Life cycle truncation in Digenea, a case study of Neophasis spp. (Acanthocolpidae)

Abstract

Truncated life cycles may emerge in digeneans if the second intermediate host is eliminated, and the first intermediate host, the mollusc, takes up its role. To understand the causes of this type of life cycle truncation, we analyzed closely related species of the genus Neophasis (Acanthocolpidae) with three-host and two-host life cycles. The life cycle of Neophasis anarrhichae involves two hosts: wolffishes of the genus Anarhichas as the definitive host and the common whelk Buccinum undatum as the intermediate host. Neophasis oculata, a closely related species with a three-host life cycle, would be a suitable candidate for the comparison, but some previous data on its life cycle seem to be erroneous. In this study, we aimed to redescribe the life cycle of N. oculata and to verify the life cycle of N. anarrhichae using molecular and morphological methods. Putative life cycle stages of these two species from intermediate hosts were linked with adult worms from definitive hosts using ribosomal molecular data: 18S, ITS1, 5.8S-ITS2, 28S. These markers did not differ within the species and were only slightly different between them. Intra- and interspecific variability was also estimated using mitochondrial COI gene. In the constructed phylogeny Neophasis spp. formed a common clade with two other genera of the Acanthocolpidae, Tormopsolus and Pleorchis. We demonstrated that the first intermediate hosts of N. oculata were gastropods Neptunea despecta and B. undatum (Buccinoidea). Shorthorn sculpins Myoxocephalus scorpius were shown to act as the second intermediate and definitive hosts of N. oculata. The previous reconstruction of the two-host life cycle of N. anarrhichae was reaffirmed. We suggest that life cycle truncation in N. anarrhichae was initiated by an acquisition of continuous morphogenesis in the hermaphroditic generation and supported by a strong prey-predator relationship between A. lupus and B. undatum.

Keywords: Buccinidae; Cercariae; Life cycle; Metacercariae; Neophasis anarrhichae; Neophasis oculata.

Graphical abstract

Fig. 1

Consensus (99 threshold) neighbour-joining tree based on the concatenated ITS1 and ITS2 860-b.p. fragment, built with Tamura-Nei genetic distance method and 1000 bootstrap resamples; support values are printed at nodes. Repeat regions of the ITS1 were excluded from the alignment. Scale bar shows substitutions per site. The ingroup includes identified and putative life cycle stages of Neophasis oculata and N. anarrhichae, the numbers of isolates are as listed in Table 1. Brachycladium goliath serves as an outgroup.

Fig. 2

Neophasis oculata intramolluscan stages: daughter redia (A), infective cercaria body structure (B) and general view (C).

Fig. 3

Neophasis oculata cercariae. (A) Infective cercaria, general structure and mucoid in the tegument (toluidine blue), differential interference contrast (DIC). (B) Ducts of the penetration glands in live cercaria. (C) Sagittal histological section of infective cercaria, Erlich's hematoxylin-eosin. (D–E) Mucoid in underdeveloped cercariae (toluidine blue, whole mount, DIC (D) and Azur II-eosin, histological section (E)). (F) SEM, ventral view. (G–I) CLSM, TRITC-phalloidin, acetylated α-tubulin and phospho Y antibody staining. (G) Infective cercaria, flame cells and nerves. (H–I) Underdeveloped cercariae, excretory ducts (H) and eyespots (I). Scale bars – 50 μm.Abbreviations: aс – anterior collecting duct; cd – caudal excretory duct; ev – excretory vesicle; fc – flame cells; ga – cerebral ganglion; mc – mucoid cytons; os – oral sucker; pс – posterior collecting duct; pe – pigmented eyespots; pg – penetration glands; pd – penetration gland ducts; ph – pharynx; t – tail; ue – unpigmented eyespot; vnc – ventral nerve chords; vs – ventral sucker. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Neophasis oculata metacercariae, inside cyst (A) and removed from cyst (B).

Fig. 5

Neophasis oculata metacercariae (A–F) and sexual adult (G). (A) General view of metacercaria (acetic carmine, DIC). (B–C) Histological sections, Mallory's trichrome stain, encysted metacercaria (B) and some of its inner structures (C). (D–F) SEM, ventral view (D) and magnified spines in the anterior (E) and posterior (F) regions. (G) Sexual adult (acetic carmine, DIC). Scale bars – 100 μm on A, B, D, G; 50 μm on C; 10 μm on E, F.Abbreviations: c – ceca; ci – cirrus; eg – eggs; ev – excretory vesicle; icy – inner cyst layer; ocy – outer cyst layer; os – oral sucker; ot – ootype; ov – ovary; pe – pigmented eyespots; ph – pharynx; sv – seminal vesicle; te – testes; ut – uterus; vi – vitelline follicles; vs – ventral sucker.

Fig. 6

Neophasis anarrhichae mother sporocyst (A) and mother redia (B).

Fig. 7

Neophasis anarrhichae successive life cycle stages. (A, B) whole mounts (toluidine blue, DIC) of the anterior end of daughter redia (A) and cercaria embryo (B). (C) Cercaria later embryo, CLSM, TRITC-phalloidin and phospho Y antibody staining. (D, E) whole mounts (toluidine blue, DIC) of cercaria (D) and metacercaria (E). (F) Anterior end of metacercaria with gland ducts, CLSM, acetylated α-tubulin and phospho Y antibody staining. (G) Sagittal section of metacercaria, Heidenhain's iron hematoxylin staining. (H) Sagittal section of metacercaria, Mallory's trichrome stain. (I) progenetic metacercaria (toluidine blue, DIC). (J) sexual adult (acetic carmine, DIC). Scale bars – 50 μm.Abbreviations: aс – anterior collecting duct; bp – birth pore canal; c – ceca; cd – caudal excretory duct; ci – cirrus; eg – eggs; ev – excretory vesicle; os – oral sucker; ot – ootype; ov – ovary; ovd – oviduct; pс – posterior collecting duct; pe – pigmented eyespots; pd – penetration gland ducts; ph – pharynx; sv – seminal vesicle; t – tail; te – testes; vi – vitelline follicles; vs – ventral sucker. Arrow indicate on site of main collecting duct division. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Phylogenetic position of Neophasis oculata and N. anarrhichae based on the concatenated 18S and 28S rDNA sequence data, inferred with Bayesian inference. Newly generated sequences are indicated in bold. Posterior probabilities are printed at nodes, followed by bootstrap values for the nodes that were also supported in the tree inferred with Maximum likelihood method. Scale bar shows the substitution rate. GenBank accession numbers for the 18S and 28S rDNA sequences are listed in the Supplementary Table S1.

Fig. 9

Proposed life cycle scheme of Neophasis oculata (A) and N. anarrhichae (B).