The mucosa of the intestine plays a vital role in digestive, resorptive, and metabolic processes in various animals. The fine structure of intestinal epithelium in Teleostei is investigated in sufficient detail (Odense and Bіshор, 1966; Yamamoto, 1966; Iwai, 1969; Gauthier and Landis, 1972; Noaillac-Dереyrе and Gаs, 1973, 1974, 1979; Krementz and Chapman, 1975; Stroband, 1977; Ezeasor and Stokoe, 1981; Kuperman and Kuz’mina, 1994; Horn et al., 2006; Dai et al., 2007; German, 2009; Naguib et al., 2011). In these studies, special attention was paid to the structure of enterocytes. A fundamental similarity of the ultrastructure of intestinal epithelium in fish belonging to different taxonomic groups and in other vertebrates was noted (Ugolev and Kuz’mina, 1993). It is important that the apical surface of the enterocyte membrane forms a brush border consisting of numerous finger-like prominences of the plasmatic membrane (microvilli) and adjacent thin mucopolysaccharide threads producing a network (glycocalyx).

The length of microvilli of enterocytes in different teleostean species varies, as a rule, within ~1−2 μm and varies in different parts of the fish intestine (Ugolev and Kuz’mkna, 1993). The depth of microvilli is especially variable in fishes having a long intestine, especially in representatives of the family Loricariidae (Siluriformes) feeding on wood. In these fish, the intestine length surpasses their body length 11.6–17.2 times, while that in some specimens of Hypostomus pyrineusi is by 40 times. In four investigated species of the family Loricariidae, the depth of intestinal folds and length and density of microvilli decrease distally. In three species (Panaque cf. nigrolineatus, P. nocturnus, and Hypostomus pyrineusi), the differences in length and density of microvilli in different regions of the intestine are significant, while it is not significant in Pterygoplichthys disjunctivus (German, 2009).

As a rule, the thickness of glycocalyx is up to 0.1 μm. However, this structure in fish is revealed in only a few studies (Huebner and Chee, 1978; Kuperman and Kuz’mina, 1994). It should be noted that not only the size of microvilli but also the presence of glycocalyx have fundamental significance. The microvilli are the structural base of membrane digestion and increase digestive and transporting surface of enterocytes (Ugolev, 1972, 1985; Ugolev and Kuz’mina, 1993; Kuperman and Kuz’mina, 1994). The glycocalyx separates molecules by the size and charge, contributes to movement of nutrients to the apical membrane of enterocytes, performs acceptory and receptive functions, including immune ones, and prevents penetration of bacteria and of some xenobiotics (Ugolev, 1985).

Information on the ultrastructure of intestinal epithelium of Chondrostei (Radaelli et al., 2000; Korneva and Bednyakov, 2011) and, especially, in Chondrichthyes (Wilson and Castro, 2011) is scarce. It is known that the intestine in Chondrichthyes significantly differs morphologically from that of Teleostei and is subdivided into two parts: the anterior one being a thin tube and a thicker part situated distally and characterized by the presence of a spiral valve—the so-called spiral intestine (Chatchavalvanich et al., 2006; Wilson and Castro, 2011). The wall of the spiral valve is formed by folds of intestinal mucosa and submucosa. The surface area of mucosa is increased due to the presence of mucosa folds on folds of the spiral valve situated along the longitudinal axis of the intestine and having a screw–shaped form. The number of spiral folds and depth of folds is different in different fish species (Wilson and Castro, 2011). In the shark Sphyrna tiburo, the activity of digestive hydrolases is maximum in the medial part of the spiral intestine (Jhaveri et al., 2015).

The present study is aimed at the investigation of the ultrastructure of the spiral intestine in three species of cartilaginous fish (spiny dogfish Squalus acanthias, common stingray Dasyatis pastinaca, and thornback ray Raja clavata) differing in foraging type.

MATERIALS AND METHODS

Adult specimens of spiny dogfish Squalus acanthias, common stingray Dasyatis pastinaca, and thornback ray Raja clavata are investigated (by five specimens of each species weighing 1700−2100 g) captured in the Black Sea in September–October. The spiny dogfish Squalus acanthias is a typical fish consumer feeding all year. Skates are benthos-eaters, facultative fish consumers. Their food consists of invertebrates, principally crustaceans, less so mollusks and polychaetes, as well as small fish (Nikol’skii, 1954).

Immediately after capture, the fish were immobilized, dissected, and the intestine was taken out and placed in an ice bath. For electronic-microscopic investigation, the spiral intestine was cut longitudinally and separated into three equal parts (proximal, medial, and distal). As the medial part is especially important for digestion, the principal attention was paid to investigation of the ultrastructure of the epithelium of this part. From the tops of crests of the spiral valve, three fragments were taken, immediately fixed, and considered as one sample further on. In the spiny dogfish Squalus acanthias, samples were taken from two parts of the medial part (closer by 1 cm from the center to the proximal and distal parts), from the medial and distal parts in the common stingray Dasyatis pastinaca, and from the middle of the medial part in the thornback ray Raja clavata. Fixation and subsequent processing of the material were made by the standard methods for electronic microcopy (Mironov et al., 1994). The material was fixed during 2 h in 2−5% glutaric aldehyde on phosphate buffer (рН 7.4), then in 1% solution of ОsО4 in the same buffer during 1.0−1.5 h. Dehydration was made in progressive graduated concentration of ethanol and acetone, then embedding in epon-araldite was made. Ultrathin sections (50−60 nm) were contrasted with 4% aqueous solution of uranyl acetate, stained with 1% solution of lead citrate, and examined in a JEM 100C transmissive electron microscope at 80 kV.

The data were processed statistically by means of Excel software of MS Office XP and Statistica 6.0. Significance of the results was estimated by Student’s test for small samples at р ≤ 0.05.

RESULTS

The epithelium of the spiral intestine’s medial part in the spiny dogfish Squalus acanthias consists mainly of elongated cylindrical enterocytes (Fig. 1a). The apical part of plasmolemma of enterocytes bears numerous microvilli producing the brush border (Figs. 1a, 1b). The apical part of microvilli is considerably compressed and contains fragments of glycocalyx (Fig. 1b). The membrane of microvilli is typically three-layered (the insert in Fig. 1a). In most cases, the enterocytes are situated closely to each other and the brush border is a continuous series. However, in the part situated closer to the distal part of the intestine, a clear distance is seen between enterocytes in their apical part (Fig. 1c).

Fig. 1.
figure 1

Thin structure of the intestinal epithelium of Squalus acanthias: (a) enterocytes of the medial part of the spiral intestine (in the insert is the transverse section of microvilli), (b) the apical part of the same enterocytes, (c) enterocytes of the distal part of the spiral intestine, (d) structures of an unknown nature, probably supranuclear bodies. Scale bar: (a) 3 (insert is 0.3), (b) 0.5, (c) 1, (d) 5 μm.

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Inside microvilli, the microfibrillae are seen whose threads extend to the apical part of cytoplasm where there is a weakly developed terminal network. Below, there are numerous polymorphic mitochondriae with a dense matrix, vesiculae, and vacuoles. In addition, the cytoplasm contains cysterns of granular endopalsmatic reticulum, ribosomes, and dense round bodies containing lipids (Fig. 1a). It is important to note the irregular distribution of the latter within cells and their different content in neighboring cells. The apical parts of epithelial cells are connected with various kinds of connections, including by desmosomes, separating electron-dense strands situated across enterocytes and containing lipid drops (Figs. 1a, 1b). The apical membrane looks slightly incurved. The presence of thinner and longer structures than microvilli should also be noted situated in the figure near to the nucleus (Fig. 1d). They are probably situated above the nucleus and are supranuclear bodies.

Ultrastructure of epithelium of the spiral intestine of rays is similar to that of sharks (Fig. 2). However, the cytoplasm in some cases is denser electronically and the form of enterocytes differs from that described for Squalus acanthias (Figs. 2a, 2e). In both species of rays, the enterocytes are shorter and more various in comparison with elongated cylindrical enterocytes of Squalus acanthias. The apical part of microvilli is less dense than that in Squalus acanthias but the glycocalyx is more expressed in Dasyatis pastinaca (Fig. 2d). Folds of the lateral membrane of enterocytes (Fig. 2a) and different density of distribution of microvilli are also remarkable (Fig. 2b). There are different form and size of nuclei in Dasyatis pastinaca & Raja clavata (Figs. 2c, 2d). Near to the nucleus, one can see mitochondria, granular reticulum, and Golgi apparatus.

Fig. 2.
figure 2

Thin structure of the intestinal epithelium of (a–d) Dasyatis pastinaca and of (e−f) Raja clavata: (a, e) enterocytes of the medial part of the spiral intestine, (b) the transverse section of microvilli of enterocytes, (c, f) nuclei and various organelles in the center of enterocytes from the medial part of the spiral intestine, (d) enterocytes of the distal part of the spiral intestine. Scale bar: (a, b, d, e) 1, (c, f) 2 μm.

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The available data offer a possibility to compare characteristics of microvilli belonging to the brush border of enterocytes (Table 1). The mean length of microvilli situated on apical surfaces of enterocytes of the intestine’s medial part is smaller in both cases in Squalus acanthias than in Dasyatis pastinaca (by 1.8 and 1.5 times) and in Raja clavataа (in 1.6 and 1.3 times). However, the diameter of microvilli in Squalus acanthias is greater than in rays. In Dasyatis pastinaca, the length of microvilli in the distal part of the spiral intestine is significantly smaller than in its medial part. The maximum number of microvilli in Squalus acanthias is somewhat higher than that in Dasyatis pastinaca. It is important to note the irregular distribution of microvilli in the latter. This makes an impression of the presence of empty places decreasing the quantity of microvilli to 37.

Table 1.   Size and quantity of microvilli on the apical surface of enterocytes of the spiral intestine in the spiny dogfish Squalus acanthias and rays Dasyatis pastinaca & Raja clavata
Full size table

DISCUSSION

The obtained results confirm the data not only on principal similarity of the ultrastructure of enterocytes in different fish species but also on variability of the depth of microvilli (Ugolev and Kuz’mina, 1993; Kuperman and Kuz’mina, 1994) and of electronic density of cytoplasm (Abaurrea-Equisoain and Ostos-Garrido, 1996). The surface area of mucosa in cartilaginous fish increases due to the presence of folds of mucosa on folds of the spiral valve (Wilson and Castro, 2011). Therefore, the length, diameter, and number of microvilli on the apical surface of enterocytes are important for digestion. Smaller length of microvilli in the distal part of the spiral intestine in comparison with the proximal part found in Dasyatis pastinaca well agrees with the data for Teleostei: the depth of microvilli in the posterior region is smaller than that in the anterior and middle regions of the intestine (Iwai, 1969; Noaillac-Dереyrе and Gаs, 1973; Stroband, 1977; Kuperman and Kuz’mina, 1994).

The differences in quantitative characteristics of microvilli in the spiny dogfish Squalus acanthias and rays may be related to the kind of foraging, in particular, to more chemically homogeneous food of the fish predator in comparison with that of benthos eaters. In previous investigations of the ultrastructure of intestinal enterocytes in fish differing in foraging, the greater length of microvilli was noted in the benthic-eater bream Abramis brama than in the fish predator pike Esox lucius (Kuperman and Kuz’mina, 1994). The presence of glycocalyx in all investigated species deserves special attention, especially expressed in rays, since this structure is rarely found in fish. Previously, a clearly expressed glycocalyx was found on the microvilli of enterocytes of the upper part of the intestine of a teleostean Hорlоsternum thorocatum (Huebner and Chee, 1978) and in microvilli of enterocytes of the medial region of the intestine of Lota lota (Kuperman and Kuz’mina, 1994). For representatives of Acipenseridae, it was noted that their glycocalyx is weakly developed (Korneva and Bednyakov, 2011). However, absence or slight expression of this structure might result from techniques of work, in particular a quick lysis due to retardation in fixation of the material.

The greater distance between enterocytes in their distal part is similar to that previously found in investigations of the ultrastructure of the brush border of enterocytes of Neogobius melanostomus. Since this distance is observed at the level of microvilli in a representative of teleosteans, this permitted the characterization of this distribution as bush-like (Ugolev, 1972). In both cases, this phenomenon is by all probability related to persorbtion—the transport of macromolecules over intercellular spaces.

The enterocytes with electron dense cytoplasm found in rays do not differ by principal ultrastructural characteristics from typical enterocytes. Earlier, the presence of enterocytes of two types was described for Anguilla anguilla (Abaurrea-Equisoain and Ostos-Garrido, 1996) and for representatives of acipenserids (Korneva and Bednyakov, 2011). The latter authors did not find any significant distinctions in the structure of cells of this type and the observed differences in cell morphology explained by their physiological state.

For teleosteans and chondrosteans, the simple contacts, desmosomes, as well as dense and intermediate contacts, were described (Kapoor еt аl., 1975; Kuperman and Kuz’mina, 1994; Korneva and Bednyakov, 2011). In addition, the simple and multidesmosomal contacts were described at the level of the terminal network (Yamamoto, 1966). In sharks and, less so, in rays various contacts are found between lateral membranes of enterocytes in the apical parts of cells, in particular, desmosomes. It is assumed that that they perform connections between neighboring cells. In the present study, the electron dense strands situated across enterocytes connected to desmosomes and containing lipid drops are found. By all probability, they contribute to rapid penetration within cells of nutrients via intercellular spaces. It seems that the presence of contacts of a different type depends on the physiological state of the investigated animals (Kuperman and Kuz’mina, 1994).

The cytoplasm of enterocytes of intestinal mucosa in the investigated fish contains organelles similar to those of other fish species (Yamamoto, 1966; Noaillac-Dереyrе and Gаs, 1973; Kapoor еt аl., 1975; Stroband, 1977; Kuperman and Kuz’mina, 1994; Khojasteh еt аl., 2009; Korneva and Bednyakov, 2011; Khadse and Gadhikar, 2017). However, the quantity and position of different organelles in cartilaginous and teleostean fish may be different depending on the functional state of the digestive system (Yamamoto, 1966; Gauthier and Landis, 1972; Noaillac-Dереyrе and Gаs, 1973, 1979; Olsen еt аl., 1999). In contrast to teleosteans, the terminal network in the terminal part of enterocytes in chondrosteans (Acipenser ruthenus,Huso huso, and in their hybrids H. huso × A. ruthenus and A. ruthenus × H. huso) is moderately developed (Korneva and Bednyakov, 2011; Bednyakov, 2014).

The presence of vesicles and vacuoles in the apical part of cytoplasm of enterocytes near the plasmatic membrane may be related to the presence of pinocytosis (Yamamoto, 1966; Iwai, 1969; Gauthier and Landis, 1972; Noaillac-Dереyrе and Gаs, 1973, 1979; Kuperman and Kuz’mina, 1994). In Teleostei, pinocytosis is accompanied by invagination of the membrane and is related to transport of fat and protein that are hydrolyzed in intracellular digestion. It is important to note that fat is transported in more proximal parts and protein in more distal parts of the intestine (Noaillac-Dереyrе and Gаs, 1973). The electron-dense strands connected to desmosomes found in Squalus acanthias situated across enterocytes and containing drops of lipids may be related to the consumption of fatty food. By all probability, they contribute to more rapid penetration of nutrients, getting via intercellular spaces inside cells in autumn when the fat content of teleosteans—food items of Squalus acanthias—increases (Shulman, 1972). This supposition needs further experimental checking.

Thus, the ultrathin structure of the apical part of enterocytes in the investigated Chondrichthyes fish is close to that of fish belonging to other systematic groups. At the same time, the differences in Squalus acanthias and in rays are found in the form of enterocytes, the size of microvilli, and in the quantity and distribution of organelles that might be related to differences in foraging of these fish species.

ACKNOWLEDGMENTS

The study was supported by Federal Agency for Scientific Organizations (FASO), project no. АААА-А18-118012690102-9.

COMPLIANCE WITH ETHICAL STANDARDS

Conflict of interests. The authors declare that they have no conflict of interest.

Statement on the welfare of animals. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.