhinus obscurus (Lesueur, 1818) (Carcharhinidae Jordan and Evermann, 1896) and a metacestode infecting Pseudorhombus arsius (Hamilton, 1822) (Paralichthyidae Regan, 1910). The second clade (OQ788334-GQ470158) was well supported (bootstrap value = 99%) and comprised sequences from three metacestodes and eight adult cestodes. These sequences were further grouped into four well-supported sub-clades (Sub-clades 2.1–2.4), including one cluster composed of three sequences of Anthobothrium caseyi Ruhnke and Caira, 2009, which represented the only taxon identified to species level.
Maximum-likelihood phylogenetic tree of Anthobothrium spp. based on 28S rDNA. Bootstrap values are indicated in black at nodes, and branch lengths > 0.01 are shown in grey. Black and coloured labels represent sequences obtained from larval and adult specimens, respectively. The newly generated sequences are indicated in bold. The scale bar indicates the expected number of substitutions per site. Lines indicate groups with a p-distance < 0.0102 among sequences.
Figure 2. Long description
The phylogenetic tree begins at a root on the left with a scale bar of 0.050 substitutions per site.
Clade 1 is the largest group at the top. It is divided into four sub-clades.
* Sub-clade 1.1 contains sequences from the Gulf of Mexico, Italy, Tunisia, and Australia, including hosts like Trichiurus lepturus and Carcharhinus species.
* Sub-clade 1.2 includes sequences from Australia, Japan, and New Zealand, with bolded sequences P Z 2 9 5 7 2 3 from Loligo vulgaris in Tunisia.
* Sub-clade 1.3 contains a single sequence P Q 4 7 0 0 4 7 from Illex coindetii in Italy.
* Sub-clade 1.4 contains two sequences from Australia.
Clade 2 is located at the bottom and is divided into four sub-clades.
* Sub-clade 2.1 includes Anthobothrium casey from the U S A, Italy, and Russia.
* Sub-clade 2.2 and 2.3 contain sequences from Australia and the Gulf of Mexico.
* Sub-clade 2.4 contains sequences from the Gulf of Mexico and Australia.
At the very bottom, Nybelina species P V 1 5 6 5 1 7 serves as an outgroup. Bootstrap values are written in black at the nodes, such as 99 and 100, while branch lengths are indicated in grey, such as 0.170 and 0.309. Blue text indicates adult specimens while black text indicates larval specimens. Bold text highlights newly generated sequences.
Pairwise mean distance matrices, calculated within and between the eight clusters (Table 4) as well as across the 41 sequences (Supplementary Table S1), supported the topology of the phylogenetic tree. Mean distances within groups ranged between 0 and 0.0034 (0.34%) in Sub-clade 1.2. The limit of the within-group genetic divergence was 0.0070 (0.70%) within Sub-clade 1.2, observed between our sequence (PZ295723) and each of LC771487, PQ146199, and PQ146220.
Estimates of evolutionary divergence over sequence pairs between and within groups.
Table 4. Long description
The table is structured with columns for Intergroup mean distance (divided into Clade 1 and Clade 2, each with four sub-clades) and a final column for Within-group mean distance. Rows are organized by Clade 1 (Sub-clades 1.1 to 1.4) and Clade 2 (Sub-clades 2.1 to 2.4).
Within-group mean distances:
* Sub-clade 1.1: 0.0019
* Sub-clade 1.2: 0.0034
* Sub-clade 1.3: n/c (not computed)
* Sub-clade 1.4: 0.0000
* Sub-clade 2.1: 0.0008
* Sub-clade 2.2: n/c (not computed)
* Sub-clade 2.3: 0.0000
* Sub-clade 2.4: 0.0011
Intergroup mean distances (p-distance):
* Sub-clade 1.2 vs 1.1: 0.0180
* Sub-clade 1.3 vs 1.1: 0.0182; vs 1.2: 0.0102
* Sub-clade 1.4 vs 1.1: 0.0278; vs 1.2: 0.0261; vs 1.3: 0.0256
* Sub-clade 2.1 vs 1.1: 0.0885; vs 1.2: 0.1081; vs 1.3: 0.0896; vs 1.4: 0.0982
* Sub-clade 2.2 vs 1.1: 0.0966; vs 1.2: 0.1206; vs 1.3: 0.0983; vs 1.4: 0.1084; vs 2.1: 0.0621
* Sub-clade 2.3 vs 1.1: 0.0945; vs 1.2: 0.1125; vs 1.3: 0.0928; vs 1.4: 0.1010; vs 2.1: 0.0538; vs 2.2: 0.0376
* Sub-clade 2.4 vs 1.1: 0.1102; vs 1.2: 0.1336; vs 1.3: 0.1134; vs 1.4: 0.1160; vs 2.1: 0.0933; vs 2.2: 0.1050; vs 2.3: 0.1069
n/c: not computed
Note: The overall mean p-distance between Clade 1 and Clade 2 is 0.1104
Mean distances between clusters ranged from 0.0102 (1.02%), observed between Sub-clade 1.2 and Sub-clade 1.3, to 0.1336 (13.36%), observed between Sub-clade 1.2 and Sub-clade 2.4.
Parasite infection analyses
In the present study, the analysis of parasitological indices showed notable differences between the two study areas, with higher values in the Gulf of Tunis. The cestode larvae Anthobothrium sp. exhibited a prevalence ranging from 25.00% to 52.94%, a mean intensity between 3.33 and 3.72, and a mean abundance from 0.83 to 1.97 (Table 1). Overall, infection in L. vulgaris was recorded in 24 hosts, with a prevalence of 41.38%, a mean intensity of 3.63, and a mean abundance of 1.50.
Discussion
Cestode larvae assigned to four orders (‘Tetraphyllidea’, Trypanorhyncha, Phyllobothriidea, Diphyllobothriidea) have been recorded in the common squid L. vulgaris from the Atlantic Ocean (Greenland and Sweden) and the Mediterranean Sea (France, Italy, and Spain) (Table 5). However, their identification and assignment to a specific genus have long been problematic, and they have frequently been referred to in the literature as members of the ‘Scolex’ group, including ‘Scolex de Bavay’ and ‘Scolex polymorphus’. As previously suggested by Tedesco et al. (Reference Tedesco, Caffara, Gustinelli, Fiorito and Fioravanti2020), our observations also indicate that the larvae examined in the present study are consistent with the ‘Scolex de Bavay’ larvae described from the stomach and digestive tract of L. vulgaris in earlier studies (Dollfus Reference Dollfus1923, Reference Dollfus1929). Given their similar geographical locality, these specimens could be conspecific.
Checklist of metacestode parasites infecting the common squid Loligo vulgaris
Table 5. Long description
The table contains four columns: Parasite species, Site of infection, Locality, and References. It is organized into five taxonomic sections.
* Tetraphyllidea: Includes Anthobothrium species found in the stomach and caecum in Tunisia (Bay of Bizerte, Gulf of Tunis).
* Trypanorhyncha: Includes Nybelinia lingualis found in the stomach (France) and intestine (Sweden, Greenland); Nybelinia species in Italy; and Heteronybelinia estigmena in the stomach (France).
* Phyllobothriidea: Includes Phyllobothrium loliginis; Phyllobothrium species found in the digestive tract, stomach, intestine, and mantle muscle across Spain and France; and Scyphophyllidium pruvoti in France.
* Diphyllobothriidea: Includes Spirometra gracilis found in the buccal cavity and intestine in Italy.
* Unaccepted larval names: Includes Scolex de Bavay found in the stomach, digestive tract, and Needham sac in France; and Scolex polymorphus in the digestive tract in France.
Footnote indicates that certain taxa are identified as nomina dubia or collective group names for larval forms of historical nomenclature according to W o R M S 2026.
‘Taxa identified as nomina dubia or collective group names for larval forms of historical nomenclature’
In recent decades, larvae of ‘Tetraphyllidea’ have remained poorly characterised, primarily because, unlike adults, they lack clear distinguishing features; consequently, using morphology alone generally allows their identification only to genus, family, or order level (Jensen and Bullard Reference Jensen and Bullard2010). The Anthobothrium larvae examined in the present study perfectly reflect this limitation of morphological identification, and thus, resorting to molecular analyses has become necessary to identify this larval lineage.
To our knowledge, eight molecular studies have been conducted on species of the genus Anthobothrium, with some focusing on molecular characterisation, while others combine it with the resolution of cestode life cycles (Bennett et al. Reference Bennett, Presswell and Poulin2023; Cutmore et al. Reference Cutmore, Bennett and Cribb2024; Jensen and Bullard Reference Jensen and Bullard2010; Nitta et al. Reference Nitta, Matsumoto and Urabe2023; Occhibove et al. Reference Occhibove, Lopez-Verdejo, Mazzella, Cusano, Palomba, Aco-Alburqueque and Santoro2025; Saad et al. Reference Saad, Justine, Jouini, Gey, Theisen, Palm and Gargouri2026; Tedesco et al. Reference Tedesco, Caffara, Gustinelli, Fiorito and Fioravanti2020, Reference Tedesco, Quadrone, Albonetti, Marcer, Caffara, Fioravanti and Gustinelli2025). However, molecular characterisation of Anthobothrium larvae at the species level remains limited, as sequences for only two species are available (28S rDNA for A. caseyi, and 18S rDNA and elongation factor-1 alpha for A. laciniatum), despite the existence of 22 valid species. In this context, our study adds new sequence data of Anthobothrium sp. larvae from a new intermediate/paratenic host record, providing an important point of comparison for further tracking of this larval lineage to its adult form.
The ML analysis, including both adult and larval Anthobothrium sequences, showed eight distinct clusters. The genetic distance between these clusters (Inter-cluster p-distance) ranged from 0.0102 (1.02%) to 0.1336 (13.36%), whereas the intra-cluster genetic divergence ranged from 0 to 0.0034 (0.34%), with the highest value observed in Sub-clade 1.2. The closest sequences to Anthobothrium sp. from the Bay of Bizerte (PZ295723) were sequences of Anthobothrium sp. from the Gulf of Tunis (PZ295724), Anthobothrium sp. (adult) infecting C. obscurus (PQ146197), and Anthobothrium sp. (larva) infecting P. arsius (PQ146218) from Australia (Cutmore et al. Reference Cutmore, Bennett and Cribb2024), all showing low genetic divergence (0.0028; 0.28%). These results are consistent with the ML topology and suggest that the observed intra-cluster variation may be related to the geographic distribution.
To the best of our knowledge, no interspecific divergence thresholds have been formally established for tetraphyllidean genera. However, comparison with other Eucestoda based on 28S rDNA indicates that the genetic variation observed in the present study is highly consistent with that reported in some taxa. For instance, in the genus Acanthobothrium Blanchard, 1848 (order Onchoproteocephalidea), little to no intraspecific variation has been reported (Fyler et al. Reference Fyler, Caira and Jensen2009), whereas in the genus Grillotia Guiart, 1927 (order Trypanorhyncha), intraspecific divergence ranges from 0.05% (0.0005) to 0.3% (0.003) (Lopez-Verdejo et al. Reference Lopez-Verdejo, Palomba, Crocetta and Santoro2024). Furthermore, interspecific divergence ranges from 0.7% to 11.3% in Acanthobothrium (Fyler et al. Reference Fyler, Caira and Jensen2009), and from 1.1% (0.011) to 6.9% (0.069) in Grillotia (Lopez-Verdejo et al. Reference Lopez-Verdejo, Palomba, Crocetta and Santoro2024). An exception to this pattern is found in the genera Rockacestus and Yamaguticestus (order Phyllobothriidea), where the interspecific divergence between morphologically distinct species is 0.00% (Caira et al. Reference Caira, Bueno and Jensen2021). Assuming that the ML analysis reflects the presence of eight distinct clusters, and given that the intra- and inter-cluster genetic variations are highly consistent with those in other orders of Eucestoda, it is plausible that some of these clusters represent separate species. However, this hypothesis is speculative and cautiously proposed until comprehensive molecular characterisation is carried out on larval and adult forms of Anthobothrium spp. from their various intermediate/paratenic and definitive hosts.
While adult Anthobothrium species were considered oioxenous, infecting carcharhiniform sharks and the Myliobatiform ray Gymnura altavela (Linnaeus, 1758) (Caira et al. Reference Caira, Jensen, Ruhnke, Caira and Jensen2017), recent data show that for some species (i.e., A. afsanae, A. cornucopia, A. samae, A. shayani, and A. spinosum) specificity can extend to a stenoxenous level (Sadeghi Vilni and Haseli Reference Sadeghi Vilni and Haseli2024). Conversely, a single carcharhiniform species can harbour a high diversity of Anthobothrium species, such as Rhizoprionodon acutus (Rüppell, 1837), which is recorded as a host for A. laciniatum, A. samae, A. shayani, and A. spinosum (Sadeghi Vilni and Haseli Reference Sadeghi Vilni and Haseli2024; Subhapradha, Reference Subhapradha1957; Yamaguti Reference Yamaguti1934).
Regarding intermediate hosts, Jensen and Bullard (Reference Jensen and Bullard2010) suggested that while host specificity for ‘tetraphyllidean’ tapeworms is euryxenous at the genus level, it might be stricter at the species level. However, recent molecular evidence nuances this hypothesis, revealing a wider intermediate host specificity range for certain larval species. For instance, Saad et al. (Reference Saad, Justine, Jouini, Gey, Theisen, Palm and Gargouri2026) demonstrated that Anthobothrium larvae collected from Sardinella aurita Valenciennes, 1847 (Clupeidae Cuvier, 1816) shared identical sequences with plerocercoids collected from Trichiurus lepturus Linnaeus, 1758 (Trichiuridae Rafinesque, 1810), proving that the same larval species can infect phylogenetically distant teleost families. Conversely, similar to the pattern observed in definitive hosts, a single intermediate host species can harbour multiple phylogenetically distinct Anthobothrium species. This is illustrated in our phylogenetic tree for T. lepturus hosting highly divergent lineages belonging to completely different clades, specifically included within Sub-clade 1.1 (GQ470162, GQ470167, GQ470168, GQ470170) and Sub-clade 2.4 (GQ470158).
The contrast in the specificity patterns of Anthobothrium species, shifting from a wide euryxenous range in intermediate hosts to a more restricted stenoxenous range in definitive hosts, represents a challenge for tracking and resolving these complex life cycles.
The migratory behaviour of intermediate/paratenic and definitive hosts has to be considered a key factor in the geographic distribution and transmission dynamics of Anthobothrium larvae. Indeed, L. vulgaris undergoes ontogenetic and reproductive migrations, moving onshore and offshore (Quetglas et al. Reference Quetglas, de Mesa, Guijarro, Massutí, Ordinas, Zaragoza and Valls2026; Worms Reference Worms and Boyle1983). In the Atlantic, long-distance horizontal migrations of up to 500 km were reported (Tinbergen and Verwey Reference Tinbergen and Verwey1945). Through these movements, squids may acquire Anthobothrium larvae in nursery or feeding areas, and subsequently transport them to definitive hosts across their different geographical distributions. In Tunisia, eight carcharhinid species have been reported, including Carcharhinus plumbeus (Nardo, 1827), C. brevipinna (Müller and Henle, 1839), C. melanopterus (Quoy and Gaimard, 1824), C. falciformis (Müller and Henle, 1839), C. limbatus (Valenciennes, 1839), C. obscurus (Lesueur, 1818), Prionace glauca (Linnaeus, 1758), and Rhizoprionodon acutus (Enajjar et al. Reference Enajjar, Saïdi and Bradai2022). The Tunisian coast is known to be a critical habitat and breeding area for several of these predators such as C. plumbeus in the Gulf of Gabès (Bradaï et al. Reference Bradaï, Saïdi, Bouaïn, Guéloget and Capapé2005; Saïdi et al. Reference Saïdi, Bradaï, Bouaïumln, Guélorget and Capapé2005). Although the phylogenetic tree places our larval sequences closest to an adult Anthobothrium sp. sequence infecting C. limbatus from Australia, it is important to note that C. limbatus is very common along the Tunisian coast (Enajjar et al. Reference Enajjar, Saïdi and Bradai2022). However, the current lack of molecular investigations of the cestode fauna of carcharhinids from Tunisia prevents any match. Furthermore, carcharhinid sharks are known for their long-distance migration. For instance, the Red Sea is considered a source of several carcharhinids in the Mediterranean, including C. brevipinna and C. limbatus (Ben-Tuvia Reference Ben-Tuvia1966; Garrick Reference Garrick1982), while occurrences in the Mediterranean of C. plumbeus originating from the eastern tropical Atlantic have been reported (Hemida et al. Reference Hemida, Seridji, Labidi, Bensaci and Capapé2002). This connectivity and overlapping migration patterns between the various hosts (intermediate/paratenic and definitive) may suggest matches between extreme geographical records. This hypothesis is supported by the study by Saad et al. (Reference Saad, Justine, Jouini, Gey, Theisen, Palm and Gargouri2026), which reported 100% sequence similarity between Anthobothrium larvae collected from the intermediate host Sardinella aurita in Tunisian waters and an adult form infecting the definitive host, C. limbatus, from the Gulf of Mexico.
Trophic studies could theoretically help clarify transmission pathways. Although L. vulgaris is preyed upon by several Mediterranean and northeastern Atlantic elasmobranchs (Squalus blainville (Risso, 1827), Scyliorhinus canicula (Linnaeus, 1758), Mustelus punctulatus Risso, 1827, M. mustelus (Linnaeus, 1758), Bathyraja brachyurops (Fowler, 1910), Aetomylaeus bovinus (Geoffroy Saint-Hilaire, 1817), Myliobatis aquila (Linnaeus, 1758), Torpedo marmorata Risso, 1810, Pteroplatytrygon violacea (Bonaparte, 1832), Raja clavata Linnaeus, 1758, and species of the genus Torpedo Duméril, 1806) (Abdel-Aziz Reference Abdel-Aziz1994; Capapé Reference Capapé1977, Reference Capapé, Crouzet, Clément, Vergne and Guelorget2007; Farias et al. Reference Farias, Figueiredo, Moura, Serrano Gordo, Neves and Serra-Pereira2006; Jardas et al. Reference Jardas, Šantić and Pallaoro2004, Reference Jardas, Šantić, Nerlović and Pallaoro2007; Kabasakal Reference Kabasakal2002; Lipej et al. Reference Lipej, Mavrič, Paliska and Capapé2013; Martinho et al. Reference Martinho, Sá, Falcão, Cabral and Pardal2012; Morte et al. Reference Morte, Redón and Sanz-Brau1997), none of these confirmed predators is a carcharhiniform species, nor do they include Gymnura altavela (Linnaeus, 1758), in which the only Anthobothrium species (A. altavelae Neifar, Euzet and Ben Hassine, Reference Neifar, Euzet and Ben Hassine2002) reported from Tunisia has been identified (Neifar et al. Reference Neifar, Euzet and Ben Hassine2002). Diet analyses focusing on the carcharhinid species represented in our tree within the Mediterranean could help to resolve some life cycles. Nevertheless, this approach is still limited as the stomach-content analyses provide only a snapshot of diets at the time of sampling.
To better resolve and accurately reconstruct the life cycles of these parasites, further investigations should address both taxonomic and ecological gaps. On the one hand, obtaining adult reference sequences remains crucial to assign sequenced larval stages to their respective species. On the other hand, expanding sampling efforts to cephalopods and teleost fishes will provide a comprehensive inventory of intermediate and paratenic hosts involved in these life cycles.
By comparison, previous studies carried out in the same region (Italy, central Mediterranean) reported lower infection indices for Anthobothrium sp. larvae in other intermediate hosts. Tedesco et al. (Reference Tedesco, Caffara, Gustinelli, Fiorito and Fioravanti2020) examined 32 specimens of Octopus vulgaris from Italy, of which four were positive for Anthobothrium sp. larvae, with infections in the intestine and caecum, with a prevalence of 13.00% and a mean intensity of 1.25. Similarly, Occhibove et al. (Reference Occhibove, Lopez-Verdejo, Mazzella, Cusano, Palomba, Aco-Alburqueque and Santoro2025) examined 200 Illex coindetii (Vérany, 1839) and reported a prevalence of 9.50%, a mean abundance of 0.20 ± 0.70, and a mean intensity of 1.80 ± 1.30 (Table 6).
Comparison of parasitological indices of collected Anthobothrium sp. larvae with previous studies
Table 6. Long description
The table consists of six columns: Host species, Locality, P (percent), m I, m A, and References.
* Row 1: Host Loligo vulgaris, Locality Tunisia, P 41.38, m I 3.63 plus or minus 4.11, m A 1.50 plus or minus 3.09, Reference Present study.
* Row 2: Host Octopus vulgaris, Locality Italy, P 13.00, m I 1.25, m A is not provided, Reference Tedesco et al. 2020.
* Row 3: Host Illex coindetii, Locality Italy, P 9.50, m I 1.80 plus or minus 1.30, m A 0.20 plus or minus 0.70, Reference Occhibove et al. 2025.
Footer notes define P (percent) as Prevalence, m I as mean intensity, and m A as mean abundance.
P (%): Prevalence; mI: mean intensity; mA: mean abundance.
These differences may be related to the higher coinfection rates observed in the previous studies, particularly with Acanthobothrium sp. (28%) and Nybelinia sp. (3%), compared with 13% of Anthobothrium sp. in O. vulgaris, and with the nematode Anisakis pegreffii Campana-Rouget and Biocca, 1955 (P = 25%) versus Anthobothrium sp. (P = 9.5%) in I. coindetii. Moreover, squids examined in the present study exhibited larger body size (TL = 26.6; TW = 163.2) compared to those reported by Occhibove et al. (Reference Occhibove, Lopez-Verdejo, Mazzella, Cusano, Palomba, Aco-Alburqueque and Santoro2025) (TL = 12.8; TW = 71.9), which may reflect an accumulation of the metacestodes over time in older hosts. Several studies have reported a positive relationship between host size and cestode larval abundance in cephalopods, consistent with cumulative trophic exposure (Marmolejo-Guzmán et al. Reference Marmolejo-Guzmán, García-The, Arjona-Cambranes, May-Sosa and Aguirre-Macedo2025; Tedesco et al. Reference Tedesco, Caffara, Gustinelli, Fiorito and Fioravanti2020).
The present study confirms, for the first time, the occurrence of Anthobothrium sp. larvae in the intermediate host L. vulgaris, supported by molecular evidence. Furthermore, the phylogenetic analysis provides initial insights into intra- and inter-cluster genetic divergence within the genus Anthobothrium and offers a preliminary framework for understanding the life cycles of some lineages represented in the phylogenetic tree.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26102314.
Acknowledgements
We acknowledge the reviewers for providing constructive comments to improve this manuscript.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
The authors declare none.
Ethical standards
Squids used in this study were sourced from commercial landings; hence, no ethics protocols were required.
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