ИСТИНА

Organization of the nervous system is traditionally used for phylogenetic and comparative analyses that are very important to establish relationships between different taxa. The phylum Brachiopoda is a monophyletic group, which is characterized by unique body plan. All brachiopods have the lophophore: a specialized part of the mesosome, which bears tentacles and provides several important functions. Among brachiopods, there are six main types of the lophophore organization, however peculiarities of the lophophore function are very similar among brachiopods. The lophophore is also known in phoronids, whose body plan is considerably different from that of brachiopods. In spite of this difference, phoronids and brachiopods are regarded as the closest relatives, which form a united clade called Brachiozoa. At the same time, modern molecular data revealed that Brachiozoa does not exist. In order to resolve this contradiction, we have studied the innervation of the lophophore and tentacles in brachiopods of two different groups with two different types of the lophophore morphology and compared this data with innervation of the phoronid lophophore. The lophophores of inarticulate brachiopod Lingula anatina Lamarck, 1801 and articulate brachiopod Hemithyris psittacea (Gmelin, 1790) are studied by method of immunocytochemistry, laser confocal microscopy, and transmission electron microscopy. In two different brachiopods, the general morphology of the nervous system of the lophophore is similar. The lophohpore contains three brachial nerves, which extend along each brachium of the lophophore: the main, accessory, and lower brachial nerves. All these nerves exhibit serotonin-like, FMRFamide-like, and α-tubulin-like immunoreactivity. All brachial nerves are located intraepidermally and have stratified structure. Thus, the uppermost layer is composed by epidermal cells, which form long thin basal projection attached to the basal lamina. The second layer is formed by perikarya of different types. These perikarya differ from each other in location, density of cytoplasm, density of karyoplasm, and characteristics of vesicles, which are the dominant organelles in the cytoplasm. Some perikarya, which are apparently sensory, contact the surface of the epidermis and bear a cilium. The third layer is formed by nerve projections. These neurites differ in diameter, orientation, and types of synaptic vesicles. Most of these neurites are 0.4 to 1 µm in diameter, are located near the basal membrane, and pass along the brachia of the lophophore. Some of neurites have the same diameter but pass in a cross direction and form an outer layer. In L. anatina, among the smaller neurites, there are one to three larger neurites, with diameters from 4 to 8 µm. The cytoplasm of these giant neurites is filled with numerous thick microtubules and small mitochondria. In both studied brachiopod species there are cross nerves, which extent in the connective tissue and connect the main and accessory brachial nerves. The cross nerves are not made visible by immunostaining. All of these cross nerves are organized in the same way. They consist of numerous neurites of different diameter. The large-diameter neurites are usually located on the periphery, whereas the small-diameter neurites occupy the central portion of the nerve. The larger neurites usually contain very electron-dense inclusions. Each cross nerve is surrounded by a thick layer of basal lamina. Each nerve is also composed of an envelope cell. The envelope cell is located on the periphery of the nerve and has long thin projections that surround the neurites. Cross nerves are often associated with outer envelope cells. The outer tentacles are innervated by neurite bundles that extend from the lower and accessory brachial nerves. The lower brachial nerve gives rise to the abfrontal and latero-abfrontal neurites, which form a thick net along the abfrontal side of each outer tentacle. These neurites exhibit serotonin-like immunoreactivity and can also be recognized by staining against α-tubulin. The abfrontal neurite bundles exhibit weak FMRF-amide-like immunoreactivity. The accessory brachial nerve gives rise to the latero-frontal and mediofrontal neurite bundles of outer tentacles. The latero-frontal neurite bundles exhibit strong α-tubulin-like immunoreactivity. On the tip of each tentacle, the latero-frontal neurite bundles contact each other and form a loop. According to TEM data, the organization of the latero-frontal neurite bundles is complex and includes numerous neurites of different types and perikarya. The perikarya usually form a basal layer. The cytoplasm of the perikarya is filled with numerous electron-lucent synaptic vesicles. Above the perikarya, large neurites form a second layer. Each neurite bundle contains several hundred neurites. The large neurites, whose diameters may reach 1 µm, have electron-lucent cytoplasm that is filled with thick microtubules. Some of the large neurites contact the basal lamina, and their cytoplasm contains many synaptic vesicles with electron-lucent content. Small neurites form a third layer and are usually located above the large neurites. The frontal neurite bundles exhibit FMRFamide-like immunoreactivity and can also be recognized by staining against α-tubulin. According to TEM data, the frontal neurite bundles are represented by several compact aggregations of neurites. The inner tentacles are innervated by the accessory brachial nerve. The abfrontal side of each inner tentacle is innervated by the medioabfrontal neurite bundle, which exhibits serotonin-like, FMRFamide-like, and α-tubulin-like immunoreactivity. These neurite bundles originate from the FMRFamide-like immunoreactive perikarya, which are grouped along the line between the two rows of tentacles. Two thick neurite bundles extend along the latero-abfrontal sides of the tentacles. They are organized in the same way as the latero-frontal neurite bundles in the outer tentacles and consist of many large neurites. The frontal side of the inner tentacles is innervated by seven to eight small neurite bundles, which exhibit serotonin-like and α-tubulin-like immunoreactivity. In both brachiopod species, tentacles of both rows contain subperitoneal neurites. They pass along the abfrontal and lateral sides of the tentacles and are located between the basal lamina and coelothelial cells. These neurites have a large diameter (about 1 µm), electron-lucent cytoplasm, and many thick microtubules. The subperitoneal neurites are abundant in tentacles of Hemithyris psittacea.In this species, subperitoneal neurites exhibit strong α-tubulin-like immunoreactivity. The subperitoneal neurites arise from cells, which are located at the base of each tentacle. The bodies of these cells can also be recognized with staining against α-tubulin. According to our data, nerve elements of the brachiopod lophophore have a stratified organization, and the nerve elements form rows: the inner row consists of neurites, the middle row is formed by perikarya and the somata of glial cells, and the outer row is formed by epidermal cells. The same stratified structure of all nerve elements is described in dorsal ganglion, tentacular nerve ring, and trunk nerve plexus of phoronids. The ultrastructure of the main nerves is very similar in phoronids and brachiopods. The main brachial nerve of brachiopods and dorsal ganglion of phoronids contain several types of perikarya, which differ in sets of organelles, types of synaptic vesicles, and cytoplasm and karyoplasm density. The giant nerve fibers have never been described in brachiopods before and are reported here for the first time. The presence of giant nerve fibers is a characteristic element of the nervous system in phoronids and some other invertebrates. These fibers are usually used for fast conduction of the nerve impulses providing escape responses. In L. anatina, the giant fibers extend along each brachium and, apparently, contribute to fast conduction of the nerve impulses along lengthy brachia. According to our data, the epidermis of the thickest zones is innervated by special latero-frontal (in outer tentacles) and latero-abfrontal (in inner tentacles) neurite bundles. These are huge aggregations of neurites with large diameters. In the outer tentacles, these aggregations of neurites have a complex organization and include perikarya and a layer of small-diameter neurites. Interestingly, the large-diameter neurites do not exhibit neither serotonin-like or FMRFamide-like immunoreactivity but do react strongly to anti α–tubulin. The innervation of the latero-frontal zones of the outer tentacles seems very similar to the innervation of the latero-abfrontal zones of the inner tentacles, and the nervous system of the inner tentacles appears to mirror the nervous system of the outer tentacles. This similarity correlates with the density of cilia along the tentacles: along both rows, the areas along tentacles with the most cilia are innervated in the same way. Using one of the homology criteria about the special quality of the structure, we can infer that the latero-abfrontal zones of the inner tentacles and latero-frontal zones of the outer tentacles are homologous. Thus, the frontal zone is very wide in inner tentacles but narrow in outer tentacles. This research is supported in part by several grants. The collection of material was done with support from the Russian Foundation of Basic Research (#14-04-00238), the TEM and 3-D investigations were done with support from the Russian Scientific Fund (#14-04-262), and the processing of the report was supported by Grants of the President of Russia (#MD-5812.2015.4; # NSH-1801.2014.4).