Pavel N. Nikolov

In search of mitochondrial markers for resolving the phylogeny of cyclophyllidean tapeworms (Platyhelminthes, Cestoda) — a test study with Davaineidae

The most species rich order of tapeworms is the Cyclophyllidea and prior to wide-scale sampling of these worms for phylogenetics, we wished to develop reliable PCR primers that would capture fragments of mitochondrial (mt) DNA with phylogenetic utility across the order. Nuclear ribosomal RNA gene sequences are well-established and valuable markers for resolving flatworm interrelationships spanning a wide range of taxonomic divergences, but fail to provide resolution amongst recently diverged lineages. Entire mt genomes of selected cyclophyllidean tapeworms are available on GenBank, and we used these to design PCR primers to amplify mtDNA from cox1, rrnL and nad1 for a range of cyclophyllideans (7 davaineids, 1 hymenolepidid and 1 dilepidid) and selected outgroups (Tetrabothrius sp. and Mesocestoides sp.). A combined nuclear and mt gene data set was used to estimate a reference phylogeny and the performance of the individual genes was compared to this. Although nuclear and mt genes each contributed to the structure and stability of the phylogenetic estimate, strongest nodal support was provided by nuclear data amongst the basal lineages and by mt data amongst the most recently diverged lineages. The apparent complementarity afforded by combining nuclear and mt data was compromised by these data partitions providing conflicting signal at poorly supported nodes. Nevertheless, we argue for a combined evidence approach. PCR primers that amplify rrnL were designed and tested successfully against a diversity of cyclophyllideans; rrnL and nad1 appeared to be more informative than the fragment of cox1. The genus Raillietina was not supported by molecular evidence. The new primers will likely provide considerable resolution to estimates of cyclophyllidean interrelationships in future studies.

Cestode parasitism in invasive and native brine shrimps (Artemia spp.) as a possible factor promoting the rapid invasion of A. franciscana in the Mediterranean region.

Artemia franciscana is an invasive crustacean expanding its range in hypersaline wetlands in the Mediterranean region and replacing native Artemia parthenogenetica and Artemia salina. Native brine shrimps are known as intermediate hosts of cestodes; infected individuals exhibit changes in their behaviour and appearance, thus facilitating the parasite transmission to the avian hosts by predation. To assess whether invasive brine shrimps participate in the cestode life cycles to the same extent as the native species, we examined the natural infections in seven populations of Artemia spp. along the southern coast of Spain and Portugal: three populations of each A. franciscana and A. parthenogenetica and one population of A. salina. Ten cestode species were found in A. parthenogenetica, while only six were recorded in each of A. salina and A. franciscana. The overall infection was consistently higher in native than in invasive populations. For a particular cestode species, the prevalence or abundance was significantly higher in a native population for 54 pairwise comparisons and only higher for an invasive population for 4 pairwise comparisons. These results suggest that cestodes may influence competitive interactions between native and invasive brine shrimps, thus partly explaining the invasive success of A. franciscana.

Red and transparent brine shrimps ( Artemia parthenogenetica ): a comparative study of their cestode infections

Although the red colour of brine shrimps has previously been supposed to be associated with cestode parasitism, no evidence has been provided that red and transparent brine shrimps differ in their role for cestode circulation. We compared the cestode infections in 100 red and 100 transparent Artemia parthenogenetica collected from the Odiel salt pans in Spain. Seven cestode species were recorded in red, and only four of them were recorded in transparent brine shrimps. The red brine shrimps were more infected with cysticercoids than transparent individuals (prevalence, 87 vs 21%; mean intensity, 1.56 vs 1.00; mean abundance, 1.35 vs 0.21). Three cestode species (Flamingolepis liguloides, Anomotaenia tringae and Confluaria podicipina) had significantly higher prevalence and mean abundance in red brine shrimps. These results suggest that red brine shrimps have a more important role for the circulation of avian cestodes than transparent individuals of the same population.

Records of cysticercoids of Fimbriarioides tadornae Maksimova, 1976 and Branchiopodataenia gvozdevi (Maksimova, 1988) (Cyclophyllidea, Hymenolepididae) from brine shrimps at the Mediterranean coasts of Spain and France, with a key to cestodes from Artemia spp. from the Western Mediterranean

Cysticercoids of two cestode species isolated from brine shrimps from the Mediterranean coasts of Spain and France are described. Fimbriarioides tadornae Maksimova, 1976 (adults known as parasites of Tadorna tadorna) was recorded from Artemia parthenogenetica and A. franciscana from Spain (Bras del Port and River Ebro Delta, respectively), and from A. franciscana from Aigues-Mortes, France (new geographical record). Branchiopodataenia gvozdevi (Maksimova, 1988) (adults known as parasites of Larus genei) was recorded from A. parthenogenetica (Bras del Port, Spain), A. salina (San Pedro del Pinatar, Spain) and A. franciscana (River Ebro Delta, Spain); this is the first record of the species in Europe. An illustrated key to cysticercoids of 12 cestode species from Artemia spp. from the Western Mediterranean is proposed.

Spermiogenesis and sperm ultrastructure of Valipora mutabilis Linton, 1927 (Cestoda, Cyclophyllidea, Gryporhynchidae)

This is the first ultrastructural study of the spermiogenesis and the mature spermatozoon of a cyclophyllidean cestode of the family Gryporhynchidae. The spermiogenesis of Valipora mutabilis begins with the formation of a differentiation zone delimited by arching membranes and containing two centrioles. One of the centrioles develops an axoneme that grows directly into the cytoplasmic protrusion. The other centriole remains situated in a cytoplasmic bud and is subsequently aborted (type IV of cestode spermiogenesis). The mature spermatozoon of V. mutabilis is a filiform cell, tapered at both extremities and lacks mitochondria. The anterior extremity is characterised by the presence of an apical cone and a single helicoidal crested body. The axoneme is of 9 + ‘1’ trepaxonematan pattern, with a periaxonemal sheath. The cortical microtubules are twisted at an angle of about 45° to the spermatozoon axis. The nucleus is electron dense and spirally coiled around the axoneme. The cytoplasm is electron lucent and contains numerous granules of electron-dense material. In contrast to a recent opinion for close phylogenetic relationships, these ultrastructural data demonstrate the distant position between gryporhynchids and the family Taeniidae. The most similar pattern in the ultrastructure of the spermiogenesis and the mature spermatozoon has been described for dilepidids, some hymenolepidids and some anoplocephalids.

Participation of metanauplii and juvenile individuals of Artemia parthenogenetica (Branchiopoda) in the circulation of avian cestodes

Adult crustaceans of the genus Artemia (brine shrimps) are intermediate hosts in the life cycle of cestode species parasitic in aquatic birds as their definitive hosts. However, there are no data on the role of larval and juvenile brine shrimps in the transmission of avian helminth parasites. In order to examine the possible role of early developmental stages (nauplii, metanauplii and juveniles) of Artemia for the circulation of avian cestodes, the natural cestode infection in the population of Artemia parthenogenetica from La Mata Lagoon, Mediterranean coast of Spain, was studied. Metacestodes (cysticercoids) of four cestode species were recorded in adult brine shrimps: Flamingolepis liguloides and Flamingolepis flamingo (hymenolepidids parasitic in flamingos), Confluaria podicipina (a hymenolepidid species parasitic in grebes) and Eurycestus avoceti (a dilepidid species parasitic in avocets, stilts, plovers and, to a lesser extent, in flamingos). No cysticercoids were found in nauplii. Two species, F. liguloides and F. flamingo, were found in metanauplii and juvenile brine shrimps. Only 36.3% of the cysticercoids of F. liguloides occurred in adult brine shrimps; the remaining 63.7% were parasitic in metanauplii (39.6%) and juveniles (24.1%). Similarly, the metacestodes of F. flamingo were also distributed among various age groups: in adults (44.4% of cysticercoids), juveniles (27.8%) and metanauplii (27.8%). These results indicate that the early developmental stages of Artemia have an important role for the circulation of certain parasite species. No cysticercoids of C. podicipina and E. avoceti were recorded in larval and juvenile brine shrimps. The selective infestation of larval brine shrimps with flamingo parasites is probably associated with the feeding behaviour of definitive hosts, which are filtering predators; in contrast, grebes and waders pick brine shrimps individually one by one. The possible underlying mechanism for selective infestation of metanauplii and adults by certain cestode species is associated with the size of parasite eggs, allowing only cestode species with small eggs to be ingested by larval brine shrimps.

Spermiogenesis and spermatozoon ultrastructure of the paruterinid cestode Notopentorchis sp. (Cyclophyllidea).

Ultrastructural characters of the spermiogenesis and mature spermatozoon of Notopentorchis sp. (Cestoda, Cyclophyllidea, Paruterinidae), a parasite from Apus affinis (Aves, Apodiformes, Apodidae) from Gabon, are described by means of transmission electron microscopy. Cytochemical analysis for detection of glycogen was applied. Vestigial striated roots associated with the two centrioles are present in the zone of differentiation. The spermiogenesis is characterized by an external growth of free flagellum followed by a proximodistal fusion of the latter with cytoplasmic protrusion, thus, corresponding to the cestode spermiogenesis of the type III pattern described by Bâ and Marchand (Mem. Mus. Natl. Hist. Nat. 166:87–95, 1995). In the final stage of spermiogenesis, a single crested body appears at the base of the forming spermatozoon. The mature spermatozoon of Notopentorchis sp. is filiform and tapering at both extremities. It consists of five regions differing in their ultrastructural characteristics. The anterior extremity of the mature spermatozoon is characterized by the presence of an apical cone and a single crested body. The cytoplasm contains one axoneme of 9 + “1” type of the trepaxonematan Platyhelminthes, a periaxonemal sheath, a layer of twisted cortical microtubules, transverse intracytoplasmic walls, and granules of glycogen. The nucleus is coiled in spiral around the axoneme. The posterior extremity of the spermatozoon is characterized by the presence of electron-dense material. This structural organization corresponds to the morphology of cestode spermatozoon of type VII as defined by Levron et al. (Biol Rev 85: 523-543, 2010). The comparison of the results with those of the two previous studies on paruterinids suggests that several characters of the spermiogenesis and the mature spermatozoon are invariable, i.e. the type III spermiogenesis and the presence of vestigial striated roots, a single crested body, a periaxonemal sheath, and intracytoplasmic walls. The main differences of the sperm cells among members of this family are the lack of dense granules (as in Triaenorhina rectangula) and the presence of electron-dense material in the posterior extremity of the spermatozoon (as in Notopentorchis sp.).

Cestodes from Hector's Beaked Whale (Mesoplodon hectori) and Spectacled Porpoise (Phocoena dioptrica) from Argentinean Waters

Abstract Single individuals of 2 little-known cetacean species, Mesoplodon hectori and Phocoena dioptrica, stranded and died on the coast of Argentina (Buenos Aires and Chubut provinces, respectively) and were studied for the presence of helminths. The cestodes found were described and illustrated using light microscopy. The following cestode taxa were recovered: Tetrabothrius (Tetrabothrius) hobergi n. sp. (several fragmented specimens, at least 1 gravid) and Tetrabothrius (s.l.) sp. 1 (several fragmented immature specimens) from M. hectori, and Tetrabothrius (s.l.) sp. 2 (single fragmented immature specimen) and 2 morphotypes of tetraphyllidean larvae from P. dioptrica. Tetrabothrius (T.) hobergi n. sp. can be distinguished from Tetrabothrius (T.) forsteri by the greater number of testes and larger eggs and oncospheres, from Tetrabothrius (T.) curilensis by the smaller testes and vitellarium, the shape and size of the ovary, and the larger oncospheres and longer embryonic hooks, and from Tetrabothrius (T.) sp. from Ziphius cavirostris by the narrower strobila, smaller scolex, and smaller number of testes. The generic designations of Tetrabothrius (s.l.) sp. 1 and Tetrabothrius (s.l.) sp. 2 were based on the scolex morphology. Tetrabothrius (s.l.) sp. 1 is closest to Tetrabothrius (T.) forsteri and Tetrabothrius (Biamniculus) innominatus based on the number of testes, while the scolex size of Tetrabothrius (Tetrabothrius) sp. 2 is within the variability range reported for Tetrabothrius (T.) forsteri. More definite identification of the 2 species was not possible due to the condition of the available material. The present study provides the first descriptions of cestodes from M. hectori and P. dioptrica, thus enriching the knowledge regarding the helminths of insufficiently studied marine mammals.

Cyclophyllidean cysticercoids from Echinogammarus tibaldii (Amphipoda, Gammaridae) from Lake Piediluco, Italy

In August 2007, 856 specimens of Echinogammarus tibaldii Pinkster et Stock, 1970 from the Lake Piediluco (Central Italy) were collected from 3 different sites and examined for larval helminths. Fourteen amphipods (1.63%) were infected with larvae of Lateriporus teres (Dilepididae) and one amphipod (0.12%) was infected with Microsomacanthus microsoma (Hymenolepididae). Adults of both species are known from waterfowl and gulls. The present results constitute new host records for both cestode species and the first finding of L. teres in Italy.

Taxonomic revision of the cestodes of the family Progynotaeniidae (Cyclophyllidea) parasitising stone curlews (Charadriiformes: Burhinidae)

The type series of Progynotaenia evaginata Fuhrmann, 1909 from Burhinus senegalensis in Sudan, P. foetida Meggitt, 1928 from B. oedicnemus in Egypt and Angularia australis Maplestone, 1921 from B. grallarius in Australia are redescribed. As a comparative material, specimens of P. evaginata from B. oedicnemus in Kazakhstan were studied. The type-series of P. evaginata and P. foetida were found to be heterogeneous due to the presence of scoleces and fragments of cestodes of the genus Stenovaria Spasskii & Borgarenko, 1973 (Dilepididae). For P. foetida, a lectotype is designated. P. foetida is recognised as a synonym of P. evaginata (new synonymy). Angularia australis, previously considered a member of the Dilepididae, is transferred to the Progynotaeniidae as a synonym of P. evaginata (new synonymy). The synonymy of P. indica Johri, 1963 with P. evaginata, proposed by Ryzhikov & Tolkacheva (1981), is supported. The host range and the geographical distribution of P. evaginata are restricted to birds of the genus Burhinus from the Eastern Hemisphere.