Morphology of the lophophore in C. grayi
C. grayi has the most complex type of lophophore among brachiopods – the plectolophe (Fig. 1A). The lophophore consists of three arms: two wing-like lateral arms and one spiral medial arm (Figs. 1A, B, 2A, 3A). The arms are formed by a strongly curved brachial axis that consists of a double row of tentacles and a brachial fold separated by a food groove. The mouth is located in the middle of the brachial axis in the food groove between the brachial fold and tentacle rows. On both sides of the mouth, in the lateral arms, the brachial axis runs along the lower part of each lateral arm, turns backward on its distal end, and passes the upper side of the lateral arm. Above the mouth, the left and right ends of the brachial axis extend into the middle arm, where they form a spiral, and end at the distal end of the middle arm (Fig. 2A). Thus, each arm consists of two brachial axes bearing two rows of tentacles, two brachial folds, and two food grooves (Figs. 1C, 3B). In the lateral arms, the brachial axes are close to each other, so that the epidermis between the two brachial folds forms a deep brachial gutter (Fig. 3B). Each row of tentacles, except for the area under the mouth, is double and consists alternating inner and outer tentacles: the inner tentacles are closer to the brachial fold than the outer tentacles (Fig. 1C).
Both the inner and outer tentacles have zones that differ in ciliation, in position relative to the other tentacles and the brachial fold, and in the histological structure of epithelium (Figs. 1D, 4A, B). Tentacles of both types have eight zones: one frontal, one abfrontal, two latero-frontal, two lateral, and two lateroabfrontal. Inner and outer tentacles differ from each other in form in transverse section and in the location of ciliated zones (Figs. 4A, B, 5A, B). In both inner and outer tentacles, the frontal zone faces the brachial fold. In outer tentacles, the frontal zone is concave and forms a deep frontal groove that is lined with cubic epithelium (Figs. 4A, 5A). In
inner tentacles, the frontal side protrudes and bears the ciliated ridge, which is formed by columnar epithelium (Figs. 4B, 5B). The abfrontal zone is opposite to the frontal zone. The abfrontal zone is wide in the outer tentacles but very narrow in the inner tentacles. Lateral zones form two densely ciliated ridges in tentacles of both types. The lateral ciliated ridges are located close to the frontal zone in the outer tentacles and are located close to the abfrontal zone in the inner tentacles. According to these locations of the lateral ridges, there are two latero-abfrontal zones in the outer tentacles, and two extensive latero-frontal zones in the inner tentacles (Fig. 4A, B).
General anatomy of the nervous system in C. grayi
Two ganglia, one subenteric and the other supraenteric, are the main elements of the nervous system in C. grayi (Figs. 2A-B, 3A). The subenteric ganglion is located under the mouth and gives rise to two thick lateral nerves, which innervate the body and muscles. The subenteric ganglion also gives rise to the lower brachial nerves, which extend into both lateral brachial arms, skirt them, and penetrate into the middle brachial arm (Figs. 2A, B, 3A). The supraenteric ganglion is located above the mouth and consists of two lateral nerve centers, which are connected to each other via a thick nerve (Figs. 2A, 3A). The supraenteric ganglion gives rise to the pair of main brachial nerves, which extend at the base of the brachial fold along the middle line of each lateral brachial arm (Fig. 3B). At the end of each lateral arm, each main brachial nerve extends back to the mouth and then penetrates the middle brachial arm. The subenteric and supraenteric ganglia are connected to each other via thick circumoral connectives. Near the subenteric ganglion, each connective gives rise to a thick nerve, which extends into the middle brachial arm and joins the lower brachial nerve (Fig. 3A). The supraenteric ganglion also gives rise to nerves in the middle brachial arm; these nerves fuse with the main brachial nerves.
Innervation of the brachial arms in C. grayi
Each lateral arm is innervated by six brachial nerves: two main, two second accessory, and two lower (Figs. 3B, 6). Because each lateral arm is formed by the looped brachial axis, only
three nerves belong to each half of the brachial axis: one main, one second accessory, and one lower.
Two main brachial nerves extend along the middle line of each lateral arm and along the middle arm, at the base of the brachial folds (Figs. 2A, 3B, 6). In each lateral arm, two main brachial nerves are located near each other (Figs. 2B, 3B, 7A). According to CLSM, each main brachial nerve is formed by longitudinal neurite bundles, which exhibit acetylated alpha-tubulin immunoreactivity (Fig. 7B). Each main brachial nerve is completely located in the epithelium of the arm and resembles a compact neurite bundle that is 20–25 µm in diameter (Fig. 8A). Neurite bundles form a large neuropile, which is divided into several portions by basal projections of supportive cells (= radial glia cells) (Fig. 8A). Transmission electron microscopy (TEM) revealed that these projections contain electron-dense intermediate filaments, which extend from the apical to the basal part of the cells and which adhere to the basal lamina via hemidesmosomes (Fig. 8B, C). Perikarya of at least two types are associated with the neuropile (Fig. 8A). Perikarya of the first type have a compact soma with a small nucleus bearing a large nucleolus and electron-dense cytoplasm, which is filled with small mitochondria and an electron-dense matrix (Fig. 8B). The perikarya of the second type have a large soma, which contains a large nucleus with electron-light karyoplasm (Fig. 8C). The glial cells are located within the nerve projections (Fig. 8B). These cells have a compact soma and projections, which are filled with large electron-dense granules (Fig. 8D). These projections are numerous in the neuropile. The neuropil consists of two types of neurites (Fig. 8D). Neurites of the first type are the most abundant; they have electron-light cytoplasm, which contains many microtubules (Fig. 8D). Neurites of second type are rare; their cytoplasm is filled with numerous electron-dense small granules and dense-core vesicles (Fig. 8D); these neurites can form large varicoses, which contain numerous dense-core synaptic vesicles (Fig. 8E).
Each main brachial nerve gives rise to numerous cross nerves (Figs. 6, 7B-D). Each cross nerve penetrates the connective tissue of the brachial arm, extends to the epidermis of the food
groove, and then extends to the base of the tentacles (Fig. 9A). As a consequence, each cross nerve has two ascending parts and one descending part (Figs. 3B, 6, 7B). The cross nerve is formed by 40–50 neurites of different diameters (Fig. 9B). Some of these neurites contain electron-dense granules and dense-core synaptic vesicles (Fig. 9B). Each cross nerve is associated with several envelop cells, which surround the nerve and have flocculent electron-light cytoplasm with numerous mitochondria, vesicles, and canals of rough endoplasmic reticulum (Fig. 9C).
At the base of the tentacles, several cross nerves are grouped together in sites between the inner tentacles and give rise to two short, thick nerves, which extend between the bases of the inner tentacles to the frontal side of the outer tentacles (Fig. 7C). These short nerves are connected to the second accessory brachial nerve (Figs. 3B, 6, 9A, D). This nerve is formed by a group of perikarya and neurites, which are located at the base of frontal side of the outer tentacles and which skirt these tentacles laterally (Figs. 7D, E, 9A, D). These frontal semicircles are connected to each other by bridges, which extend along bases on the abfrontal sides of the inner tentacles (Fig. 7D, E). The ultrastructure of the accessory brachial nerve is similar to that of the main brachial nerve. TEM revealed two types of perikarya in the accessory brachial nerve. One type, which has a lot in common with perikarya of the main brachial nerve, is the most abundant (Fig. 9A, D). They are large, have electron-light cytoplasm, and contain a large nucleus that lacks a nucleolus (Fig. 10A). Their cytoplasm is filled with numerous Golgi apparatuses and vesicles with electron-lucent content (Fig. 10B). The perikarya of the other type (i.e., perikarya of the third type – pkIII) are located at the base of the epithelium and are not abundant (Fig. 9A). These perikarya have electron-dense cytoplasm and contain vesicles with electron-dense content (Fig. 10A). A specific feature of these perikarya is the presence of two centrioles (Fig. 10A).
The third large brachial nerve is the lower brachial nerve (Figs. 2B, 3A, 6). It extends along both outer sides of each brachial arm and extends about 350 µm from the tentacle base (Fig. 11A). The lower brachial nerve is represented by a thick aggregation of neurites and neurite
bundles, which abut each other and form a very thick compact nerve tract (Figs. 10C, 11C). The large basal neuropil is penetrated by long and thin basal projection of supportive cells (= radial glia cells) (Fig. 10C). The lower brachial nerve is associated with many perikarya, which form long projections that extend along the nerve (Fig. 11D). According to TEM, there are two types of perikarya, which are identical to the perikarya of the second accessory nerve. In the lower brachial nerve, the basal perikarya have the same peculiarities as perikarya from the second accessory brachial nerve: their cytoplasm contains numerous vesicles with electron-dense content and centrioles (Fig. 10D). These basal perikarya form the projections, which contain vesicles with electron-dense content, dense-core vesicles, and vesicles with electron-light content (Fig. 10E). The lower brachial nerve gives rise to the many radial nerves of the arm. These nerves form a thick nerve net along the outer surface of the brachial arm (Fig. 11A). A pair of radial nerves extend between the lower brachial nerve and the abfrontal side of each outer tentacle (Fig. 11A).
Innervation of tentacles
Corresponding to their difference in morphology, the outer and inner tentacles differ in innervation. The differences concern the connection of tentacle nerves and brachial nerves and the location of tentacles nerves in the outer and inner tentacles.
The outer tentacles contain seven groups of longitudinal nerves: one frontal, two lateral, two latero-abfrontal, and two abfrontal (Fig. 4C). The frontal nerve originates from the groups of perikarya of the second accessory brachial nerve (Figs. 6,7E). The frontal nerve is formed by many thin neurite bundles, each of which consists of 5–7 neurites of small diameter (Fig. 5C). These thin neurite bundles are scattered in the epithelium of the frontal grove, and the frontal nerve therefore has weak acetylated alpha-tubulin immunoreactivity (Fig. 4C). Lateral tentacle nerves arise from the second accessory nerve (Figs. 6, 7D, E). Each lateral nerve is formed by three thick neurite bundles, which extended into the epithelium of the lateral ciliated ridges (Fig. 5G). Each neurite bundle consists of 10–20 neurites, which have large diameters and are filled with electron-light cytoplasm (Fig. 5G). Latero-abfrontal nerves originate from the radial nerves of the lophophoral arm and arise from the lower brachial nerve (Figs. 6, 11A). Each latero-
abfrontal nerve is formed by one or two thick compact neurite bundles, which consist of > 50 neurites (Fig. 5H). The abfrontal zone is innervated by two abfrontal nerves, which originate from the radial nerves of the outer side of the lophophore arm (Figs. 6, 11B). According to immunocytochemistry, each abfrontal nerve consists of three neurite bundles (Fig. 4C), each of which is formed by 8–10 neurites (Fig. 5D).
In the inner tentacles, the frontal nerves originate directly from the cross nerves (Figs. 6, 7C). In one tentacle, the frontal nerve contains neurites from different cross nerves (Fig. 7C). According to immunocytochemistry, each frontal nerve consists of 3–5 separate neurite bundles (Fig. 4D). TEM revealed a continuous layer of neurites in the basal part of the frontal epithelium (Fig. 5F). All other nerves of the inner tentacles originate from the second accessory nerve (Figs. 6, 7D). In the inner tentacles, the lateral and abfrontal nerves are organized in the same way as in the outer tentacles except that the inner tentacles lack the latero-abfrontal nerve.
Both outer and inner tentacles are innervated by peritoneal neurites (Fig. 4E). These neurites originate from perikarya located at the base of the tentacles (Fig. 4E). Peritoneal neurites extend between the basal lamina and the cells of the coelomic lining (Fig. 5E). They have electron-light cytoplasm and contain bundles of microtubules (Fig. 5E).