Veneriserva, Rossi, 1984

Tilic, Ekin & Rouse, Greg W., 2024, Hardly Venus’s servant-morphological adaptations of Veneriserva to an endoparasitic lifestyle and its phylogenetic position within Dorvilleidae (Annelida), Organisms Diversity & Evolution 24 (1), pp. 67-83 : 71-74

Position of Veneriserva within Aphrodita

The µCT scan of a parasitized A. longipalpa allowed visualizing in 3D the position of Veneriserva inside their host without dissection and in their natural condition ( Fig. 3 ). In the Aphrodita specimen scanned, a single juvenile and a single female V. pygoclava were present. The juvenile is likely to be a male, as two females were never observed together in a single host. Veneriserva pygoclava resided within the coelomic cavity of the host, largely occupying the lateral and ventral coelomic spaces ( Fig. 3 ). The larger female was approximately 77.1 mm long, 10 times the size of the juvenile (± 7.2 mm), and about twice the length of the host (± 42.7 mm) ( Fig. 3A ). The female was positioned in a U-shaped coil with the posterior and anterior ends both located near the anterior of the host. The juvenile was located near the anterior end of the female ( Fig. 3A, B, E, F ). Other female Veneriserva with an even larger parasite to host body length ratio were observed in some of the dissected specimens ( Fig. 1C, G ). In these, the parasites could be observed making multiple coils within the ventral coelomic space, and also extending dorsally ( Fig. 1A–C ), taking up a very large area within the host’s body. Neither the musculature nor the gut of the host appeared to be substantially damaged by the parasite ( Fig. 3B–D ). Despite the relatively large size of many of the A. longipalpa collected, no gonads or gametes of any stage were observed in any sampled hosts, infected or not. Specimens were observed from the months December, March, May, July, September, and October over the period of 2017 to 2023; so, the breeding season may be over the winter/spring months.

Interestingly, the two juvenile A. longipalpa specimens examined (both less than 3 cm in length) did not show any signs of infection upon external inspection of the ventral side. However, histological sectioning of these specimens revealed a single juvenile V. pygoclava in both ( Fig. 4A, B ). These V. pygoclava specimens were juveniles themselves and did not show any signs of gametogenesis ( Fig. 4B ), so sex determination was not possible.

Oogenesis

Oogenesis in Veneriserva pygoclava occurs in segmentally repeated ovaries. The oogonia of V. pygoclava proliferated from the ventral surface of the dorsal blood vessel ( Fig. 5A, C ) and the gonads were attached to the intersegmental septa (mesenteries) ( Fig. 5C ). Each developing oocyte was directly connected to a nurse cell by intercellular cytoplasmic bridges ( Fig. 5D ). The nucleus of the oocyte appeared to undergo numerous morphological changes during oogenesis, as it appeared heterochromatic in early stages and enlarged and became euchromatic with a single prominent nucleolus during the vitellogenic phase ( Fig. 5B, C ). The nurse cells appeared to undergo a striking morphological change after the onset of vitellogenesis, making them easy to distinguish from their neighboring oocytes. During vitellogenesis, the volume of the nurse cell nucleus rapidly increased and was always dense and heterochromatic, whereas the nuclei of the oocytes were all euchromatic at this stage ( Fig. 5C ). Another difference between vitellogenic oocytes and nurse cells was the size and distribution of yolk platelets. Nurse cells never contained ripe yolk bodies, but only small-sized yolk platelets arranged in clusters ( Fig. 5D ). While oocytes kept growing reaching a final diameter of about 100 µm, nurse cells had already reached their final diameter of about 30 µm during vitellogenesis. The nurse cells then appear to undergo a decrease in diameter until they are finally incorporated into the oocytes ( Fig. 5B ).

Spermiogenesis

In the histologically sectioned male specimen, spermiogenesis occurred along the peritoneal lining ( Fig. 5F ). Spermatogonia were found ventrally associated with the coelomic lining, near the dorsal blood vessel ( Fig. 5F ). Spermatocytes and mature sperm were found released into the coelomic cavity. Upon dissection, liberated sperm cells could be observed under the light microscope. Mature sperm of V. pygoclava had a large conical acrosome (~ 12 µm long) and a spherical nucleus (6 µm in diameter) with no evidence of an emergent flagellum ( Fig. 5E ).

Pharyngeal apparatus and lack of an intestine

The alimentary tract of V. pygoclava was found to be unique and aberrant among Annelida, in that it consisted of a functional muscular axial proboscis with a jaw apparatus but with no through-gut ( Figs. 3E , and 4B–H ). The pharynx ended blindly ( Fig. 4G, H ) leaving a completely hollow body cavity only occupied by developing gametes in mature specimens ( Fig. 5A, F ). The complete reduction of the gut was observed in both juvenile ( Fig. 4B–H ) and adult specimens of V. pygoclava ( Fig. 5A, F ) we sectioned histologically. Even though a jaw apparatus is present, and the animals can evert and move the maxillae in a pinching motion ( Fig. 1H ). The maxillae and the fused mandibles appear reduced when compared to free-living Dorvilleidae . The pygidium of V. pygoclava was confirmed as being club shaped, which inspired the species epithet. An anus, rudimentary or not, was not confirmed.

Epidermal ultrastructure

The entire body surface of V. pygoclava was densely covered with microvilli of the epidermal cells that pierced through the cuticle ( Fig. 6A–C ). The cuticle was less than 500 µm in thickness. A thin electron-dense epicuticle was present and had an inner lighter and an outer denser zone. The outer electron dense zone of the epicuticle was covered with a thin layer of darkly stained particles, giving it a fuzzy appearance ( Fig. 6B ). The unbanded collagen fibers of the cuticle were embedded in an electron-light glycocalyx matrix and were more densely arranged towards the epicuticle ( Fig. 6B ). Distally, the epidermal cells contained numerous transport vesicles (inclusion bodies) ( Fig. 6E ). Individual mucosecretory cells were abundant in the epidermis and were situated between supportive cells ( Fig. 6A ). Patches of multiciliated epidermal cells were present scattered around the body ( Fig. 6D ). These multiciliated cells contained larger amounts of mitochondria and were also covered with microvilli ( Fig. 6D ). Microvilli were branched ( Fig. 6E ) and had an enlarged, inflated tip covered with electron-dense droplets ( Fig. 6B ). The inflated portion of a microvillus was bulbous and had a maximum diameter of ± 300 nm.

Phylogenetic placement of Veneriserva

The ML analysis of the three mitochondrial and two nuclear genes for Dorvilleidae showed two main clades (labeled A and B) containing all Ophryotrocha terminals as well as members of other genera such as Exallopus , Iphitime , Pseudophryotrocha , and Veneriserva . Veneriserva pygoclava was in clade B with a relatively long branch and formed a poorly supported clade with the three included terminals of Iphitime ( Fig. 7 ), which are all symbiotic with Crustacea ( de Paiva and Nonato, 1991). It was sister taxon to Iphitime paguri Fage & Legendre, 1934 , making Iphitime paraphyletic, though there was little support for this. This Iphitime / Veneriserva clade formed a well-supported clade together with a sister clade of Ophryotrocha mainly associated with hydrothermal vents (6 species) ( Zhang et al., 2023) and one with a whale fall O. clava Taboada et al., 2013 . Another clade of symbiotic dorvilleids, also living with Crustacea ( O. mediterranea Martin et al., 1991 and O. geryonicola Esmark, 1874 ), formed a clade with the free-living O. vivipara Banse, 1963 ( Fig. 7 ) and was also in clade B. Five specimens of V. pygoclava exhibited four CO1 haplotypes, each differing by a few mutational steps ( Fig. 7 ).

Figures

Fig.3 µCT visualization of parasites within Aphrodita longipalpa. A 3D rendering of parasites shown within the projection of the host body. B–D Virtual dissections of surface renderings, showing crosssections of the host across three consecutive body regions, from anterior to posterior. Raw image data from the micro-CT stack, illustrating a horizontal section through the host (E) and a sagittal section (F). Head of the juvenile parasite is magnified to display the prominent jaws in white. Abbreviations—ja jaws, ne nephridia, pha pharynx. Female Veneriserva pygoclava is shown in yellow or with yellow arrowheads and the juvenile V. pygoclava in blue or with blue arrowheads

◂Fig. 1 Live photos and dissection of parasitized Aphrodita longipalpa and Veneriserva pygoclava. A Ventral view of A. longipalpa. B Dorsal view of A. longipalpa with removed feltage chaetae, revealing the parasite visible through the body wall. C Ventrally dissected A. longipalpa, exposing the sizable female parasite. Veneriserva pygoclava individuals within the host are indicated by arrowheads. D Juvenile female V. pygoclava, with developing oocytes visible through the body wall along the mid-dorsal orange line. E Female V. pygoclava showing the mid-dorsal orange pigmentation and the white mark at the base of the prostomium. F Male V. pygoclava. G A large female and smaller male V. pygoclava, extracted from the same host. The pygidium is club-shaped in both males and females and juveniles. H Juvenile V. pygoclava shown from multiple angles, characterized by a complete white coloration; black jaws are magnified in panel

Fig. 4 AZAN-stained paraffin histology. A Histological cross-section of a juvenile Aphrodita longipalpa featuring an endoparasitic immature Veneriserva pygoclava (denoted by a star). B Longitudinal section of V. pygoclava, highlighting the absence of a through gut, and continuous uninterrupted mesenteries. C–H Cross-sections through the anterior region of V. pygoclava, showing the muscularized pharynx with jaws culminating in blind termination at section G. Abbreviations—ac acicula, br brain, df dorsal felt, el elytra, ja jaws, mo mouth, ne nephridium, pha pharynx, vnc ventral nerve cord

◂Fig.5 Gametogenesis in male and female Veneriserva pygoclava. A–D Semi-thin histological sections of female Veneriserva pygoclava, stained with toluidine blue. A Cross-section of a female Veneriserva. B Close-up of large mature oocytes without discernible nurse cells. C Developing oocytes attached to mesenteries (mes), and oogonia proliferating from the ventral side of the dorsal blood vessel (bv). D Details of vitellogenic oocytes and nurse cells. Arrowheads indicate brownstained yolk platelets and yolk bodies. E Live sperm cells captured in a light micrograph. F–G Cross-sections of male Veneriserva. Note the absence of a gut in the cross-sections. Abbreviations—ac acicula, acr acrosome, bv blood vessel, coe coelomic cavity, mes mesentery, nc nurse cell, nn nurse cell nucleus, nu sperm cell nucleus, Oo oocyte, on oocyte nucleus, sp spermatogonia, vnc ventral nerve cord

Fig.6 Epidermal ultrastructure of Veneriserva pygoclava. A–D TEM images of the epidermis revealing the presence of dense, modified microvilli (mv) that cover the body surface. A mucosecretory gland cell (gl) is discernable in A. D shows details of a multi-ciliated epidermal cell. B depicts the microvilli (mv) covering the cuticle (cu). Note the inflated tips of the microvilli and the electron-dense droplets. E Apically the epidermal cells display an abundance of transport vesicles (v). Arrowheads mark the branching microvilli piercing through the cuticle in all images. Abbreviations—ci cilia, m mitochondria, nc nucleus

Fig.7 Maximum likelihood (ML) tree of Dorvilleidae. The tree depicts the phylogenetic relationships within Dorvilleidae inferred through concatenated 16S, COI, Cytb, 18S, and H3 sequences. Bootstrap support values are provided for each node. Nodes with complete support are indicated with an asterisk (*); values below 50% are not shown. Branches of parasitic/symbiotic species highlighted in blue. Haplotype network for 5 Veneriserva pygoclava specimens is shown next to the tree