Organization of the blood system of rhynchonellid brachiopod Hemithiris psittacea (Brachiopoda: Rhynchonelliformea).

The brachiopods are sessile invertebrates with an unusual blood system, which consists of a long-branched dorsal vessel. It is still unknown how blood circulates in this system. In the present study, for the first time we propose the circulation of blood in brachiopod Hemithiris psittacea based on morphological and experimental data. The main heart is located on the dorsal side of the stomach and divides the dorsal vessel into anterior and posterior parts. The anterior part enters the lophophore, where it gives off blind branches to each tentacle. The posterior part passes by the funnels of the nephridia and forms a blindly closed network in the gonads. We suggest that the circulation of blood includes three successive stages. During the first phase of systole of the main heart, blood flows through the anterior dorsal vessel. During the second phase of systole, blood flows through the posterior dorsal vessel. During diastole, blood flows from the anterior and posterior vessels and fills the main heart. The origin of a peculiar blood system in brachiopods can be explained by reduction of the ventral vessel, which is probably correlates with the reduction of the ventral side of the brachiopod ancestor's body. Another peculiarity of brachiopod blood system is the presence of an ampullar heart, which functions as a blood depot and allows blood to move in the vessels in two directions in an oscillatory mode. The brachiopod blood system contains vessels lacking true endothelium and can be classified as an "incompletely closed" type.

Structure of the oral tentacles of early ontogeny stage in brachiopod Hemithiris psittacea (Rhynchonelliformea, Rhynchonellida).

Brachiopods have the most complex lophophore in comparison with other lophophorates, i.e., phoronids and bryozoans. However, at early ontogenetic stages, brachiopods have a lophophore of simple morphology, which consists of the oral tentacles. Data on the ultrastructure of the oral tentacles is mostly missing. Nonetheless, it has recently been suggested that the structure of oral tentacles is ancestral for all lophophorates in general, and for brachiopods in particular. The fine structure of the oral tentacles in the brachiopod Hemithiris psittacea is studied using light microscopy, transmission and scanning electron microscopy, cytochemistry and confocal laser scanning microscopy. The oral tentacles have a round shape in transverse section, and four ciliary zones, i.e., one frontal, two lateral, and one abfrontal. Latero-frontal sensory cells occur among the frontal epithelium. Four basiepithelial nerves in the ciliary epithelium are colocalized with ciliary zones. Lophophores of simple morphology in phoronids and brachiopods are characterized by non-specified round forms of tentacles. In phoronids and bryozoans, tentacles have additional latero-frontal ciliary zones that function as a sieve during filtration. In most brachiopods, lateral cilia are involved in the capture of food particles, whereas latero-frontal cells are retained in the frontal zone as sensory elements. The oral tentacles of H. psittacea contain a coelomic canal and have distinct frontal and abfrontal longitudinal muscles, which are separated from each other by peritoneal cells. A similar structure of tentacle muscles occurs in all bryozoans, whereas in phoronids, the frontal and abfrontal tentacle muscles are not separated by peritoneal cells. We suggest that the lophophorates' ancestor had tentacles, which were similar to the tentacles of some phoronids with lophophore of simple morphology. We also assume that the structure of the oral tentacles is ancestral for all brachiopods and the specialization of brachiopod tentacles correlates with the appearance of the double row of tentacles.

Tentacle muscles in brachiopods: Ultrastructure and relation to peculiarities of life style.

Although the morphology of the brachiopod tentacle organ, the lophophore, is diverse, the organization of tentacles has traditionally been thought to be similar among brachiopods. We report here, however, that the structure of the tentacle muscles differs among brachiopod species representing three subphyla: Lingula anatina (Linguliformea: Linguloidea), Pelagodiscus atlanticus (Linguliformea: Discinoidea), Novocrania anomala (Craniiformea), and Coptothyris grayi (Rhynchonelliformea). Although the tentacle muscles in all four species are formed by myoepithelial cells with thick myofilaments of different diameters, three types of tentacle organization were detected. The tentacles of the first type occur in P. atlanticus, C. grayi, and in all rhynchonelliforms studied before. These tentacles have a well-developed frontal muscle and a small abfrontal muscle, which may reflect the ancestral organization of tentacles of all brachiopods. This type of tentacle has presumably been modified in other brachiopods due to changes in life style. Tentacles of the second type occur in the burrowing species L. anatina and are characterized by the presence of equally developed smooth frontal and abfrontal muscles. Tentacles of the third type occur in N. anomala and are characterized by the presence of only well-developed frontal muscles; the abfrontal muscles are reduced due to the specific position of tentacles during filtration and to the presence of numerous peritoneal neurites on the abfrontal side of the tentacles. Tentacles of the first type are also present in phoronids and bryozoans, and may be ancestral for all lophophorates.

The nervous system of the most complex lophophore provides new insights into the evolution of Brachiopoda.

The lophophore is a tentacle organ unique to the lophophorates. Recent research has revealed that the organization of the nervous and muscular systems of the lophophore is similar in phoronids, brachiopods, and bryozoans. At the same time, the evolution of the lophophore in certain lophophorates is still being debated. Innervation of the adult lophophore has been studied by immunocytochemistry and confocal laser scanning microscopy for only two brachiopod species belonging to two subphyla: Linguliformea and Rhynchonelliformea. Species from both groups have the spirolophe, which is the most common type of the lophophore among brachiopods. In this study, we used transmission electron microscopy, immunocytochemistry, and confocal laser scanning microscopy to describe the innervation of the most complex lophophore (the plectolophe) of the rhynchonelliform species Coptothyris grayi. The C. grayi lophophore (the plectolophe) is innervated by three brachial nerves: the main, second accessory, and lower. Thus, the plectolophe lacks the accessory brachial nerve, which is typically present in other studied brachiopods. All C. grayi brachial nerves contain two types of perikarya. Because the accessory nerve is absent, the cross nerves, which pass into the connective tissue, have a complex morphology: each nerve consists of two ascending and one descending branches. The outer and inner tentacles are innervated by several groups of neurite bundles: one frontal, two lateral, two abfrontal, and two latero-abfrontal (the latter is present in only the outer tentacles). Tentacle nerves originate from the second accessory and lower brachial nerves. The inner and outer tentacles are also innervated by numerous peritoneal neurites, which exhibit acetylated alpha-tubulin-like immunoreactivity. The nervous system of the lophophore of C. grayi manifests several evolutionary trends. On the one hand, it has undergone simplification, i.e., the absence of the accessory brachial nerve, which is apparently correlated with a reduction in the complexity of the lophophore's musculature. On the other hand, C. grayi has a prominent second accessory nerve, which contains large groups of frontal perikarya, and also has additional nerves extending from the both ganglia to the medial arm; these features are consistent with the complex morphology of the C. grayi plectolophe. In brachiopods, the evolution of the lophophore nervous system apparently involved two main modifications. The first modification was the appearance and further strengthening of the second accessory brachial nerve, which apparently arose because of the formation of a double row of tentacles instead of the single row of the brachiopod ancestor. The second modification was the partial or complete reduction of some brachial nerves, which was correlated with the reduced complexity of the lophophore musculature and the appearance of skeletal structures that support the lophophore.

The Modulation of Functional Status of Bovine Spermatozoa by Progesterone.

The aim of this study is to identify the effects of progesterone (PRG) on the capacitation and the acrosome reaction in bovine spermatozoa. The fresh sperm samples were incubated with and without capacitation inductors (heparin, dibutyryl cyclic adenosine monophosphate (dbcAMP)), hormones (prolactin (PRL), PRG), inhibitors of microfilaments (cytochalasin D) and microtubules (nocodazole) during capacitation and acrosome reactions. The functional status of spermatozoa was examined using the chlortetracycline assay. Supplementation of heparin stimulated capacitation in the presence and absence of PRG. Cytochalasin D blocked the stimulating effect of heparin on capacitation. The addition of PRL during capacitation (without PRG) did not affect the functional status of spermatozoa, while in PRG-treated cells PRL stimulated the acrosome reaction. PRL (with and without PRG) increased the acrosome reaction in capacitated cells. These PRL-dependent effects were inhibited by nocodazole. During the acrosome reaction, in presence of dbcAMP, PRG decreased the proportion of acrosome-reacted cells compared to PRG-untreated cells. This effect in PRG-treated cells was canceled in the presence of nocodazole. In conclusion, PRG under the action of PRL and dbcAMP determines the changes in the functional status of native sperm cells, which indicates PRG modulating effect on the indicators of post-ejaculatory maturation of spermatozoa.

Patients with functional bowel disorder have disaccharidase deficiency: A single-center study from Russia.

BACKGROUND: Functional bowel disorder (FBD) may be caused by a decrease in disaccharidase activity. Thus, the timely diagnosis of disaccharidase deficiency could lead to a better prognosis in patients with this condition. AIM: To determine the potential value of intestinal disaccharidases glucoamylase, maltase, sucrase, and lactase in understanding the etiology and pathogenesis of FBD. METHODS: A total of 82 FBD patients were examined. According to the Rome IV criteria (2016), 23 patients had diarrhea-predominant irritable bowel syndrome (IBS), 33 had functional diarrhea, 10 had constipation-predominant IBS, 4 had functional constipation, and 12 had mixed IBS. The Dahlqvist method was used to measure disaccharidase activity in the brush-border membrane of mature enterocytes of the small intestine, in duodenal biopsies obtained during esophagogastroduodenoscopy. RESULTS: Lactase deficiency was detected in 86.5% of patients, maltase deficiency in 48.7%, sucrase deficiency in 50%, and glucoamylase deficiency in 84.1%. The activities of all enzymes were reduced in 31.7% of patients, and carbohydrase deficiency was detected in 63.5% of patients. The low activity of enzymes involved in membrane digestion in the small intestine was found in 95.2% of patients. CONCLUSION: In 78 of the 82 patients with FBD, gastrointestinal symptoms were associated with disaccharidase deficiency.

In vitro Regeneration of Clematis Plants in the Nikita Botanical Garden via Somatic Embryogenesis and Organogenesis.

The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars 'Alpinist,' 'Ay-Nor,' 'Bal Tsvetov,' 'Crimson Star,' 'Crystal Fountain,' 'Kosmicheskaya Melodiya,' 'Lesnaya Opera,' 'Madame Julia Correvon,' 'Nevesta,' 'Nikitsky Rosovyi,' 'Nikolay Rubtsov,' 'Serenada Kryma,' and 'Vechniy Zov' were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3-0.4% Cl2 (15 min), and 1% thimerosal (10 min), 1-cm long segments with a single node were introduced to an in vitro culture. The explants were established on the basal MS medium supplemented with BAP (2.20-8.90 µM) and 0.049 µM NAA, or TDZ (3.0; 6.0, and 9.0 µM) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 µM BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 µmol m-2 s-1, at a temperature of 24 ± 1°C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 µM BAP, or 6.0 µM TDZ were optimal cytokinin concentrations for micropropagation. The explants of 'Alpinist,' 'Ay-Nor,' 'Crimson Star,' 'Crystal Fountain,' 'Nevesta,' and 'Serenada Kryma' cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 µmol m-2 s-1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 µM BAP with 0.09 µM IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in 'Crystal Fountain' (100%), 'Crimson Star' (100%), 'Nevesta' (97%), and 'Ay-Nor' (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.

Spermatogenesis in the deep-sea brachiopod Pelagodiscus atlanticus and the phylogenetic significance of spermatozoon structure.

Details of spermatogenesis and sperm organization are often useful for reconstructing the phylogeny of closely related groups of invertebrates. Development in general and gametogenesis in particular usually differ in shallow water and deep-sea invertebrates. Here, the spermatogenesis and ultrastructure of sperm were studied in the deep-sea brachiopod Pelagodiscus atlanticus. The testes of P. atlanticus are voluminous sacs located along the lateral sides of the body. Germ cells develop around the blood capillaries, contact the basal lamina, and contain germ plasm, numerous mitochondria, Golgi apparatus, lipid droplets, and centrioles of the rudimentary cilium. During spermatogenesis, several proacrosomal vesicles appear at the posterior pole of the cell; these vesicles then fuse and migrate to the anterior pole. The spermatozoon has a head with an acrosome, nucleus, eight mitochondria, proximal and distal centrioles orthogonally arranged, and a long tail. Comparative analysis suggests that the spermatozoon of P. atlanticus can be considered the most ancestral among all brachiopods. Such an organization indicates that fertilization is external in this deep-sea species. Spermatozoa of other brachiopods should be regarded as derived from this ancestral type. The transformation of brachiopod spermatozoa might have occurred in three different ways that correspond to the three main clades of recent brachiopods: Linguliformea, Craniiformea, and Rhynchonelliformea.

The first data on the innervation of the lophophore in the rhynchonelliform brachiopod Hemithiris psittacea: what is the ground pattern of the lophophore in lophophorates?

BACKGROUND: The nervous system in brachiopods has seldom been studied with modern methods. An understanding of lophophore innervation in adult brachiopods is useful for comparing the innervation of the same lophophore type among different brachiopods and can also help answer questions about the monophyly of the lophophorates. Although some brachiopods are studied with modern methods, rhynchonelliform brachiopods still require investigation. The current study used transmission electron microscopy, immunocytochemistry, and confocal laser scanning microscopy to investigate the nerve system of the lophophore and tentacles in the rhynchonelliform Hemithiris psittacea. RESULTS: Four longitudinal nerves pass along each brachium of the lophophore: the main, accessory, second accessory, and lower. The main brachial nerve extends at the base of the dorsal side of the brachial fold and gives rise to the cross nerves, passing through the extracellular matrix to the tentacles. Cross nerves skirt the accessory brachial nerve, branch, and penetrate into adjacent outer and inner tentacles, where they are referred to as the frontal tentacular nerves. The second accessory nerve passes along the base of the inner tentacles. This nerve consists of Ʊ-like parts, which repetitively skirt the frontal and lateral sides of the inner tentacle and the frontal sides of the outer tentacles. The second accessory nerve gives rise to the latero-frontal nerves of the inner and outer tentacles. The abfrontal nerves of the inner tentacles also originate from the second accessory nerve, whereas the abfrontal nerves of the outer tentacles originate from the lower brachial nerve. The lower brachial nerve extends along the outer side of the lophophore brachia and gives rise to the intertentacular nerves, which form a T-like branch and penetrate the adjacent outer tentacles where they are referred to as abfrontal nerves. The paired outer radial nerves start from the lower brachial nerve, extend into the second accessory nerve, and give rise to the lateroabfrontal tentacular nerves of the outer tentacles. CONCLUSIONS: The innervation of the lophophore in the rhynchonelliform Hemithiris psittacea differs from that in the inarticulate Lingula anatina in several ways. The accessory brachial nerve does not participate in the innervation of the tentacles in H. psittacea as it does in L. anatina. The second accessory nerve is present in H. psittacea but not in L. anatina. There are six tentacular nerves in the outer tentacles of H. psittacea but only four in all other brachiopods studied to date. The reduced contribution of the accessory brachial nerve to tentacle innervation may reflect the general pattern of reduction of the inner lophophoral nerve in both phoronids and brachiopods. Bryozoan lophophores, in contrast, have a weakened outer nerve and a strengthened inner nerve. Our results suggest that the ancestral lophophore of all lophophorates had a simple shape but many nerve elements.

The periesophageal celom of the articulate brachiopod Hemithyris psittacea (Rhynchonelliformea, Brachiopoda).

The celomic system of the articulate brachiopod Hemithyris psittacea is composed of the perivisceral cavity, the canal system of the lophophore, and the periesophageal celom. We study the microscopic anatomy and ultrastructure of the periesophageal celom using scanning and transmission electron microscopy. The periesophageal celom surrounds the esophagus, is isolated from the perivisceral cavity, and is divided by septa. The lining of the periesophageal celom includes two types of cells, epithelial cells and myoepithelial cells, both are monociliary. Some epithelial cells have long processes extending along the basal lamina, suggesting that these cells might function as podocytes. The myoepithelial cells have basal myofilaments and may be overlapped by the apical processes of the adjacent epithelial cells. The periesophageal celom forms protrusions that penetrate the extracellular matrix (ECM) of the body wall above the mouth and the ECM that surrounds the esophagus. The canals of the esophageal ECM form a complicated system. The celomic lining of the external circumferential canals consists of the epithelial cells and the podocyte-like cells. The deepest canals lack a lumen; they are filled with the muscle cells surrounded by basal lamina. These branched canals might perform dual functions. First, they increase the surface area and might therefore facilitate ultrafiltration through the podocyte-like cells. Second, the deepest canals form the thickened muscle wall of the esophagus and could be necessary for antiperistalsis of the gut.