Microscopic Anatomy and Ultrastructure of the Nervous ... · Microscopic Anatomy and Ultrastructure...

ISSN 1063-0740, Russian Journal of Marine Biology, 2009, Vol. 35, No. 5, pp. 388–404. © Pleiades Publishing, Ltd., 2009.Original Russian Text © E.N. Temereva, V.V. Malakhov, 2009, published in Biologiya Morya.

388

Phoronids (Phoronida) constitute an isolated phy-lum of invertebrates, which is characterized by a uniquestructural pattern. The studies of this group are veryimportant in terms of comparative anatomy and phy-logeny, as taxonomic position of phoronids in the ani-mal kingdom still remains uncertain. Most classicalpapers consider phoronids and a central group of archi-coelomate animals related to deuterostomes [30, 38,45]. In the recent years, proceeding from molecularbiological data, the phoronids, as well as other lopho-phorates are drawn together with trochophorous ani-mals and placed into the group Lophotrochozoa [21].All these facts are suggestive of further studies on themicroscopic anatomy and ultrastructure of phoronids.

Classical studies on the microscopic anatomy ofphoronids showed that they have no organized nerveganglia or nerve trunks, except for one or two giantnerve fibers [17, 44]. A more detailed study of the ner-vous system in phoronids [46] showed that it representsa nerve plexus. In recent decades some papers havebeen published in which the elements of the phoronidannervous system were studied at the histochemical andultrastructural levels [3–6, 19]. However, the availabledata cannot provide an answer for some importantquestions about general cytological organization ofnervous systems in phoronids; namely, what cell ele-ments compose certain nerve structures and how they

are arranged relative to each other. Moreover, it is stilluncertain what the cause is for such a structure of thenervous system in phoronids, i.e., whether it is theresult of a secondary simplification due to tubicularmode of life or whether the plexus-type nervous systemis the initial type of organization of the nervous systemin Bilateria. The target of this study was to provide adetailed description of the microscopic anatomy andcytoarchitectonics of all components of phoronidannervous system in example of

Phoronis harmeri.

MATERIAL AND METHODS

As the material for this study we used adult speci-mens of

Phoronopsis harmeri

collected in Vostok Bayof the Sea of Japan around the Vostok Marine Biologi-cal Station of Zhirmusky Institute of Marine BiologyFEB RAS and in Puget Sound (Pacific coast of NorthAmerica), around Friday Harbor Marine BiologicalStation.

Prior to fixation the animals were removed fromtubes. Studies of the general anatomy of the nervoussystem were performed using histological techniques.For the latter purpose, the whole animals were fixedwith 4% formalin (prepared with filtered seawater),rinsed from the fixative in distilled water and preservedin 70% ethyl alcohol. Fragments of the body (head,

ZOOLOGY

Microscopic Anatomy and Ultrastructure of the Nervous System of

Phoronopsis harmeri

Pixell, 1912 (Lophophorata: Phoronida)

E. N. Temereva and V. V. Malakhov

Moscow State University, Moscow, 119991 Russiae-mail: [email protected]

Received May 21, 2009

Abstract

—The microscopic anatomy and ultrastructure of the nervous system of

Phoronopsis harmeri

wasinvestigated using histological techniques and electron microscopy. The collar nerve ring is basically formedby circular nerve fibers originating from sensitive cells of tentacles. The dorsal nerve plexus principally consistsof large motor neurons. It is shown for the first time that the sensitive collar nerve ring immediately passes intothe motor dorsal nerve plexus. The basic components of the nervous system have similar cytoarchitectonics anda layered structure. The first layer is formed by numerous nerve fibers surrounded by the processes of glia-likecells. The bodies of glia-like cells constitute the second layer. The third layer consists of neuron bodies over-arched by the bodies of epidermal cells. The giant nervous fiber is accompanied by more than one hundrednerve fibers of a common structure and, thus, marks the true longitudinal nerve. The phoronids possess one ortwo longitudinal nerves. It is supposed that the plexus nature of the nervous system in phoronids may be relatedto their phylogenesis. A comparison of the nervous system organization and body plans among the Lophopho-rata suggests that the nervous system of phoronids cannot be considered as a reductive variant of the brachiopodnervous system. At the same time, the structure of the nervous system of bryozoans can be derived from that ofphoronids.

Key words

: Phoronida, Lophophorata, nervous system, plexus.

DOI:

10.1134/S1063074009050046

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MICROSCOPIC ANATOMY AND ULTRASTRUCTURE OF THE NERVOUS SYSTEM 389

anterior and posterior trunk regions, and ampulla) weretreated in ascending alcohol series, butyl alcohol,xylene, and paraplast. After embedding in paraplast,5

μ

m thick sections were prepared with a Leica RM2125 rotation microtome. The sections were thenstained with Carracci hematoxylin and covered withCanada balsam. Altogether 3 series of sagittal sections,2 series of frontal sections and 18 series of transversesections were prepared. The sections were examinedunder a Zeiss AxioPLAN2 light microscope and photo-graphed with an AxioCam HRm digital photo camera.

The fine morphology and ultrastructure of all ele-ments of the nervous system were studied using meth-ods of scanning (SEM) and transmission (TEM) elec-tron microscopy. When using SEM techniques, frag-ments of animal bodies fixed with formalin weredehydrated in an ascending alcohol series and in ace-tone, dried in a critical point dryer, fixed onto stubs andsputter covered with platinum–palladium alloy; thenthe samples were examined under a CamScan S2 scan-ning electron microscope.

For TEM the fragments of animal bodies were fixedwith 2% glutaraldehyde solution in 0.1 M cacodylatebuffer, with addition of sucrose (100 mM/l) and pH = 7.2.After rinsing in cacodylate buffer, the material waspostfixed with 1% solution of osmium tetroxide andpreserved in 70% alcohol. Then the samples wereembedded into an araldite mixture using standard tech-niques; fine sections were prepared with an ultratome,stained and examined under a JEM 100B transmissionelectron microscope in the Interdepartmental Labora-tory of Electron Microscopy of the Biological Facultyof Moscow State University.

The following designations are used in figures:(

a

) anus; (

abn

) ascending branch of nephridial canal;(

afz

) abfrontal zone of tentacle; (

am

) ampulla; (

ap

) analpapilla; (

abp

) anterior body part; (

bc

) blood capillary;(

bgc

) body of glia-like cell; (

bm

) basal matrix;(

bmp

) process of basal matrix; (

bnf

) big nerve fiberswith electron-light cytoplasm; (

c1

) preoral coelom;(

c2

) tentacular coelom; (

c3

) trunk coelom; (

cf

) collar;(

cm

) circular musculature; (

d

) diaphragm; (

dnp

) dorsalnerve plexus; (

e

) epidermis; (

ef

) fold of covering epi-thelium; (

ec

) cells of giant nerve envelope; (

ep

) epis-tome; (

fz

) frontal zone of tentacles; (

GC

) Golgi com-plex; (

gc

) glandular cells of epidermis; (

gnf

) giantnerve fiber; (

in

) ascending loop of intestine; (

lac

) leftanal chamber of trunk coelom; (

llm

) left lateral mesen-tery; (

lm

) longitudinal musculature; (

loc

) left oralchamber of trunk coelom; (

m

) mitochondria;(

mi

) microvilli; (

mlb

) myelin body; (

mnf

) nerve fibersof small diameter with cytoplasm of medium electrondensity; (

mv

) medial blood vessel; (

n

) nucleus;(

nb

) neuron body; (

ne

) nephridiophore; (

nf

) nervefibers; (

np

) neuropile; (

nr

) collar nerve plexus; (

nu

)nucleolus; (

pbp

) posterior body part; (

t

) tentacle; (

to

)tonofilaments; (

v

) vacuole.

RESULTS

General Anatomy and Histology

The nervous system of

Phoronopsis harmeri

occu-pies a basiepidermic position and lies in between thebasal parts of epithelial cells and basal lamina (Fig. 1a).The following elements can be distinguished in the ner-vous system of phoronids: dorsal (Fig. 2a) and collar(Figs. 2a, 2b) nerve concentrations, left giant nervefiber (Figs. 2a, 2c), intraepidermal nerve plexus of thebody (Fig. 1a), and certain isolated bundles of nerve fibersrunning within the epithelium of tentacles (Fig. 2d) andinternal organs.

Dorsal and collar nerve plexuss are major elementsof the nervous system interconnected to each other andpassing to each other at the dorsal side of the epistome(Figs. 1b, 2b). The collar plexus passes along the outerlayer of tentacles and is covered by an epithelial fold(the collar), which is the most pronounced in frontalsections through the head body region (Fig. 3b). Thecollar plexus primarily comprises circular nerve fibers,which is clearly seen in transverse sections (Figs. 2b, 4b).These fibers arise from sensory cells of tentacles andare concentrated along both the frontal and abfrontalsides (tentacles) (Fig. 2d). The most numerous fibersare the nerve fibers running along the frontal side ofeach tentacle. They form there a compact longitudinalbundle, 20–25

μ

m in diameter, comprising, besidescommon thin fibers, also a few (two to three) unusuallylarge nerve processes, 5–8

μ

m in diameter, which couldbe revealed even under an ordinary light microscope(Fig. 2d).

The dorsal nerve plexus lies in the column of cover-ing epithelium, on the dorsal side of the animal,between the mouth and anus (Figs. 1a, 1b).

The giant nerve fiber arises from the dorsal nerveplexus (Figs. 2a, 3a, 3d), where it has small diameter(no more than 8

μ

m and dark granulated cytoplasm)(Fig. 3d). Remaining in the column of covering epithe-lium, above the layer of basal matrix, the giant nervefibers runs along the ascending branch of the canal ofleft nephridium (Fig. 3e); in this area its diameterincreases up to 13–15

μ

m. In the anterior trunk regionthe giant nerve fiber passes through the attachmentpoint of the left lateral mesenterium (Fig. 2c). There ithas the greatest diameter, namely, 90–95

μ

m in

Ph. harmeri

members from Puget Sound Bay and 40–50

μ

m in the phoronids from Vostok Bay. Both thediameter and the shape of the transverse section changein the giant nerve fiber passing along the anterior trunkregion; the latter changes from flattened (Fig. 3g) torounded (Fig. 3i). The giant axon was successfullyretraced up to the anterior one third of the posteriortrunk region.

The epidermal nerve plexus is unevenly developedalong the entire body of the animal. It shows the great-est thickness in the anterior body region and the small-est thickness in the posterior region (Fig. 1a).

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TEMEREVA, MALAKHOV

Ultrastructure

Nerve plexus in the integument.

The thickness ofnerve plexus in the anterior body region equals 7–10

μ

m. The nerve fibers run in between basal processesof epithelial cells; the latter are arranged into groups.The gaps between these groups, 1.5–2

μ

m are filledwith nerve fibers (Fig. 5a). The fibers always make upa more or less homogeneous layer, whose upper bound-ary is rather smooth. On the other hand, the layer ofnerve fibers is the thickest close to thin areas of basalmatrix, at the bottom of epidermal folds (Fig. 4c). Nonerve fibers occur between the bodies of the epidermalcells. From behind, the fibers are applied to the basallamina of the extracellular matrix; but direct contactswith the latter can be observed primarily for fibers ofmedium diameter, with loose granulated cytoplasm ofmedium electron density (Fig. 5b). In these fibers syn-aptic vesicles usually are extremely numerous. Theseare coated vesicles or vesicles with contents of mediumdensity; usually they are concentrated along the cellmembrane, which is applied against basal lamina.

Above the medium diameter fibers there are fibers of upto 1.3

μ

m in diameter, with electron-light cytoplasmcontaining sparsely scattered synaptic vesicles withcontents of medium density. The large fibers also comein contact with basal lamina, although not throughouttheir length, but only in certain areas (Fig. 5c). The bulkof the nerve processes is located above the layer oflarge fibers; in this area thin and relatively thick fibersrun chaotically in different directions.

Nerve fibers are separated from epidermal cells byprocesses and bodies of glia-like cells (Fig. 5a, d). Insections it could often be seen that the cytoplasmic pro-cesses of these cells surround the bundles of axons(Figs. 5a; 6b, c). The nuclei of the glia-like cells arecup-shaped; each nucleus comprises a nucleolus(Figs. 5a, 5d, 6c). Around the nucleus there are cisternsof the rough endoplasmic reticulum (RER) and Golgiapparatus; centrioles can often be seen close to the lat-ter (Fig. 5e). The cytoplasm of the cells contain numer-ous microtubules, multivesicular bodies, glycogengranules, mitochondria (Fig. 6c), and electron dense

cfnr

t

ep

ne gnfacf

nr

ep

a

t

dnp

abp

pbp

am

(a)

(b)

dnp

Fig. 1.

Scheme of the organization of nervous system in

Phoronopsis harmeri

: (a) relative density of nerve plexus (indicated byblack dots) in different parts of body; (b) head end of the body, view from above (the structure of lophophore is somewhat schema-tized; tentacles are removed; their bases are shown schematically).


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granules 150–300 nm in diameter. The latter granulesrepresent a characteristic feature of the organization ofthe glia-like cells.

The nerve plexus shows the smallest thickness in theposterior trunk region. In this area it is composed of iso-lated nerves (bundles of nerve fibers) running throughthe cell labyrinth in between the basal processes of epider-mal cells (Fig. 6a). Each bundle comprises 5–15 fibers ofapproximately the same diameter (150–200

μ

m), con-sistency of cytoplasm, and type of synaptic vesicles.

Isolated nerve fibers are stretched between the basalmembrane of epidermal cells and basal lamina of extra-cellular matrix (Fig. 6b). The nerves comprise pro-cesses of glia-like cells surrounding nerve fibers alongalmost the entire perimeter, making up a semicircle,broken at the side of extracellular matrix (Figs. 6a, 6b).The cytoplasm of these processes contains characteris-tic electron-dense inclusions.

In both the ampulla and posterior trunk region thenerve plexus is constructed of isolated nerves, which

nr

ep

gnf

afz

c2

cf

bc

fz

ap

gnf

dnp

t

t

(a) (c)

(d)(b)

t

Fig. 2.

Structure of nervous system in

Phoronopsis harmeri

in longitudinal (a) and transverse (b–d) histological sections: (a) a sec-tion through the head end of body; (b) a section at the level of collar (asterisk indicates the position of dorsal nerve plexus; arrow-heads point to nerve plexus); (c) a section through the middle of the anterior trunk region of a specimen from Puget Sound (arrowspoint to left lateral mesentery); (d) a section through a tentacle of a specimen from Vostok Bay, Sea of Japan (arrowheads and arrowsshow large and small nerve fibers respectively). Scale bar: (a) 0.1 mm; (b, c) 0.2 mm; (d) 50

μ

m.

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TEMEREVA, MALAKHOV

50

μ

m

cfc2

d

c3c1np

c3

gnf

c3

dnp

(b)

(a)10

μ

m

(d)

c2

c3

d

np

20

μ

m

cfnr

c3

abn

in

(c)

50

μ

m

20

μ

m

ec

e

gnf

cmllm

l

m

e

gnf

ec

c3

loc

lac(f)

(e)

(g) 20 μm20 μm

lm

Fig. 3. Details of histological organization of major elements of nervous system in Phoronopsis harmeri in longitudinal (a–e) andtransverse (f, g) sections: (a) dorsal nerve plexus (arrows indicate somata of large neurons); (b) a frontal section through collar andcollar nerve plexus; (c) collar nerve plexus; (d–g) a giant nerve fiber: d, close to dorsal nerve plexus; e, close to ascending nephridialbranch (arrowheads point to the nerve fiber); (f) and (g), in the anterior part of the trunk. Scale bar: (a) 10 μm; (b, e) 50 μm;(c, d, f, g) 20 μm.

RUSSIAN JOURNAL OF MARINE BIOLOGY Vol. 35 No. 5 2009


never make up a solid layer characteristic of the ante-rior trunk region. The diameter of these nerves varies invery wide limits, from 1.4 to 6–7 μm (Fig. 6c). Smallnerve bundles comprise six to seven nerve fibers ofsmall diameter, up to 230 nm each (Fig. 6c). Such nervefibers show medium cytoplasm density and the pres-ence of sparse synaptic vesicles with electron-light con-tent. Large nerve bundles include 12–16 fibers of smalldiameter (200–250 nm) and one to two fibers of up to750 μm in diameter each (Fig. 6c). The cytoplasm oflarge nerve fibers contain numerous synaptic vesiclesof different types, with electron-light and electron-dense contents, contents of medium electron density,and coated vesicles. Large bundles of nerve fibers aresurrounded by processes of glia-like cells along theentire perimeter (Fig. 6c). The bodies of these cells arelocated above the bundles and are applied to the bodiesof epidermal cells.

Dorsal nerve plexus. The epithelium comprisingneurons and nerve fibers of the dorsal plexus is made ofcolumnar cells (Figs. 3a, 7a) with strongly narrowedbasal parts and expanded apical parts. The apical sur-face bears microvilli and a flagellum (Figs. 7a, 8a). Bun-dles of tonofilaments are running in cytoplasm of basalparts of the cells; they are attached to basal lamina ofextracellular matrix with hemidesmosomes (Fig. 7b).

Neurons are located between the processes of sup-porting cells and are large (7–8 μm in diameter)rounded cells with a large nucleus and light cytoplasm(Figs. 3a, 7c). Their cytoplasm looks loose due to thepresence of numerous vesicles, whose diameter rangesfrom 10 to 90 nm. The content of these vesicles is eithertransparent or shows medium electron density. Thenucleus is usually displaced into the periphery of thecell, almost entirely devoid of membrane-associatedchromatin and bears a nucleolus (Fig. 7c). The bulk ofthe cell cytoplasm is occupied by long mitochondriawith an electron-dense matrix. Sometimes the cellscontain lipid droplets of medium electron density (up to500 nm in diameter) and large (up to 1.5 μm in diame-ter) myelin bodies. The cytoplasm of the neurons alsocontains Golgi bodies and cisterns of smooth and roughendoplasmic reticulum.

Beneath the neurons there are glia-like cells, whosecytoplasm contains numerous electron-dense granules150–300 nm in diameter (Fig. 8a). Processes of theglia-like cells run between the nerve fibers, which con-stitute the bulk of the dorsal nerve plexus.

Bundles of nerve cell processes lie immediatelyabove the basal lamina and stretch in between the basalprocesses of supporting cells (Figs. 7a, 8a). The heightof the neuropile reaches 13 μm in certain areas(Fig. 3a). The diameter of axons ranges from 150 nm to1 μm (Fig. 7d). As a rule, large processes have electron-light cytoplasm and are almost entirely devoid of syn-aptic vesicles (Fig. 7d). However, in some fibers oflarge diameter we found synaptic vesicles of differenttypes: coated vesicles (the most numerous), vesicles

with contents of medium electron density and a few(one to two) vesicles with transparent contents(Fig. 7b). The large fibers are the most numerous closeto basal lamina; however, they occur also in other parts

c1 mv

c3

cf

c2

cm

(a)

(b)

(c)

ef

bm

c3

npc3

Fig. 4. Fine morphology of some components of nervoussystem in Phoronopsis harmeri in longitudinal (a, c) andtransverse (b) sections, SEM data: (a) dorsal nerve plexus;(b) collar nerve plexus; (c) nerve plexus in the integument ofthe anterior part of trunk. Scale bar: (a) 40 mm; (b, c) 30 mm.

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TEMEREVA, MALAKHOV

bnf

bnfmnf

gcgc

n

bm

cm

bnf

bgc

bm

GC

bgc

to

bnf

tobm

(c)

(a) (d)

mnf

bm(b) (e)

Fig. 5. Fine structure of nerve plexus in the anterior part of trunk of Phoronopsis harmeri in longitudinal ultrathin sections: (a) a gross viewof the basal part of epidermis (arrowheads point to processes of glia-like cells); (b) nerve fibers with the multitude of synaptic vesicles locatedclose to the basal lamina; (c) stratified structure of the neuropile: nerve fibers of medium diameter applied to the basal matrix and fibers ofgreater diameter located above them; (d) the body of a glia-like cell, with a nucleus and characteristic electron-dense inclusions; (e) a cen-triole (indicated by the arrow) in the cytoplasm of a glia-like cell. Scale bar: (a) 2 μm; (b, c, e) 0.5 μm; (d) 0.3 μm.



gc

gc

bm

cm

gc

(a)

bm

(b)

bgc

bm

(c)

Fig. 6. Nerve plexus in the posterior part of trunk region (a, b) and in ampulla (c): (a) a gross view of a part of plexus constituted ofisolated bundles of nerve fibers; (b) two nerve bundles, each consisting of 6–12 nerve fibers and surrounded (partially or entirely)by processes of glia-like cells. Arrows show nerve bundles and isolated nerve fibers; arrowheads indicate processes of glia-like cells.Scale bar: (a, b) 2.5 μm; (c) 1.25 μm.

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TEMEREVA, MALAKHOV

(b)bm

bnf

mnf

mi

n

nb

nu

n

mlb

m

mnb

nb

nb

mnf

bnf

to

(c)

(a)(d)

bm

Fig. 7. Details of the fine structure of dorsal nerve plexus (longitudinal sections, TEM): (a) gross view of a section of the dorsalplexus, from apical surfaces of supporting epidermal cells to basal matrix (figured arrowheads show processes of glia-like cells);(b) nerve fibers and processes of supporting cells close to basal lamina (arrows indicate hemidesmosomes); (c) soma of a neuron(straight arrowheads point to canals of rough endoplasmic reticulum); (d) an area of neuropile, with nerve fibers of small diameterand large nerve fibers. Scale bar: (a–c) 1 mm; (d) 0.7 mm.



bnfbnf

mi

to

to

bgc nf

gnf

v

to

(a) (b)

(c)

nb

nb

bgc

bm

ecec

Fig. 8. Scheme of the organization of major components of nervous system in Phoronopsis harmeri (reconstruction from TEMdata): (a) dorsal nerve plexus; (b) collar nerve plexus; (c) a giant nerve fiber (arrowheads show hemidesmosomes).

Fig. 9. Details of fine structure of collar nerve plexus (TEM, longitudinal sections): (a) a gross panoramic view of the sectionthrough the nerve concentration, from apical surfaces of supporting cells to basal lamina; (b) basal part of neuropile in that part ofnerve concentration, where no evaginations of the basal matrix are present; (c) central part of neuropile with numerous synapticcontacts (indicated by arrowheads); (d) basal part of neuropile in the part where the basal matrix makes up evaginations; (e) a nervefiber of big diameter located close to basal lamina (adjacent to basal lamina nerve fibers of small diameter are pointed out by arrow-heads); (f) a hemidesmosome between a supporting cell and an evagination of the basal matrix (indicated by the arrow). Scale bar:(a) 1.25 μm; (b–e) 1 μm; (f) 0.5 μm.

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TEMEREVA, MALAKHOV

bnf

bnf

bm

mnf

bgc

bnf

to

bmp

mi

bnf

bnfnb

bm

to

mnf

bm

bnf

nb

(a)

(b)

(c)

(d)

(e)

(f)



bm

gnf

bnf

bnf

gnfm to

bgc

bnf

gnf

m

v

ec

bm

gnfgnf

nf

nf

bm(a) (d)

(c)(b)

(a)

400


TEMEREVA, MALAKHOV

of the neuropile (Figs. 7a, 8a). Fibers of small diameterhave cytoplasm of medium electron density and containsparse vesicles with transparent content (Fig. 7d).

Collar nerve plexus. The epidermis comprising thenerve ring is made of nonflagellate cells, whose apicalsurface bears numerous microvilli (Figs. 8b, 9a). Thebasal parts of the epidermal cells constitute the bulk oftheir height (about 20 μm) (Fig. 8b); in the cytoplasmof these cells there are strong bundles of tonofilamentsattached by hemidesmosomes to outgrowths of basalmatrix (Figs. 8b, 9f).

Between the basal parts of epidermal cells there aresparse neurons showing a structure similar to those inneurons of the dorsal nerve plexus (Figs. 8b, 9a).Beneath the neurons, closer to basal lamina, there areglia-like cells, whose cytoplasm comprises characteris-tic electron-dense granules about 150 nm in diameter(Figs. 8b, 9a).

The entire space between the bodies of glia-likecells and basal lamina is filled with numerous processesof nerve cells. The height of neuropile reaches 30 μm(Fig. 3c). Over a significant stretch of circular concen-tration, basal lamina is directly adjacent to nerve fibersof small diameter, with cytoplasm of medium electrondensity (Fig. 9b). In the part of the collar plexus, wherethe basal matrix makes invaginations toward the epider-mis, the basal parts of the neuropile are made of largenerve fibers with electron-light cytoplasm (Fig. 9d).However, these fibers almost never come into directcontact with basal lamina, being separated from the lat-ter by a thin (about 100 nm) layer of small nerve pro-cesses with granulated cytoplasm (Fig. 9e).

It is pertinent to note the presence of numerous syn-aptic contacts in the circular nerve concentration(Fig. 9c); for example, in a section of 25 μm2 in area weregistered up to eight synaptic contacts.

Giant nerve fiber. As for all elements of phoron-idan nervous system, the giant nerve fibers run in thecolumn of the epidermis (Figs. 3f, 3g). The structure ofthe giant nerve fiber has already been described else-where [10].

In Ph. harmeri the giant nerve fiber is a giant axonarising from one of the neurons of the dorsal nerveplexus. As has been mentioned above, the proximal partof the giant fiber has a small diameter and is filled withloose cytoplasm of medium electron density (Fig. 10a).Farther from the anterior end of the body, the cytoplasmof the fiber becomes more electron-light (Fig. 10a) andcontains large vacuoles up to 10 μm in diameter filledwith transparent contents (Fig. 10b). Throughout thelength of the nerve fiber its cytoplasm contains small

mitochondria with an electron-dense matrix (Figs. 10a,10b, 10d). In the giant axon there are almost no synap-tic vesicles. Only close to the dorsal plexus, in the gran-ular parietal cytoplasm of the giant fiber are theresparse synaptic vesicles of different types, coated andfilled with content of medium electron density(Fig. 10e). The most numerous are vesicles 90–120 μmin diameter with electron-light contents (Fig. 10e); theyalso occur in some other parts of the fiber (Fig. 10b).

Around the nerve fiber there are sheath cells that aremodified cells of the integument (Figs. 8c; 10c, d). Thecells and their nuclei are strongly flattened; cell mem-branes make up a kind of myelin sheath around the fiber(Fig. 10c). The giant axon has no direct contacts withthe basal lamina and is separated from the latter bysheath cells and nerve fibers of the common structure(Figs. 8c, 10d). The cytoplasm of the sheath cells con-tains numerous mitochondria with electron-densematrix, cisterns of the rough endoplasmic reticulum,and rounded inclusions of medium electron density,about 500 nm in diameter (Fig. 10c). The basal parts ofsheath cells are narrowed into thin processes compris-ing bundles of tonofilaments attached to the basal lam-ina by hemidesmosomes (Figs. 8c, 10d). Thus, thesheath cells reinforce the perimeter of the giant nervefiber and fasten it to the basal lamina.

Throughout its length the giant nerve fiber is accom-panied by nerve fibers of a common structure. In thehead part of the body (close to the dorsal plexus) sheathcells around the giant axon are absent and it comes intodirect contact with the nerve fibers of the commonstructure (Fig. 10e). As in other parts of the nervoussystem, among these fibers we could distinguish fibersof greater diameter with light cytoplasm and those ofsmaller diameter with cytoplasm of medium electrondensity (Fig. 10a, 10d, 10e). The fibers of greater diam-eter are the most numerous in the anterior body region(Fig. 10d).

DISCUSSION

As is shown in classical papers [17, 44, 46], the ner-vous system of phoronids is lacking nerve ganglia andnerve trunks and represents a plexus, which is moredense in certain areas of the body. This fact makesphoronids a unique group among all coelomic Bilateria.A plexus-type nervous system is characteristic of coe-lenterate animals and is known in some parenchyma-tous Bilateria, for example Xenoturbellida [35], andalso in Acoelomorpha (in the latter case it is combinedwith nerve trunks and cerebral ganglion) [36, 37]. Inthis connection there seemed to be no escape from the

Fig. 10. Details of fine structure of a giant nerve fiber (TEM; a, e, longitudinal sections; b–d, transverse sections): (a) giant axon atthe place, where it arises from dorsal nerve plexus (the most proximal part of the fiber is seen in the lower left corner; arrowheadsindicate aggregations of synaptic vesicles); (b) an area of cytoplasm, with a part of a large vacuole and mitochondria; (c) sheathcells; (d) panoramic view of the basal part of giant fiber and adjacent neuropile; (e) an area of the parietal cytoplasm of a giant fiberat the area where the latter arises from the dorsal nerve plexus. Scale bar: (a, b) 1 μm; (c) 2 μm; (d) 1.67 μm; (e) 0.5 μm.



question of whether such an organization of nervoussystem in phoronids represents the initial organizationpattern or whether it is a result of a secondary simplifi-cation? To answer this question we have to analyze thestructure of the nervous system in phoronidan larvaeand in other lophophorate animals, the closest relativesof the phoronids.

The nervous system of phoronidan larvae has beenstudied with ultrastructural and histochemical methods[22–24, 28, 41, 43]. It has been shown to include an api-cal nerve ganglion, anterior median and marginalnerves, two ventrolateral and two dorsolateral nerves ofpreoral lobe, circular nerves located in the base of thetentacular ring, and the radial nerves of tentacles.Moreover, according to our unpublished data, there is acircular nerve running beneath the telotroch. Thus, thenervous system of phoronidan larvae has both nerveganglia and nerve trunks, which are reduced later, in thecourse of metamorphosis [25, 45].

The larvae of another group of tentaculate animals,the brachiopods, also possess an apical nerve ganglionand a primordium of a ventral (subesophageal) gan-glion [7]. In the course of metamorphosis the apicalganglion is reduced and adult animals retain only awell-developed ventral (subesophageal) ganglion [11,12].

The nervous system in the larvae of bryozoans com-prises an apical nerve ganglion and a pyriform organ,connected to each other with a median nerve, and anerve ring located beneath the prototroch [42, 47]. Dur-ing metamorphosis all nerve structures are prone toreduction [48]. The nervous system of adult bryozoansconsists of a nerve plexus and a so-called cerebral gan-glion, which gives rise to a pair of lophophoral nerves[26, 29, 31]. Thus, the reduction of the apical ganglionin the course of metamorphosis is a common develop-mental pattern characteristic of all lophophorate ani-mals.

As regards the differences in the organization of thenervous system in adult phoronids and brachiopods,they are due to the profound differences in body planbetween these two groups. It has been shown that in thecourse of metamorphosis the larva of the brachiopodNovocrania anomala is folded onto the ventral side[33]. The metamorphosis of Craniida obviously reflectsthe ancestral body plan indigenous of all brachiopods.In this case, the subesophageal ganglion retains its posi-tion within the mantle cavity, on the ventral side ofbody.

The metamorphosis of phoronids is known to pro-ceed in a different way [2, 25, 45]. First, a deep invagi-nation develops on the ventral side of the larva; later onit everts so that the body of the phoronid develops as anoutgrowth of the ventral side of the larva. Thus, thedeepest part of the phoronidan body inserted into thetube, the ampulla, corresponds to the middle part ofventral side, i.e., to the location of the ventral ganglion.This fact obviously is a cause of the total reduction of

ventral ganglion in phoronids, as they are attached tothe substrate by exactly the area where the ventral gan-glion should be located. Phoronids have lost both nervecenters that existed in common ancestors of lophophor-ate animals, the apical ganglion (this is a common lossin all lophophorate animals) and ventral ganglion(which is reduced, because the phoronids are attachedto the substrate by an outgrowth of the ventral side ofbody). Thus, the nervous system of phoronids is not justa simplified version of the nerve system patternobserved in brachiopods, but rather represents a resultof a different (compared with the brachiopods) reorga-nizations of the initial body plan. The profound differ-ences in body plans between phoronids and brachio-pods do not permit us to hold the opinion that thephoronids are not a particular phylum of the animalkingdom, but are rather a daughter taxon within theBrachiopoda [14–16]. The absence of nerve ganglia inphoronids should be considered as a secondary phe-nomenon. The development of the definitive nervoussystem of adult phoronids takes part on the base ofintraepithelial nerve plexus. Within the group, the nerveplexus becomes denser in certain areas, resulting in theformation of dorsal and circular concentrations of nerveelements.

Bryozoans are similar to phoronids in terms of bodyplan [1]. It is possible to suppose that characteristic ele-ments of the nervous system in the bryozoans (cerebralganglion and lophophoral nerves) originated at theexpense of further concentration of structures thatalready began arising in phoronids. The lophophoralnerves of bryozoans evidently correspond to the some-what more concentrated circular nerve concentration ofphoronids. As regards the cerebral ganglion of bryozo-ans, it is known to develop (see [27]) as a result of sink-ing of an area of epidermis located dorsally of the epis-tome into the body. This area corresponds to the areawhere the dorsal nerve plexus is located in phoronids.Thus, the nervous system of bryozoans could bederived from the nervous system structural pattern ofphoronids.

However, the existence of a plexus-type nervoussystem by no means excludes its structural differentia-tion and the presence of highly specialized elements.For example, in the nervous system of phoronids thestructural differentiation of the nerve plexus is mani-fested in the stratification of the latter, while the spe-cialized features are revealed as giant nerve fibers. Aswe have shown, all components of the nervous systemin phoronids (except the giant nerve fiber) are orga-nized as an intraepithelial nerve plexus with the strati-fication of constitutive elements. In the direct vicinityof the basal lamina there are bundles of nerve fiberswith synaptic vesicles and synapses (the neuropile) sur-rounded by processes of glia-like cells. The somata ofthe glia-like cells are located mostly above the neuro-pile, while the perikaryons of neurons make up the nextlayer. Above the latter there are densely arranged bod-ies of epidermal cells, whose processes penetrate this

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entire stratified structure and are fastened to the basallamina. The observed differences refer to the relativethickness of the entire plexus and certain of its constit-uents. For example, the density of neurons is the great-est in the dorsal plexus, while the best developed neu-ropile is revealed in the collar plexus. As for the trunkregion, the nerve plexus is the best developed in theanterior trunk area; it is weaker in the ampulla andweakest in the posterior trunk area.

The dorsal nerve plexus obviously represents thecentral organ within the nervous system of phoronids(although its structure still remains within the confinesof more or less dense nerve plexus). According to ourdata, the neurons of the dorsal plexus belong to thesame structural type; these are relatively large cellswith great cytoplasmic volume. If we follow the termi-nology of Fernández et al. [19], their structural organi-zation is the most similar to unipolar neurons of the firsttype. Taking into account that the dorsal nerve plexusgives rise to giant nerve fibers (or a single nerve fiber),we can suppose that it comprises motor neurons besidesother nerve elements. Silen [46] described three nervecells whose processes are fused together to make up agiant nerve fiber.

The circular nerve concentration was described bysome authors [17, 44, 46] in members of the genusPhoronis. According to the data of the cited authors, itruns outside the bases of tentacles and consists mostlyof circular fibers and nerve cells, which Fernández et al.[19], refer to as the fourth type of neurons, i.e., smallbipolar cells. A specific feature of the circular nerveconcentration in Phoronopsis harmeri is that it issunken down to the bottom of the collar fold. Accordingto Mamkaev [9], this could be considered as an initialstage of the sinking of the nervous system into underly-ing tissues; this situation is therefore similar to thatobserved in hemichordates and chordates, where thedorsal nerve cord sinks to the inside of the body in theform of a nerve tube.

In Ph. harmeri circular concentration mostly con-sists of circular fibers and small neurons. The functionsof the circular concentration have not been revealedyet; however, it can take processes from the sensorycells located in the tentacles. If we take into account thegreat number of sensory elements in phoronidan tenta-cles [10, 34], the great thickness of the neuropile layerwithin the circular concentration can easily beexplained. Thus, it is possible to suppose that the circu-lar nerve concentration is mostly a sensory part of thenervous system, whereas the dorsal nerve plexus(which gives rise to motor giant fibers) comprisesmotor elements.

The presence of giant nerve fibers is a characteristicelement of the nervous system in phoronids. Thesefibers usually are used for fast conduction of the nerveimpulses providing escape responses. For example, insquid, the giant nerve fibers conduct impulses that ini-tiate contraction of the muscle wall musculature; as a

result a jet stream is ejected from the mantle cavity[13]. On the other hand, giant nerve fibers are a com-mon feature of the nerve system organization in tube-dwelling animals. For example, in annelids the ventralnerve chain, along with the fibers of common structure,contains giant axons in the neurochords [13]. These arededicated to rapid conduction of nerve impulses initiat-ing contraction of longitudinal musculature and retrac-tion of the animal into the tube. The intraepithelial loca-tion of the giant nerve fiber is characteristic of the gianttube-dwelling animals, the vestimentiferans [8]. Thesesimilarities could be considered as convergences aris-ing independently in different animal groups as a resultof adaptation to the tube-dwelling mode of life.

In the burrowing members of the genus Phoronisthere are two symmetrically located giant nerve fibers,although species inhabiting soft bottoms have only one(left) nerve fiber developed [18]. The members of thegenus Phoronopsis also have only one (left) giant fiber.According to our data, in Ph. harmeri the giant axon isaccompanied by more than 100 nerve fibers of commonstructure. This fact allows us to consider the giant nervefiber to be passing within a longitudinal nerve. The lat-ter nerve is bound to the ectodermal epithelium; there-fore, it can be considered as the result of the denseningof the intraepidermal nerve plexus of the body. Accord-ing to photos presented in a paper by Fernández et al.[19], in Phoronis australis the giant nerve fiber is alsoaccompanied by numerous longitudinal nerve fibers ofcommon structure. Obviously, the interpretation of thegiant nerve fiber as running within a longitudinal nervecould be used with reference to all phoronids. There-fore, we can conclude that phoronids possess one ortwo lateral basal–apical nerves.

The presence of only one (left) giant nerve fiber inall members of the genus Phoronopsis and some spe-cies of the genus Phoronis makes the nervous system ofthese animals asymmetrical. In all likelihood, this con-dition should be considered as a secondary one if it iscompared with the paired symmetrically located giantfibers. Ruppert et al. [40] called our attention to the factthat the asymmetry in the organization of the nervoussystem in phoronids agrees with the absence of the leftmesentery in Phoronis muelleri and compared thesefacts with the well-known asymmetry in deuterostomeanimals.

According to our data, the giant nerve fiber inPh. harmeri is surrounded by modified epidermal cellsmaking up a multilayer envelope resembling a myelinsheath. A similar sheath was also described around thegiant fiber of Ph. australis (see [19]); the latter authorsconsidered the sheath cells to be glial cells. In our opin-ion, the sheath cells retain all the characteristic featuresof epidermal cells: they are fastened to the basal lamina,their cytoplasm contains tonofilaments, and they aredevoid of the characteristic electron-dense granules thatare present in the glia-like cells and other regions of thenervous system of phoronids.



The so-called glia-like cells have been described inmany invertebrates that retain nerve elements in theintegument, namely, in vestimentiferans [20], polycha-etes [39], mollusks [32], and nemerteans [49]. A char-acteristic feature of the fine structure of these cells isthe presence of electron-dense granules, 150–300 nm indiameter, in their cytoplasm. Glia-like cells are locatedin basal parts of the epithelium and often surround, par-tially or entirely, the fibers of the nerve plexus. Thefunctions of these cells are not known and it is notentirely understood to what extent they correspond totrue glial cells.

ACKNOWLEDGMENTS

The authors are deeply indebted to V.V. Yushin,Yu.V. Mamkaev, Yu.P. Lagutenko and all colleaguesfrom Interdepartmental Laboratory of Electron Micros-copy of Moscow State University.

The project was partially supported by Russian Foun-dation for Basic Research (grant no. 08-04-00991).

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