ultrastructure of the sinus gland and lateral cephalic nerve plexus in the isopod ligia oceanica...

GENERAL AND COMPARATIVE ENDOCRINOLOGY 52, 38-50 (1983)

Ultrastructure of the Sinus Gland and Lateral Cephalic Nerve Plexus in the lsopod Ligia oceanica (Crustacea Oniscoidea)

GILBERT MARTIN,*JRENI~E MAISSIAT,? AND PATRICK GIRARD*

Service Universitaire de Microscopic e’lectronique applique’e d la biologie, Poitiers; *Laboratoire de Biologie animale, ERA 230 “Physiologie et Ge’ne’tique des Crusta&,” Universite’ de Poitiers, Poitiers; and

TLaboratoire de Biologie cellulaire, Facultt de Mkdecine et de Pharmacie, Poitiers, France

Accepted September 24, 1982

Isopod crustaceans have two distinct cephalic neurohemal organs: the sinus gland (SG) and lateral cephalic nerve plexus (LCNP). The present study of Ligia oceanica was designed to ascertain the ultrastructure, during the moulting cycle, of the terminals constituting the SG and LCNP, both of which store and release neurosecretory material, and to trace these terminals to their probable origin in neurosecretory perikarya. The SG was observed to contain four types of terminals (I, II, III, and IV) assigned, on the basis of the appearance of their neurosecretory granules, to four types of neurosecretory cells in the protocerebrum (pi, l32, Bi, and B,,). When the same morphological criteria were applied to the LCNP, two types of terminals were found-III’ and IV’. Type III’ was thought to originate in the B, plexus cells and in the Bz cells of the suboesophageal ganglion. The origins of Type IV’ terminals were believed to be the B, and B, cells of this ganglion. Release from both the GS and LCNP occurred by exocytosis. The discussion attempts to relate the ultrastructural variations observed in the SG and LCNP with existing data on the neuroendocrine regulation of the moult. Such regulation involves the two antagonistic hormones (moult-inhibiting and mot&accelerating) which determine the circulating ecdysteroid level. It is also suggested that the plexus cells are the site of synthesis of a factor controlling the release of the exuviation factor.

Two distinct cephalic neurohemal organs are at present known to exist in isopod crustaceans: the sinus gland (SG) or pseudo- frontal organ of Graber (1933), joined to the optic lobe of the protocerebrum where the lobe’s outer medulla meets the ganglionic lamella; and the lateral cephalic nerve plexus (LCNP) described by Besse and Le- grand (1964), a nerve junction very heavily loaded with neurosecretions and joined to the moulting gland or Y organ (Gabe, 1953).

Sinus gland ultrastructure was studied in Porcellio dilatatus by a member of our group (Martin, 1972), who observed that it contained neurosecretory cell terminals inter- mixed with glial cells. The following three types of terminal were defined on the basis of morphological and cytochemical criteria:

* TO whom reprint requests should be addressed: Biologie animale, 40 Av. du Recteur Pineau, 86022 Poi- tiers CCdex, France.

Type I, containing granules 2000 to 2700 A in diameter; Type II, with granules 1000 to 1650 A in diameter! and Type III, with granules 1000 to 1350 A in diameter.

By studying the repercussions on sinus gland structure of the removal of the central region of the brain, it was possible to es- tablish links between the three types of terminal on the one hand and the four types of neurosecretory cell in the protocerebrum on the other. Thus, Type I terminals were found to come from l3 and A cells, and Type II terminals, from B cells; consequently, it was concluded that Type III terminals come from y cells (Martin 1972, 1981).

The anatomy and histology of the LCNP have been studied in seven species of isopod crustacean: Porcellio scaber and On- iscus asellus (Messner, 1963, 1966), Por- cellio dilatatus (Besse and Legrand, 1964), Anilocra frontalis (Juchault and Legrand,

38 0016~6480/83 $1.50 Copyright 0 1983 by Academic Press, Inc. AU rights of reproduction in any form reserved.

ULTRASTRUCTURE OF ISOPOD NEUROSECRETORY ORGANS

L oceanica .

FIG. 1. Diagram of Ligia oceanica central nervous system: SG and LCNP. Md, MXr, 2, PMX cortical areas of mouth appendages nerves. LG: Lamina ganglionuris, ME: medulla externa, MI: medulla interna. nMd, nMXr, nMX2, and nMXp, which come from the suboesophageal ganglion (SOG), nAz, originating in the tritocerebrum; nGS, connected to the protocerebrnm via the moulting gland; nST, which comes from the stomach ganglion and is part of the stomatogastric system; and nRi, which comes from the ipsilateral cardiac nerve, itself originating in the stomach ganglion. (Prom Mocquard et al., 1971, and modified by us.)

1963, Sphaeroma serratum (Chaigneau, 1966), Helleria brevicornis (Delaleu and Giry, 1967), and Ligia oceanica Mocquard et aZ., 1971; Juchault and Kouigan, 1975). Maissiat and co-workers (1979) observed the ultrastructural changes of the LCNP in S. serratum males after the puberal moult. In L. oceanica, light microscopy showed that the LCNP is formed by the junction of 8 nerves (Fig. 1).

Transport of neurosecretory products was

observed in the LCNP, especially along nMXr, nGs, and nSt. In the first of these three nerves Juchault and Kouigan (1975) noted the presence of acidophihc and ba- sophilic products, but only of b’asophific products in the other two. One of the neurosecretory substances in the LCNP was observed to be synthesized by two neurosecretory plexus cells. The entire LCNP, which adheres closely to the Y or bathed by the hemolymph.

40 MARTIN, MAISSIAT, AND GIRARD

We thought it of interest to examine by electron microscopy the cyclical changes of the LCNP and sinus gland during the moult in L. oceanica, in order to clarify the part each organ plays in regulating the moult.

MATERIALS AND METHODS

We chose to study L. oceanica because we had al- ready identified the criteria which permit this animal to be observed during different phases of its moulting cycle (Maissiat, 1978). The present report deals essen- tially with males weighing about 200 mg during the following stages: diecdysis (Cz and C,), proecdysis (Do-i.e., apolysis-and Dz+), interecdysis, i.e., the period between posterior and anterior exuviation (Dz to A), and postecdysis (AB).

Sinus glands were dissected out and individually fixed. To study the plexus, we fixed portions of the head and later delimited the plexus on semithin sections stained with toluidine blue.

The fixation procedure consisted of quickly removing the organs from the animals into a fixative comprising 3% giutaraldehyde in 0.4 M cacodylate buffer (pH 7.3), fixing for 2 hr at a cold temperature in the same solution, postfiiing for.1 hr at room temperature in 1% osmium tetroxide buffered with cacodylate, and embedding in Araldite. Ultrathin sections were cut with a Reichert OMU 3 ultramicrotome, mounted on bare grids, stained with uranyl acetate and lead citrate according to Reynolds (1963), and examined with a Hitachi HU 11 Cs electron microscope. Glycogen cytochemistry (PATAg) was processed as described by Thiery (1967). Diameters or axes of neurosecretory granules were measured with a Polaron micrometer.

Data were processed using an Olivetti P 602 com- puter, programmed to give the mean diameter and standard error for the neurosecretory granules observed in each terminal.

RESULTS

Sinus Gland (SG)

I. Structure at Diecdysis

In Ligia, the sinus gland was observed to be joined to the ventral surface of the optic lobe; it consisted of numerous diverticula (Figs. 2 and 3) which gave it a fringe-like appearance unusual in Oniscoidea, whose sinus gland is generally more compact. Pe- ripheral diverticula stained intensely with paraldehyde-fuchsin and hematoxylin. In

addition to glial cells, a few terminals stained with phloxine.

As visualized by electron microscopy, each diverticulum, wrapped in a sheath or neurilemma, included different types of terminals, as well as glial cells-the only nu- cleated elements. A branched hemolym- phatic sinus was joined to the outer surface of the neurilemma at different points along the diverticula.

The terminals inside the diverticula contained mainly neurosecretory granules and mitochondria, as well as glycogen revealed by PATAg (Fig. 4). The morphology of the neurosecretory granules enabled us to dis- tinguish the following four types of terminal.

Type I. Terminals containing large, spherical, electron-dense granules with a diameter between 1600 and 1900 A.

Type II. Terminals containing very large granules which were electron-dense, mostly spherical and more than 2000 A in diameter.

Type ZZZ. Terminals with medium-sized, electron-dense spherical granules with a diameter ranging from 1000 to 1400 A.

Type IV. Terminals with irregularly shaped granules of different sizes, with different degrees of electron density. The diameter of the largest of these granules varied from 650 to 1000 A.

Also in the sinus gland, we found terminals comprising mainly “vesicles” (or “vesiculae”) with contents of low electron density. The few dense granules indicated that these terminals belonged to Types III and IV.

ZZ. Sinus Gland Variations during the Moulting Cycle

During the moulting cycle, variations were observed in the load of all terminals constituting SG diverticula. It was small after anterior exuviation and gradually increased until the Cd stage. During Cs and Do, a few granules were released but this had little effect on the overall SG load which

FIG. 2. Sinus gland. Note neurosecretory terminals (arrows) with granules of different sizes and glial cells (GC). Ne: Neurilemma. x 7920.

FIG. 3. Diverticula of sinus gland (SG). x7920. FIG. 4. Particles of glycogen revealed by PATAg (circles). x 15680. FIG. 5. Cf stage. Release by exocytosis of a neurosecretory granule (arrow). x 36,000.

41


continued increasing until Dz + . There is thus reason to think that more granules are transported than released. Release occurred by exocytosis (Fig. 5) chiefly from Dz- to the end of Cl.

At exuviation, the release of granules re- duced their numbers in the terminals, where we generally noted the presence of a population of small synaptoid vesicles, about 300 A in diameter. During stages C2 and D2+, very large granules-the largest had a diameter of over 2500 A-appeared in the Type II terminals. These granules were often irregular in shape and did not appear to be released but probably degenerated in the terminals. We took their increased size as an indication of their nonrelease.

The Lateral Cephalic Nerve Plexus (LCNP) and the Origin of Its

Axon Terminals

We began by studying the neurosecretory cells of the suboesophageal ganglion, whose terminals help to form the LCNP, and then examined the structure of the LCNP, especially its intrinsic neurosecretory cells during diecdysis, as well as the variations in these structures during the moulting cycle.

I. The Neurosecretory Cells of the Suboesophagkal Ganglion

The existence of Type A neurosecretory perikarya at the base of the mandibular and maxillipede nerves was reported in light microscopy studies (Martin, 1971; Juchault and Kouigan, 1975) but was not confirmed by the present electron microscopic observations. On the other hand, in the course of these observations, the individuals visualized during diecdysis enabled us to describe three types of B cell-B, B2, and B,- also located near the mandibular and maxillipede nerves and displaying slight affinity for the stains usually applied to neurosecretory material.

B, cells (Fig. 6). Electron microscopy

showed these cells to be ovoid (17 x 13 km) with a spherical nucleus about 9 pm in diameter. Within their cytoplasm, there was slight development of the rough endoplasmic reticulum (RER) in the form of small cisternae dilated in varying degrees and ar- ranged concentrically in relation to the nu- clear envelope. The neurosecretory granules were subspherical and electron dense. Their mean size was 1012 t 40 A and their granular load was always small.

Bz cells (Fig. 7). These ovoid cells were larger than the B, cells and measured 25 X

15 pm; they contained many vacuoles caused by the dilatation of a large number of RER cisternae. Aggregates of neurosecretory granules were distributed between the stacks formed by these cisternae. Gran- ules were subspherical and electron dense and measured 1064 f 44 A. Multivesicular bodies were sometimes visible near the granules. Axonal transit of these granules was observed in the nMXl nerve which helps to form the LCNI?

B, cells (Fig. 8). These cells were also ovoid (20 x 16 pm) with a spherical nucleus about 8 km in diameter. They were characterized by the presence of stacks of six or seven slightly dilated RER cisternae. Their neurosecretory granules were spherical, consisting of a dense matrix, and their mean size was small (673 +- 20 A).

II. LCNP Structure during Diecdysis

Axon terminals constituting the LCNP (Fig. 9). The enlarged part of the LCNP, adjacent to the Y organ, was seen to be a fairly large neurohemal organ enclosed in a thin neurilemma-type sheath, which did not penetrate the Y organ but was 380 A away from it.

The LCNP was observed to be formed by a large number of axon terminals of various sizes, sometimes with glial cells run- ning along them. Most of these terminals contained large numbers of neurosecretory granules which were roughly spherical, in-

FIG. 6. B,- cell of suboesophageal ganglion (SOG). Note the small cisternae of rough endoplasmic reticulum (RER) and few number of neurosecretory granules (NSG). N, nucleus; G, golgi apparatus. x 16,000.

FIG. 7. Bz- cell of SOG. Note abundance of vacuoles (V) caused by the dilatation of RER cistemae and NSG (arrows). x 16,000.

FIG. 8. B,- cell of SOG. The stacks of cisternae are slightly dilated and the size of NSG is small (arrows). x 10,000.

43


dicating the presence of a large load during diecdysis. The axoplasm also contained rod- like and occasionally annulate mitochondria, as well as mitochondria with lamellar crests, glycogen, microtubules, and a few clear vacuoles. Some terminals contained no neurosecretory material at all.

If axon terminals are classified according to the size and density of their elementary granules, the lateral cephalic nerve plexus may be said to include two types of neurosecretory terminals: Type III’, which contains granules whose diameter varies from 1000 to 1400 A and corresponds to the Type III observed in the sinus gland; and Type IV’, which consists of granules with a diameter of 650 to 1000 A and corresponds to Type IV in the sinus gland.

Granules which were only slightly electron dense or seemed optically empty were often observed next to the above two electron-dense types. However, they will not be considered as a separate category, as they were present in both types of LCNP terminal. This question will be raised in the discussion.

Plexus cells (Fig. 13). In the area near the Y organ, the LCNP was seen to contain two neurosecretory cells measuring 18 X 13 Frn, earlier observed by Kouigan (1973) using light microscopy. This author showed that their cytoplasm included clumps of neurosecretory material which stained more easily with paraldehyde-fuchsin than chrome hematoxylin. On the ultrastructural level, these two cells with clear, ovoid nu- clei, 9 or 10 pm in diameter, displayed very diffuse chromatin. These cells contained many RER cisternae, mostly dilated and dispersed throughout the cytoplasm, abundant mitochondria, dictyosomes displaying little activity, and only a few neurosecretory granules in diecdysis. The latter granules were dense and spherical, with a diameter of the order of 1300 ? 42 A. No lysosome-type structures were observed.

The staining affinity of the two neurosecretory plexus cells near the Y organ indicated them to be Type B of the Matsumoto

classification (1959). However, this type of B cell seems distinct from all the others, both for its location and ultrastructural fea- tures-larger granules and less developed RER-hence the term B, chosen to desig- nate these two cells.

III. Variations in the LCNP during the Moulting Cycle

During the period extending from the Do stage until the end of the AB phase, exo- cytotic figures as well as a very large number of clear synaptoid-type vesicles appeared in both types of LCNP terminal (Figs. 10-12). The diameter of these vesicles was about 300 A. Electron micrographs thus revealed indications of an intensive phase of hor- monal release. The number of neurosecretory granules in the plexus cells increased gradually but considerably from stages D,-, to Dz- (Fig. 14). This development is connected with the intense activity of the dictyosomes, which were observed to bud off, releasing large numbers of vesicles with electron-dense contents.

These vesicle accumulations in the cytoplasm reached their maxima during DZ-, gradually diminished during the AB period, and remained small throughout diecdysis. The functioning of B, cells therefore appears to be cyclic, which distinguishes them from other known B-cell types.

DISCUSSION

The present morphological study shows that there are four types of terminal in the sinus gland of L. oceanica, in which granule release, occurring abundantly during inter- and postecdysis, appears to take place by exocytosis.

Despite the uncertainty of morphological criteria, based on the appearance of the granules, we suggest that the various neurosecretory perikarya correspond to the types of terminal in the SC. In this connection, research on the neurosecretory cells in the protocerebrum of L. oceanica (Martin, 1981) showed that, like other Iso- poda examined (Chataigner et al., 1978;

FIG. 9. Lateral cephalic nerve plexus (LCNP) ules and low electron density granules (arrows).-

FIG. 10. Do stage: Note large number of synap FIGS. 11 and 12. Dz- stage. Exocytosis figures

x 60,000.

. Neurosecretory endings with electron-dense gran- M, mitochondria; V, vacuole. x 20,000. ‘toid vesicles in LCNP terminals (arrows). x 20,000. in LCNP terminals (arrows). SV, synaptoid vesicle.

45


FIG. 13. CJ stage. Plexus cell. RER cisternae are dilated and there are few MSG. X 24,000. FIG. 14. Dz- stage. Plexus cell. Note abundance of NSG. ~24,000.

ULTRASTRUCTUREOFISOPODNEUROSECRETORYORGANS 47

TABLE 1

Sinus gland

Cell types Perikary a 5Pe of terminal

Size of granules (A)

Protocerebrum Pl

P2

Bl

BIJ

Front region of the protocerebrum and lateral Hanstrom organ (1450-1600 A)

Front region of the protocerebrum (cortex I) (>1600-2600 A)

Front region of the protocerebrum (cortex I) (1000-1320 A)

All cortical areas (650-1000 A)

I 1600-19

II >20

III lOOO-14OQ

IV 65O-!OOO

Martin, 1981) this species exhibited at least four types of neurosecretory cell, desig- nated as pi, &, B, (B ubiquitous), and Bi, respectively. Table 1 assigns each of these cell types to a type of cell terminal in the SG.

According to the morphological criteria applied here, the terminal population making up the LCNP can only be divided into two groups containing granules with diameters of either more or less than 1000 A, respectively.

This separation according to granule size might reflect different chemical composi- tions-aminergic for the smallest granules and peptidergic for the largest (Knowles, 1965).

The presence of neurosecretory A cells reported in the suboesophageal ganglion when examined by light microscopy (Ju- chault and Kouigan, 1975) was not confirmed here by electron microscopy. On the other hand, we did observe that this gan-

glion exhibited first a B2-cell type eharac- terized by its RER and large granule load, and second, microgranular B, cells (673 rt 20 A) whose terminals, like those of the type, were found both in the SG and LC (Table 2).

Comparative ultrastructural st SG and LCNP showed that des atomical link between the two neu organs via the nSG nerve (Fig. l), LCNP terminals differ, inasmuch a and Type II terminals in the sinus gland (corresponding to gi and p2 cells) are not found in the LCNP. Further, the sirni~~r~ty between terminal Types III and IV in organs does not necessarily mean the identical, since in the sinus gland Type III terminals probably originate from rosecretory cells. Similarly, in the Type III’ terminals must corn plexus cells, and consequently would correspond to the composi uitous cell type which we describe

TABLE 2

LCNP

Type Perikary a 5Pe of terminal

Size (A,

SW B2 BU hn

Plexus cells BP

md and mXr, ganglions 1064 f 44 A III’ 1000-1400 id 1012 & 40 A IV’ 650-1000 id 613 rt 20 zk IV’ 65~-iO~~

PNCL 1300 i 42 A III’ lOOO-14QO


nection with the suboesophageal mass (cortex of the mandibular and maxillular ganglions).

The existence of clear granules in certain Type III and IV terminals of the SG, as well as in both these types in the LCNP, seems to correspond to a fixation artifact connected with granule maturation.

At the present time, various investigators (Vernet, 1976; Maissiat, 1978) consider that the moult in crustaceans is determined by the maintenance of a balance between MIH (moult-inhibiting hormone) and MH (moult hormone, i.e., ecdysone). The existence of an MAH (moult-accelerating hormone) seems to be supported, in Decapoda, by the results of MacWhinnie (1962), MacWhinnie et al. (1972), and Farges (1973) and in the Isopod S. serratum by the observations of Charmantier (1978). In the oniscoid P. di- Zatatus, we suggested that the length of the moulting cycle is controlled by the two antagonistic neurohormones, MIH and MAH. However, the opposite results were ob- tained in L. oceanica and P. dilatatus after both were subjected to “decerebration” (removal of the median region of the protocerebrum), so that no general pattern can be suggested for endocrine regulation of the moult.

Nevertheless, Table 3 summarizes our observations in L. oceanica regarding the changes in neurosecretory cells in the SG and LCNP, on the basis of the variations noted in circulating ecdysteroid levels. In this connection, “decerebration” short- ened the intermoult in Ligia; in Porcellio, it caused anecdysis in males and lengthening of the intermoult in females. In both species, the PI cells in the protocerebrum may be assumed to have the same function, namely, to secrete MAH, which stimulates ecdysone synthesis by the Y organ, since both the structure of these cells and their location in the median part of the protocerebrum are similar in Porcellio and Ligia. The neurosecretory load admittedly reaches its maximum later in Ligia (at D2 +) than in Porcellio (end of the C period), but the ex-

ocytosis of the large Type I granules corresponding to these p1 cells in the sinus gland occurs chiefly in Ligia during phases C3 and Do, i.e., at points in time-just be- fore the ecdysone secretion peaks--at which MAH secretion may logically be taken to occur (Table 3).

To explain the contradictory results ob- tained in Ligia and Porcellio by removing the median part of the protocerebrum (respectively, shortening of the moult-Moc- quard et al., 1971-and anecdysis in males or lengthening of the intermoult in females-Martin et al., 1979, 1980), the ana- tomical differences between the two species must be taken into account. Ligia, un- like Porcellio, has two separate symmetrical locations for its PI cell stacks, one in the median area of the protocerebrum (as in Porcellio) and the other at the junction of the optic lobe medullae (lateral Hanstr(im organ). Removal of the median region of the protocerebrum therefore completely deprived Porcellio of PI cells but left one of Ligia’s pair of p1 cell stacks intact. If, in addition, the chief source of MIH in both species is considered to be a protocerebral cell type (B) (Martin, 1981), depletion of MIH could conceivably shorten the intermoult in Ligia, while in Porcellio males, the moulting cycle would be blocked by the ab- sence of MAH. In Porcellio females, where the intermoult is only lengthened, the non- functioning of the Y organ might be pal- liated by an ovarian source of ecdysone.

In the SG of Ligia, the large number of synaptoid vesicles mostly observed in Type III and IV terminals from phases Dz+ to Cl might partly be due to abundant release of MIH. As regards the plexus, its advanta- geous position next to the Y organ might mean that by releasing large amounts of MIH from Type III’ or IV’ terminals, the LCNP could control the fast drop in circulating ecdysteroid levels observed after stage Dl”’ (Maissiat, 1978). During inter- and postecdysis, simultaneous release of MIH by the SG and LCNP would enable this level to be adjusted to the very low level indis-

ULTRASTRUCTURE OF ISOPOD NEUROSECRETORY ORGANS

TABLE 3 OUR OBSERVATIONS AND HYPOTHESIS ON SG AND LCNP FUNCTIONING, REGARDING THE CIRCULATING

HAEMOLYMPH ECDYSTERO~DS LEVELS DURING THE MQLTING CYCLE

- AB . cl . cz . c3 . G . Do . d .D;' ~3,~' . &* . D; . +++ t A++/

+++/++++~+++ S.G I

t t t ? Exocytosis EXQcytOSiS Exocytosis (endings III, IV) (endings I) (endings III, IV:

+MIH ? +MAH? -'MIH?

F.Xocytosis Exocytosis (endings III', Iv') (endings III', Iv'

-+ MIH ? .- ._I--.-... -.- -...-............................-........" . ..-.............-...................... rt..MIH-.t -.--...........

+-H--b i-k+ L.C.N.P. B cells

k+++

$ EHRF ?

IOOb

Ecdysteroids level of

IS, Ligia oceanica d CPP,tWlJ

/

'8

\ *

50 w * \

* 25 *

/\ /

S---x +--*

0 L

. AB . C, . q . Cj . C4 . Do . D; SD;' .?

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ultrastructure of the sinus gland and lateral cephalic nerve plexus in the isopod ligia oceanica...

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