The unusual cytoarchitecture of “vitelline follicles” in freshwater blood flukes of the genus Sanguinicola (Digenea, Aporocotylidae)

This is the first study assessing the cytoarchitecture of the vitellarium of members of the freshwater, teleost-infecting lineage of blood-flukes (Aporocotylidae). The vitelline cytoarchitecture of two innominate species of Sanguinicola from freshwater fishes in Russia showed that vitelline cells at different stages of maturation are widely distributed throughout much of the body and are mixed with other cell types. The latter feature indicates that use of the term “follicular vitellarium” is inappropriate for species of this genus. An additional characteristic of the vitelline cells in these Sanguinicola spp. is their ability to form long, pseudopodia-like extensions of the peripheral cytoplasm that contact neighbouring vitelline cells and sarcoplasmic extensions, forming both heterologous and homologous intercellular junctions. Within the vitelline duct lumen, the cytoplasm of mature vitelline cells is filled with regular clusters (0.5–1.0 μm in diameter), comprising 10–30 vitelline globules, which have heterogeneous contents and electron-lucent lipid droplets (1.1–1.7 μm in diameter), but no apparent modifications of vitelline globules occur within the vitelline duct. The flattened, ciliated, epithelial lining of the common vitelline duct contains intra-epithelial nuclei, its luminal surface bears shallow lamellae and adjacent cells are adjoined by apical septate junctions. All of these observations, when compared to the marine Aporocotyle simplex, likely represent additional characteristics supporting the divergent evolutionary lineages of marine and freshwater aporocotylids.


Introduction
In the Digenea, the vitellarium is morphologically diverse; variations include single or double compact masses, a range of lobed or tubular structures and few to numerous isolated or linked follicles. Its configuration has been used as an important taxonomic or even phylogenetic criterion at several levels [4]. Furthermore, judging from the existing literature on ultrastructure, some characteristics of the vitelline cytoarchitecture have been used as discriminatory traits for digenean lineages [24,25]. The latter traits include the presence of one or two types of cell components within the vitellarium, i.e., vitelline cells (vitellocytes) at different stages of development and interstitial cells; the occurrence of junctional complexes between cells within the vitellarium; and the presence or absence of a specialized sheath of the basal matrix, which isolates the vitellarium from the surrounding cells and organs. Most of such ultrastructural studies on digenean species have targeted the process of vitellogenesis, but, in a number of studies, the cytoarchitecture of the vitellarium itself has been investigated [6-8, 11-13, 24, 25, 30, 35, 36].
It is challenging to understand the evolution of the Digenea without detailed studies on their early branching groups. Although tree topologies fluctuate, depending on the number and identity of taxa included in the analysis, the fish blood flukes are typically and routinely recovered as an early-branching monophyletic group within the Digenea [21,23]. Species of this family, especially the freshwater members/lineages, have been little studied using the transmission electron microscope (TEM) [18-20, 28, 29]. The phylogenetic relationships between the three postulated aporocotylid evolutionary lineages (chondrichthyan-infecting, freshwater teleost-infecting and marine teleost-infecting) [1] are only partly supported by morphology and genetics [1,[21][22][23].
There is only one detailed ultrastructural study on the cytoarchitecture of the vitellarium of an aporocotylid. This concerns the marine, teleost-infecting species Aporocotyle simplex Odhner, 1900 [27]. The present study of blood flukes of the genus Sanguinicola Plehn, 1905 is the first TEM investigation of the vitellarium of members of the freshwater, teleost-infecting lineage. Within this study, we will examine the phylogenetic relationships between freshwater and marine aporocotylids from teleosts based on the ultrastructural characteristics of their vitellarium.

Ethics statement
All necessary permits for fishing were obtained for the authors by the Institute for Biology of Inland Waters, Russian Academy of Sciences, from the relevant authorities of the Russian Federation. The treatment of the collected fishes and the investigations of Sanguinicola spp. infections are in compliance with all institutional, national, and international guidelines on the care and use of animals.

Specimens and species
Specimens of Sanguinicola sp. 1 were collected from the ventral aorta leading from the heart of naturally infected pike Esox lucius (Linnaeus, 1758) (Esocidae) and specimens of Sanguinicola sp. 2. were collected from the ventral aorta of the heart of naturally infected ides Leuciscus idus (Linnaeus, 1758) (Cyprinidae) from the Rybinsk Reservoir in the Upper Volga River Basin, Russia. Using scanning electron microscopy (SEM), these two Sanguinicola spp. have been shown to differ morphologically (Figs. 1A-1D).
The status of species of Sanguinicola in European waters is in need of revision. This is especially so for the type-species, S. inermis Plehn, 1905, which has been reported from a wide range of hosts but especially from cyprinids. In this work, we are treating the two species studied as innominate, pending a detailed taxonomic study. Sanguinicola sp. 1 is the same species as that previously referred to as Sanguinicola inermis [28] and Sanguinicola sp. [29].

Electron microscopy
For electron microscopy, live blood flukes were fixed using 3% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.2) for 7 days at 5°C, rinsed three times for 10 min periods in the same buffer and postfixed in 1% osmium tetroxide for 1 h. For SEM observations, fixed specimens were dehydrated in a graded ethanol series, with a final change to absolute acetone and then critical-point dried with liquid CO 2 . Later, the specimens were mounted on stubs, sputter-coated with gold-palladium and examined under a JEOL-JSM-6510LV microscope operating at 30 kV. For transmission electron microscopy (TEM), fixed specimens were later embedded in a mixture of Araldite and Epon using an Araldite/Embed-812 EM Embedding kit (EMS). Ultrathin sections were then stained with uranyl acetate and lead citrate, and examined using a JEM 1011 microscope operating at 80 kV.  The common vitelline duct of Sanguinicola spp. runs medially along the body and is lined by a flattened, ciliated epithelium (Figs. 3D-3F), whose intra-epithelial nuclei are flat, large  and contain scattered areas of heterochromatin (Fig. 3E). The luminal surface of each epithelial cell is protuberant, forming shallow lamellae (Figs. 3E-3H), and its cytoplasm contains ciliary basal bodies, numerous free ribosomes, a few electron-lucent vesicles and mitochondria (Fig. 3G). Cilia are unevenly distributed both singly and as closely associated pairs on the luminal surface (Figs. 3E-3G). Adjacent cells are adjoined by apical septate junctions (Fig. 3H). Sparse muscle fibres are found beneath the duct epithelium (Figs. 3D, 3F). Isolated, mature vitelline cells are present within the lumen of the duct, aligned in a single row along its entire length (Figs. 3D, 3F). Their cytoplasm is filled with regular clusters of globules and lipid droplets (Figs. 3D, 3F). No apparent modifications of these vitelline globules were observed.

Discussion
The present study has revealed some unusual features in the cytoarchitecture of the vitellarium of two innominate species of Sanguinicola (from pike Esox lucius and ide Leuciscus idus). An unusual characteristic of the vitellarium in these species is the presence of single vitelline cells, or a gathering of a small number of vitelline cells, at different stages in their development, which are widely distributed throughout much of the body and mixed with other cell types rather than comprising follicles. Consequently, the term vitelline "follicle" should not be used for these, and probably other, species Sanguinicola. Traditionally, the term "follicular vitellarium" in digeneans has been used to describe the accumulation of vitelline cells at different stages of maturation, situated close together and formed into small, usually numerous, compact vitelline masses. In the present material, each follicle contains only a single cell type, i.e., vitelline cells, which are not isolated from the surrounding tissue. The cytoarchitecture of vitelline follicles proper has previously been described for another aporocotylid, Aporocotyle simplex, which belongs to the marine, teleost-infecting aporocotylid lineage [27]. Vitelline follicles with the usual cytoarchitecture have also been described in digeneans of the orders Diplostomida, i.e., the diplostomatid Pharyngostomoides procyonis [5], and Plagiorchiida, i.e., the microphallids Maritrema linguilla [11] and M. feliuli [35], the cryptogonimids Aphallus tubarium [6] and Metadena depressa [7], the pleurogenid Brandesia turgida, [25] and the opecoelid Cainocreadium labracis [36]. However, in the gorgoderids Gorgoderina vitelliloba [12] and Phyllodistomum angulatum [24], the fasciolid Fasciola hepatica [10,13], the gyrabascid Allassogonoporus amphoraeformis [30] and the azygiid Azygia lucii [24], a different kind of vitelline cytoarchitecture has been indicated, where both vitelline and interstitial cells are found surrounded by a basal matrix. In the case of the blood flukes, i.e., members of the superfamily Schistosomatoidea, which includes the Aporocotylidae, Spirorchiidae and Schistosomatidae, apart from the abovementioned aporocotylids, A. simplex [27] and two Sanguinicola spp. (present study), the ultrastructure of proper vitelline follicles has been studied in Schistosoma spp. [2,3,15]. In the Aspidogastrea, the sister group to the Digenea [21,23], observations on Aspidogaster limacoides (Aspidogastridae, Aspidogastrinae) [17,26], Rohdella amazon-ica (Aspidogastridae, Rohdellinae) [37] and Rugogaster hydrolagi (Rugogastridae) [31] indicate different vitelline patterns. The vitelline follicles of the rugogastrid R. hydrologi are isolated and surrounded by a "basal lamina and fibrous matrix" [31], whereas in the aspidogastrid A. limacoides there is no special limiting basal matrix and sarcoplasmic extensions of muscle cells penetrate deep into the follicles [26]. Based on the limited available data and the above examples of the vitelline ultrastructure in members of the Trematoda, it may be that the cytoarchitecture of the vitellarium could prove useful as an additional characteristic feature at family and lower levels. Evidence for this comes from the family Microphallidae, where the species studied both possess the same level of vitelline organization [11,35]. However, variation in the vitelline cytoarchitecture of studied members of the Aporocotylidae ( [27], present study) may reflect a lower (than family) level affiliation with the different lineages within the Aporocotylidae, as outlined in molecular studies [1].
An additional feature of the vitelline cells of Sanguinicola spp. is their ability to form long pseudopodia-like extensions of the peripheral cytoplasm, the limiting plasma membrane of which may be in contact with neighbouring vitelline cells or their sarcoplasmic extensions. Both heterologous (i.e., those occurring between adjacent membranes of vitelline cells and the sarcoplasmic processes of the muscle cells) and homologous (i.e., those occurring between adjacent membranes of vitelline cells) intercellular junctions have been shown to be present in the two Sanguinicola spp. studied. Interestingly, these intercellular junctions have been observed between vitelline cells at the same stage and at different stages of maturation. There are previous reports of the presence of heterologous and homologous contact sites between the vitelline follicles, i.e., in the monopisthocotylean monogenean Ancyrocephalus paradoxus [26], the aspidogastrid aspidogastrean Aspidogaster limacoides [26] and digeneans of the families Diplostomatidae [5], Microphallidae [11,35], Pleurogenidae [25] and Aporocotylidae ( [27], present study). The presence of intercellular junctions in digeneans possessing a proper follicular vitellarium may suggest a close relationship between vitelline cells and between vitelline and muscle cells. Despite the visible isolation of vitelline cells in freshwater aporocotylids indicated above, they maintain their intercellular interconnections due to their ability to form cytoplasmic pseudopodia-like extensions.
The present study has shown that the dominant inclusions in mature vitelline cells in both species of Sanguinicola studied herein, as in the marine Aporocotyle simplex [27], are clusters of vitelline globules. Individual vitelline globules of these aporocotylids have a heterogeneous matrix possessing central and peripheral areas of greater density (Present study, [27]). Judging from the available literature on the morphology of vitelline globules in the Digenea, similar globules have not been found in other studied taxa [2, 9-13, 24, 35, 36]. In the studied aporocotylids, the clusters are small, with a diameter of 0.5-1.0 lm, and contain 10-30 globules in the case of Sanguinicola spp. and 0.8-1.8 lm with 10-20 globules in A. simplex (Present study, [27]). Differences in the morphology, size and number of digenean vitelline clusters and globules within the clusters may be related to variations in the structure, size and number of the eggs and the size of the egg-shell [3,11,24,25,[34][35][36]. Vitelline globules represent building material for eggshell formation [34], which is deposited on the developing eggshell [14,40]. For the marine aporocotylid species, A. simplex, we have provided morphological evidence for eggshell formation from modified vitelline globules in the form of a discontinuous, thin layer (0.07 lm in thickness) of electron-dense shell material around the fertilized ovum and associated vitellocytes [27]. Taking into account the fact that the eggshell of species of Sanguinicola and Aporocotyle is very thin [16,18,27,32,33,38], this may explain the presence of a rather small number of globule clusters and globules within the clusters in their mature vitellocytes. The proportions of lipid droplets and glycogen in digenean vitelline cells, both of which represent nutritive material available to the developing embryo, vary between different digenean species [3,11,24,25,35,36,39]. Such differences may reflect variations in the retention of the eggs within host tissues or their development in the external environment, where they may survive for long periods waiting for transmission of the miracidium to the new host [2,9,10,24,35,38]. In the case of Sanguinicola, Scheuring [33], Schell [32] and Kirk and Lewis [16] studied the life-cycle in Sanguinicola spp. and showed that eggs are released into the blood, carried to the branchial arteries and arterioles and ultimately become lodged in gill capillaries. Within seven days hatched miracidia pierce the capillary walls and the gill epithelium to be liberated into the environment, where they search out their molluscan intermediate host.
It seems likely that most nutrients can pass through the thin eggshell while the eggs are developing in the blood vessels of the host. This is the first study to examine the cytoarchitecture of the vitellarium of members of the freshwater lineage of aporocotylid blood-flukes. The study highlights differences in their vitelline cytoarchitecture compared with that of a member of the marine, teleost-infecting lineage of the Aporocotylidae. Since no member of the chondrichthyan-infecting lineage of the family, which likely occupy a more basal position in the aporocotylid tree [1], has been studied, it is not possible to assess the morpho-evolutionary steps in the formation of the aporocotylid vitellarium. However, the present data do appear to provide further morphological evidence supporting the divergent evolutionary lineages of marine and freshwater, teleost-infecting members of the family. The study supports our previous belief [25,26] that the cytoarchitecture of the vitellarium represents an additional characteristic feature of the Digenea useful at family and lower taxonomic levels.