The study presents fine structure data on the ovipositor of four bitterling species (Rhodeus ocellatus, Rhodeus amarus, Paratanakia himantegus and Acheilognathus barbatulus). In A. barbatulus and P. himantegus, ovipositor structure has never been studied. Novel data on the structure of the ovipositor were collected using two complementary methods, light- and transmission electron microscopy. The new findings relate to the covering and lining epithelia, basal laminae, cell junctions and supporting/connective tissue layer. All examined fish shared the same basic ovipositor structure and the newly reported details, regardless of their species affiliation. Evaluation of structure modifications related to the passage of eggs through the ovipositor revealed a range of transitional tissue changes, corroborating their presumable role inferred from the study of fine structure.
Bitterling fishes (Acheilognathinae, Cyprinidae) parasitise unionid mussels by laying eggs into their gills. A characteristic ovipositor, an accessory organ of the female gonads, facilitates this unusual reproductive strategy. Ovipositor length varies cyclically and may reach over 50 mm on the day of spawning, shortening to approximately 20% of its length outside the reproductive period (Kitamura 2007). The changes in ovipositor length occur rapidly, typically overnight (i.e. within a few hours). During the reproductive period, the ovipositor is a long, flaccid tube that waves along the female body as she swims. To lay the eggs, the female inserts the proximal part of the ovipositor into the exhalant siphon of the mussel and sweeps quickly forward and down. During this action, the eggs are forced through the ovipositor, which stiffens and unfurls deep into the mussel's gills as the eggs travel down. When the eggs are deposited (in less than one second after ovipositor insertion for a clutch of 2-10 eggs), the female rises up and withdraws her flaccid ovipositor (Smith et al. 2004).
The opinions on the structure of the bitterling ovipositor and the terms used in its description have varied from the early descriptions (Shirai 1964) to recent accounts by (Khlopova & Varaksin 2011, Khlopova et al. 2011, Khlopova & Kul'bachnyi 2013). The study of Rhodeus ocellatus ovipositor histology by Shirai (1964) and a hypothesis about the role of the urinary bladder in female ovulation (Bretschneider & Duyvené de Witt 1947) influenced an elaborated study design by Matsubara (1994), which included a description of ovipositor structure. The experimental results of the role of the urinary bladder in bitterling oviposition were convincing but were not, unfortunately, accompanied by an adequate histological documentation. Problematic evidence included especially the presence of a single unpaired ovary in R. ocellatus, the description of the stratified epithelium lining the lumen of the ovipositor, and the lack of muscle tissue in the ovipositor. The weak points of Matsubara's (1994) results can be attributed to the histological techniques of that time, especially the thickness of paraffin sections examined.
The histological study of Korean oily bitterling Acheilognathus koreensis ovipositor by Park & Kim (2006) compared the structure of “inner and outer ovipositor” in the females in the breeding and non-breeding season and, surprisingly, searched for analogies in the histology of the ovipositor and rectum. Lamina muscularis, well developed in the “inner ovipositor”, was considered the structure responsible for rhythmic contractions propelling mature oocytes to the “outer ovipositor”, a passive transmitting tube since it lacks lamina muscularis.
The most comprehensive histological study of bitterling ovipositors published to date by Khlopova and co-authors (Khlopova et al. 2011, Khlopova & Varaksin 2011, Khlopova & Kul'bachnyi 2013) complemented the findings of previous authors with data on six bitterling species. Unfortunately, the authors mainly used their own descriptive terms and burdened their studies with measurements of cells taken from paraffin sections. Despite this, their studies provided new data and highlighted structural similarities among species. Nevertheless, they also demonstrated the limits of paraffin histology in the study of delicate structures.
The only attempt to study ovipositor structures in detail using electron microscopes (transmission and scanning electron microscopy were performed by Jin et al. 2009), who used only a tiny excision from the mid portion of the ovipositor tube for comparison of 20 striped bitterling (Acheilognathus yamatsutae) females collected during spawning season.
The aim of our contribution is (i) to present a condensed and well-documented study of the fine structure of ovipositors in four bitterling species, (ii) to align descriptive terminology to the standard in textbooks of general histology, and (iii) to evaluate a possible functional relevance of the described structures.
Material and Methods
Material from 19 individuals of four bitterling species, R. ocellatus (9), Rhodeus amarus (5), Paratanakia himantegus (3) and Acheilognathus barbatulus (2), was used in the study. They were selected to represent phylogenetic diversity (three genera) and include a pair of closely related species (Rhodeus species) from a set of available captive species. All fish were from captive populations and were collected from outdoor experimental tanks during their reproductive season. Fish were in spawning condition (with fully extended ovipositors), except in two cases (one R. amarus, one P. himantegus, with shorter ovipositors) when fish were purposely selected to better visualise internal structure in the proximal ovipositor part (conical organ). Material designated for paraffin histology was fixed either with 10% neutral buffered formalin (NBF) or Davidson's fixative. Histological sections routinely stained with haematoxylin and eosin (H & E) and selectively with Van Gieson, Masson's trichrome, Verhoeff and Grocott methods were examined using an Olympus BX60 microscope fitted with a digital camera. As a minimum, 20 semi-serial sections were examined from each paraffin block. For transmission electron microscopy, the ovipositors were fixed with 2% osmium tetroxide in its whole to prevent damage to their structures when cut in pieces when fresh. An ascending series of acetone (70 to 100 %) was used for dehydration, and pure acetone for impregnation mixtures of Spurr resin (2:1, 1:1). Ovipositors were cut in pieces at the impregnation phase before embedding in Spurr resin. The individual segments (five in short ovipositors and up to ten in long ones) were divided into two subsamples, of which the anterior (proximal) one was used for transverse and the posterior (distal) one for longitudinal semithin and ultrathin sectioning. Semithin sections were stained with toluidine blue, and ultrathin sections were double stained (with uranyl acetate and led citrate) and examined with a Jeol 1400 microscope fitted with a digital camera. Selected ovipositor samples from each fish were documented with more than 100 electronograms, the details of which were carefully compared.
Results and Discussion
The macroscopic appearance of ovipositors of different bitterling species suggests variability in the structure of this organ across bitterling species. However, our data on four species demonstrate broad similarities across bitterling lineages. This finding prompted us to summarise fine structure data for all our study species into a single account.
The ovipositor has two parts, the proximal of which is a robust structure called the conus (conical organ sensu Khlopova et al. 2011, Khlopova & Kul'bachnyi 2013). The distal part is delicate and protrudes as a free tube of narrowing diameter when relaxed. The principal difference between the structure of these two parts is the presence/absence of muscle tissue; however, there is no sharply delimited transition point.
Proximal/conus part of ovipositor
The conus/conical organ is a prominent structure protruding from the belly of the female. It contains the rectum and the most proximal part of the ovipositor. Both these tubes are surrounded by striated muscle tissue, the rectum up to the anal opening, whereas the ovipositor is only in the most proximal part of the conus (Figs. 1-4). The oviduct (examined in spawning season) forms multiple folds close to its confluence with the tube of the proximal part of the ovipositor (Figs. 1, 5). Oviduct folds probably have a similar function as the infundibulum in some other fish species. The epithelium lining oviduct folds has differentiated apical cell domains. The nuclei are in a subapical position, an unusual phenomenon in lining epithelia (Fig. 5). The conspicuous apical/surface structures resemble those presented mostly as stereocilia, although the presence of kinetosomes (typical of cilia) is known neither in mammals nor in teleosts. The term stereovilli is appropriate for this apparently stable differentiation of the oviduct epithelial cell surface.
Although the recent structural studies (Khlopova et al. 2011, Khlopova & Varaksin 2011, Khlopova & Kul'bachnyi 2013) did not cite the study by Matsubara (1994), his opinion on the involvement of the urinary bladder (Fig. 6) in the oviposition process may not be dismissed with no further consideration. Matsubara (1994) believed that egg movement is principally triggered by the pressure of urine accumulated in the overfilled bladder. Based on the assumption that the ovipositor lacks a muscle layer, the rigidity of the ovipositor for its insertion into the mussel siphon was considered a result of urine pressure. There is no doubt about the assisting role of urine in its coincident passage with eggs through the ovipositor. However, the principal role of the bladder in triggering egg transport via the pressure of urine remains debatable. Details of the fine structure of the ovipositor, including the presence of striated muscles (capable of inducing peristaltic waves) in the conus part of the ovipositor, rich innervation of the conus as well as supporting tissue in the distal part of ovipositor contradict Matsubara's hypothesis (Matsubara 1994). The urinary bladder overfilling, taken by Matsubara as an indicator of its role in egg shedding, could result from temporary urine retention due to mechanical obstruction in its outflow. Our study of the ovipositor structure has limited power to contribute to understanding the cause of urine accumulation, its effect and possible function. Urine in the overfilled bladder could activate pressure sensors that initiate bladder emptying and help in egg deposition. However, a structural study like ours cannot clarify the cause of urine accumulation and would require a detailed study of blood and urine composition from physiological and endocrinological aspects. However, sampling blood and urine with the appropriate timing is likely technically challenging.
The distal part of the ovipositor
The wall of the distal tubular part of the ovipositor consists of three easily distinguishable tissue layers: the outermost layer of stratified epithelium (SE), the mid layer formed by supporting/connective tissue (CT) and the layer of the epithelium lining the lumen (L) (Figs. 1, 3).
The covering epithelium is of squamous type. It has one row of typical squamous cells (Fig. 7), the profiles of which are not always seen flat in ultrathin sections. Squamous cells exhibit micro protrusions that form part of the surface pattern known as “fingerprints” that have been documented in 3D views several times (Hibiya 1982, Yasutake & Wales 1983, Jin et al. 2009, Elliot 2011). They are considered important in keeping mucus on the surface of the fish epidermis. The mucus-secreting cells (Fig. 7) are scattered among squamous cells almost regularly. Deeper layers of the covering epithelium consist of cells that differ substantially from the squamous epithelia in having tonofilaments as a fundamental component of cytoplasm (Fig. 8). Considerable concentration of tonofilaments causes displacement of cell organelles and their concentration around the cell nuclei. Tonofilaments, uniform in size and density, closely resemble those documented in the epidermis of larval Petromyzon fluviatilis (Fawcett 1966). The primary function of the extensive internal cytoskeleton is, in our opinion, to reinforce the suprabasal cells of squamous epithelium by increasing their tensile strength.
The observation of cell-to-cell attachment revealed cell junctions of the common types (i.e. desmosomes, hemidesmosomes or gap junctions) but also numerous lateral interdigitations within cells of stratified epithelium (Fig. 9). Despite that, there is no solid base to quantify whether or not lateral infoldings of these cells are more numerous than in other stratified epithelia (epidermis of the body), it is evident that the covering epithelium of ovipositors also is subjected to traction and pressure. Interdigitation seemingly facilitates shape changes of epithelial cells during oviposition.
The suprabasal layer of stratified epithelium contains, in addition to common migrating cells (Fig. 10), individually scattered cells morphologically identifiable with Langerhans cells. They were found in all bitterling species examined, most frequently in A. barbatulus. Langerhans cells are well known as residents of the epidermis in humans and murine mammals. They have also been recognised in fish (Lovy et al. 2006, 2008, Alesci et al. 2020) by their specific organelles called the Birbeck granules (Fig. 10). These are considered to be specialised antigen-presenting and processing cell compartments identifiable by expression of the type II lectin Langerin CD207 (Valladeau et al. 2003, Lovy et al. 2009). Birbeck granules of Langerhans cells detected in bitterlings (Fig. 10) do not differ substantially from those considered morphological markers of Langerhans cells in mammals (Wolf 1967) and those documented by Lovy et al. (2006, 2008, 2009) in salmonids.
Basal cells of stratified epithelium are firmly attached to the underlying basal lamina, which does not run straight but follows the highly folded surface of basal cells (Fig. 11). Filaments of hemidesmosomes are arranged in parallel and follow the contour of the basal cell membrane. Fine anchoring filaments radiate in parallel from the outer aspect of the cell membrane into the basal lamina. A layer of collagen fibrils connects basal lamina to underlying connective tissue. All types of cell junctions, including hemidesmosomes, provide the covering epithelium of the ovipositor with structural continuity and mechanical strength/resistance.
Considering that both the plane of sectioning and thickness of paraffin sections much affect the dimensions of individual cell profiles, we do not consider it appropriate to identify cell “types” within squamous epithelium based on their dimensions taken from histological sections (Khlopova & Varaksin 2011). We respect definitions of basic types of tissues, including features characteristic of the covering, lining and glandular epithelia. The basic definition of multi-layered squamous epithelium considers different shapes and sizes of cells in basal, suprabasal and superficial layers.
The connective/supporting/tissue layer of ovipositor belongs (according to the generally accepted rules for classification of connective tissues) to the loose subtype characterised by low cellular density, loosely arranged fibres and abundant ground substance. Supporting cells are fibroblasts responsible for synthesising and maintaining an extracellular matrix composed of ground substance and organised bundles of fibrils (Figs. 12, 13). Collagen fibres with cross banding pattern characteristic of Type I collagen that display approximately the same periodicity as those described in humans are the most frequent. Much thinner elastin fibres are less frequent (Fig. 13). Longitudinal paraffin sections of the ovipositor taken in its luminal plane revealed the presence of connective tissue with a longitudinal arrangement in a layer parallel with the lumen (Figs. 1, 3, 5). The signs of a more or less circular organisation of much looser connective tissue were observed in longitudinal sections taken off the luminal plane (Fig. 3). This organisation is relevant to the function of the ovipositor and corroborates Park & Kim's (2006) opinion that there is a certain similarity with the structure of the digestive tube, also equipped with longitudinal and circular layers of tissue. We assume that this arrangement of connective tissue permits a wave produced by conus muscle contraction to pass along the ovipositor tube, propelling the eggs forward. The opinion expressed in the relevant literature on the presence of muscle tissue in distal ovipositors differs. Park & Kim (2006) claim the absence of muscle tissue in the “outer ovipositor”. In contrast, Jin et al. (2009) report the presence of “some muscle fibers” within the connective tissue of a short segment of the distal ovipositor. The extensive studies by Khlopova et al. (2011) and Khlopova & Kul'bachnyi (2013) are mainly focused on the proximal, conus part of the ovipositor, where the presence of muscle tissue is evident (Figs. 1, 4) but do not inform about the distal ovipositor. Despite using selective Van Gieson and Masson's trichrome staining to distinguish connective- from muscle tissue and having supplemented these methods with an evaluation of ultrathin sections, we failed to find muscle tissue in the distal part of the ovipositor. Since Park and Kim's (2006) ultrastructural study did not document the presence of muscle tissue, it would be worthwhile to try and verify this conclusion by immunohistochemical reaction using, for example, anti-alpha smooth muscle actin.
Transverse and longitudinal paraffin sections also provided basic information on the vasculature of the ovipositor connective tissue layer. Long vessel segments running parallel to the long axis of the distal ovipositor (Figs. 4, 14) were observed to supply the ovipositor up to its most distal point. Blood vessels seen in ultrathin sections of the loose connective tissue have a thick layer of endothelium embraced with pericytes or a thin layer of adventitia and bundles of embracing collagen fibrils (Fig. 14). Semi- and ultrathin sections through thin-walled vessels of various diameters revealed a surprising absence of erythrocytes and other blood cells (Figs. 4, 14). The cell-free content of blood vessels indicates that the ovipositor circulatory system regulates the exchange of fluid between plasma and extracellular space, which is essential for morphological changes in the ovipositor tissues, including those participating in the erection of the ovipositor tube. Unfortunately, molecular components of cell-free content of blood vessels cannot be demonstrated by the light- or electron microscope. As for the conus part of the ovipositor, the functional implications of the main blood vessels remain obscure. Another method (probably corrosive) will have to be used to clarify this problem.
The supporting tissue framework of the distal ovipositor is equipped with numerous myelinated and non-myelinated nerve fibres. Non-myelinated small-diameter axons are simply enveloped in the cytoplasm of Schwann cells. In contrast, large-diameter nerve fibres are wrapped in concentric layers of Schwann cell plasma membranes, forming a myelin sheath (Fig. 15). In general, conduction velocity is much higher in myelinated axons compared with those non-myelinated of the same diameter. Cross sections through large peripheral nerves were seen in the conus part of the ovipositor.
Our study of the ovipositor fine structure did not substantially contribute to recognising pigment cell organisation in this organ. The specific distribution of pigment cells is visible to the naked eye, and taxonomists made its macroscopic descriptions part of generic/species definitions. Dark pigmented strips seen in histological sections of ovipositor supporting tissue were subjected to a bleaching procedure (Bancroft & Layton 2019) which proved the presence of melanin pigment. The latter was seen in ultrathin sections as electron-dense ellipsoidal granules (melanosomes) accumulated in the cytoplasm of melanocytes (Fig. 16). Other deposits of electron-dense material, seen in cells resembling fibroblasts, could not be safely determined. It could not be excluded that these structures were dendritic processes of melanophores.
Lining epithelium in the distal ovipositor
The thinnest paraffin sections, as well as semithin and ultrathin resin sections, revealed that a simple layer of cylindrical or cuboidal epithelial cells lines the lumen of the distal ovipositor. The luminal surface of these cells possesses conspicuous protrusions (Fig. 17). Low power magnifications show that these cell protrusions do not form a dense layer and that their length is different within longitudinally sectioned ovipositor segments. Microfilaments form the core of the protrusion body. Tips of the protrusions are coated with radiating delicate pili-like structures. High magnifications revealed the absence of kinetosomes and axial filament complexes, which precludes the assignment of the protrusions to the category of motile cell processes, i.e. they are neither cilia nor flagella. Instead, their fine structure qualifies them as stereovilli associated with undulatory movement and the presence of liquids. In general, they represent transient differentiation associated with the surface activity of epithelial cells.
Stereocilia/stereovilli of the inner ear and lateral line cells of fishes were used as model structures for highly sophisticated studies of mechanotransductive receptors and molecular signalling (Monroe et al. 2015). Our finding of stereovilli differentiation in the lining epithelium of bitterling ovipositors may be utilised in general studies as a convenient source to obtain study material.
Within the epithelium lining the lumen of the distal ovipositor, we detected rodlet cells with fine structure features well known in many other teleosts (Fig. 18), including cells with signs of degradation. This finding complements the long list of fish species with rodlet cell records published by Manera & Dezfuli (2004), who summarised a vast number of studies that differ in the interpretation of the nature and function of rodlet cells. Currently, rodlet cells are designated as inflammatory cells whose role is similar to that of eosinophil granule cells. Due to their reaction to many environmental stimuli, the latter are considered potential biomarkers.
Our data demonstrate the shared fine structure of the ovipositors of four species (which come from three bitterling lineages). We described how particular layers and their cellular and subcellular structure contribute to the functional significance of the bitterling ovipositor.
List of abbreviations
A – squamous cell
AB – Acheilognathus barbatulus
B – mucus cell
BV – blood vessel
C – cell with tonofilaments
CF – collagen fibrils/fibres
CT – connective tissue
E – egg
EC – epithelial cell
L – lumen of ovipositor
N – nucleus
O – ovipositor
OF – oviduct fold
OV – oviduct
P – pericyte
PH – Paratanakia himantegus
PN – perineurium
R – rectum
RA – Rhodeus amarus
RO – Rhodeus ocellatus
SC – Schwann cell
SE – stratified squamous epithelium
SM – striated muscles
U – urinary tract
UB – urinary bladder
Funding for this study came from the Czech Science Foundation (21-00788X). The authors wish to express their thanks for the opportunity to work in the Laboratory of Electron Microscopy at the Biological Centre of the Czech Academy of Sciences, which takes part in Czech-Bioimaging, supported by the Ministry of Education, Youth and Sports, Czech Republic (Project no. LM2015062). The care and use of experimental animals complied with Czech and EU animal welfare laws, guidelines and policies as approved by the ethical committee of the Ministry of Education (MSMT 18809/2019-5, individual licence: CZ01285).
I. Dyková and M. Reichard conceived and designed the study. I. Dyková analysed and interpreted data. M. Reichard provided material. I. Dyková drafted the manuscript. I. Dyková and M. Reichard prepared the final version of the text.