Neophasis anarrhichae (Nicoll, 1909) Bray, 1987

Kremnev, Georgii, Gonchar, Anna, Krapivin, Vladimir, Uryadova, Alexandra, Miroliubov, Aleksei & Krupenko, Darya, 2021, Life cycle truncation in Digenea, a case study of Neophasis spp. (Acanthocolpidae), International Journal for Parasitology: Parasites and Wildlife 15, pp. 158-172 : 166-167

3.3.4. Intramolluscan stages of Neophasis anarrhichae ( Fig. 6 , 7 A-I)

Locality: White Sea; Keret Archipelago (Kandalaksha Bay).

Host: Buccinum undatum .

Sites: Reproductive and digestive gland (mother and daughter rediae); kidney (mother sporocyst and mother rediae). In heavily infected whelks, daughter rediae were also located in kidney, gill and mantle.

Prevalence: 5% (69 of 1376).

Vouchers: Hologenophore VG 5.3 (mother sporocyst) and isogenophores (NA004-NA010) corresponding to isolates No 29, 32, 34, 35 and 36 deposited in the collection of the Department of Invertebrate Zoology of Saint Petersburg University .

Description:

Mother sporocyst ( Fig. 6A ).

In single found specimen body elongated, 994 × 211, anterior end wide, posterior end pointed. Brood cavity occupying almost all inner space, containing nine embryos of mother rediae, with one embryo much more advanced than others. Birth pore not identified with certainty but probably located at anterior end. Germinal mass at posterior end, embedded in parenchyma.

Mother rediae ( Fig. 6B ).

[Measurements based on ten specimens from kidney fixed with 96% ethanol.]

Rediae elongated, 272–836 (595) × 61–146 (104), capable of slight contraction, with pointed posterior end. Mouth terminal, pharynx small, oval, 34–69 (48) × 25–56 (42). Cecum oval, 27–92 (66) × 22–61 (41). Brood cavity occupying almost all inner space of rediae, containing embryos of daughter rediae at different stages of development. Birth pore with distinct birth canal posterior to pharynx. Germinal mass at posterior body end, embedded in parenchyma. In younger rediae cecum occupying nearly one third of body length; brood cavity indistinct, without embryos.

Daughter rediae, cercariae and metacercariae ( Fig. 7A–I ).

These life cycle stages of Neophasis anarrhichae have been described elsewhere ( Lebour, 1910; Chubrik, 1966; Køie, 1969, 1971). Nevertheless, we would like to specify several details based on the material examined in our study.

In the daughter rediae of Neophasis anarrhichae , the birth canal and birth pore are present but are more distinct in younger specimens ( Fig. 7A ). Pigmented eyespots are first seen in the cercariae when the tail bud becomes slightly longer than the body ( Fig. 7B ); they are less prominent than in the cercariae of N. oculata (compare Figs. 7D and 3A ). An unpigmented eyespot initially emerges as a small dot in the cercarial embryo, staining only with TRITC-labeled phalloidin. We did not trace its further development. The digestive system includes the prepharynx, the pharynx, the oesophagus and the ceca primordia ( Fig. 7C ). The latter are composed of rows of cells, which do not reach the posterior body end. The cercariae have 13 penetration glands and 11 ducts which are arranged similarly to N. oculata . Excretory vesicle is thin-walled, I-shaped or egg-shaped ( Fig. 7C and D ). Excretory system is of “Mesostoma” type ( Fig. 7C ). Main collecting ducts divide into anterior and posterior parts near the anterior edge of the ventral sucker ( Fig. 7C ). Caudal excretory duct is present ( Fig. 7C ). Mucoid cytons and mucoid glands are absent ( Fig. 7B, D ).

In the metacercariae of Neophasis anarrhichae only nine ducts of penetration glands are evident ( Fig. 7F ). Glandular cells just behind the oral sucker, which were mentioned by Lebour (1910), are in fact a closely packed bunch of ducts. These ducts open at the bottom of the buccal cavity, whereas the glands itself are located laterally to the pharynx. The ceca never reach the posterior body end. Excretory vesicle is egg-shaped ( Fig. 7E, I ). The metacercariae often retain a short tail, in which the caudal excretory duct is absent. All elements of the reproductive system are clearly distinguishable ( Fig. 7E, G, H ). There is a lumen in the female and male reproductive ducts ( Fig. 7H ) and early stages of gametogenesis are commonly observed in the ovary and the testes ( Fig. 7G ). Formed sperm is found regularly in male gonads. The

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metacercariae can start egg production while they are still within the daughter rediae ( Fig. 7I ), which was observed in 15 host specimens in 2019–2020.

Remarks: We found a mother sporocyst just once during the observation period. Alongside with it, several mother rediae were present in the kidney of that whelk, while its other organs were free from digenean infection. It seems that the mother rediae are initially located only in the kidney and later move into the digestive and the reproductive gland, where they can be found besides the daughter rediae. The key distinction between them is the content of the brood cavity: mother rediae produce rediae of the next generation whereas daughter rediae produce cercarial embryos.

Figures

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. 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.)