GENERAL AND COMPARATIVE ENDOCRINOLOGY 45, 125- 130 (1981)

A Somatostatin-like Antigen in the Nervous System of an lsopod Porcellio dilatatus Brandt

GILBERT MARTIN’ AND MAURICE P. DUBOIS*

Laboratoire de Biologic Animule, ERA CNRS no 230, Universiti de Poitiers, 86022 Poitiers CPdex; und *Institut Nutionul de la Recherche Agronomique, Station de Physiologic de la Reproduction,

37380 Nouzilly, Frunce

Accepted May 24, 1981

Immunohistological investigations were performed with an antiserum to protein-linked somatostatin (SRIF) in the CNS of a crustacean Porcellio difututus. Discretely located perikarya and fibers containing a SRIF-like substance are completely negative with usual staining. The functional role of this SRIF-like substance is still unknown.

Since the isolation of somatostatin (SRIF)2 and the elucidation of its chemical structure by Brazeau et al. (1973), many papers have been devoted to this neuro- peptide and to its prominent part in verte- brate endocrine regulation. Many immuno- cytological investigations have been made on the central nervous system (CNS) and on various glandular tissues (Dubois, 1980).

Although CNS structure in invertebrates is much simpler than in vertebrates, the existence in the former of substances re- lated immunoactively to vertebrate poly- peptide hormones has been proven with antisera for these specific antigens. These peptide-like substances have been found in protochordates (Fritsch et al., 1978, 1979; Georges and Dubois, 1979, 1980), in insects (Tager et al., 1976; Kramer et al., 1977; Patton et al., 1977; Remy et al., 1977, 1978, 1979, 1980; Doerr-Schott et al., 1978; Gros et al., 1978; Strambi et al., 1978, 1979; Duve and Thorpe, 1979), in gastropods (Grimm-Jorgensen et al., 1975), in pelecy- pods (Fritsch et al., 1975), in annelids (Remy et al., 1979; Alumets et al., 1979),

’ To whom reprint requests should be addressed. * Abbreviations used: CB, central body; CHP,

Chrome hematoxylin phloxin; CNS, central nervous system; FITC, fluorescein isothiocyanate; rG gam- maglobulin; HSA, human serum albumin; PF, paral- dehyde fuschin; PSC, posterior superior cells: SRIF, somatotropin release inhibiting factor.

and in platyhelminths (Schilt et al., 1979). Schaller et al. (1977) found a peptide from Hydra demonstrable in rat embryos; this product isolated from rat embryos has the same effect on hydra cells as the original peptide from Hydra.

The present study contains the first re- port on the immunofluorescent localization of a SRIF-like antigen in the CNS of an isopod, Porcellio dilatatus.

MATERIAL AND METHODS

Adult male P. dilututus (120- 150 mg) in diecdysis or in the C period of Drach (1939) (about 2 weeks after exuviation) were used. They were kept under labora- tory conditions (temperature 20°C and natural illumi- nation).

Dissection und histology. Animals were killed by decapitation. The brains and the ventral nerve cords were fixed overnight, after dissection, in Bouin -Hol- lande fixative without acetic acid, to which a saturated mercuric chloride solution (lo%, v/v) was added. Pieces were washed in running tap water, put in small blocks of agar (2%), then dehydrated in graded ethanols and embedded in paraffin. Five-micrometer serial sections were cut in the transverse plane. Sec- tions were mounted in two alternate sets: one for im- munoreaction, the other for staining with PF or CHP.

Zmmunojluorescence. The immunogen used to ob- tain rabbit anti-SRIF antisera was prepared with cyclic SRIF (a gift of Dr. R. Guillemin, the Salk Institute) linked with HSA (ratio: 2/10, w/w) by way of glutaral- dehyde, as previously described concerning the im- munization of rabbit against LH-RH (Dubois, 1976). The procedure for immunization, indirect immunoflu- orescence reaction, and controls of specificity were

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126 MARTIN AND DUBOIS

reported elsewhere (Dubois, 1975, 1976). In brief, the anti-SRIF serum was diluted for use at the appropriate concentration (l/100- l/200) in a Verona1 buffer (0.1 M ph 7.4) to which had been added sufficient 0.5% HSA to saturate anti-HSA antibodies.

A counterstain (Evans blue 0.01%) was added to increase contrast and eliminate any green background. Controls of specificity included: (1) the use of (nonim- mune) normal rabbit serum instead of the specific anti-SRIF antibody: (2) the use of the fluorescent conjugate (sheep anti-rabbit rG immunoglobulins labeled with FITC) alone, omitting the specific anti- SRIF in a previous step; (3) the preincubation of anti- SRIF with either somatostatin (1 mg/ml undiluted anti- serum), other neuropeptides (LH-RH, TSH-RH, ocytocin. vasopressin, neurophysin), or crude porcine brain acetone extracts.

Srtrini~g. Sections were stained with PF or CHP (Gomori).

RESULTS

Positive immunoreactions were observed mainly in the protocerebrum, the trito- cerebrum, the subesophageal ganglion, and the ganglia of the ventral cord. No spe- cific fluorescence was observed either when using the anti-SRIF antiserum previ- ously saturated with somatostatin, or in the absence of the specific anti-SRIF in the first step, or when using sheep FITC conjugate Ig alone. Preincubation of anti-SRIF with

other neuropeptides and porcine brain ex- tracts did not inhibit the specific immuno- reaction.

1. Irnmunoreactive Cells and Fibers of the Protocerebrum

The protocerebrum is composed of corti- cal areas (LSC, PSC) and of different neuropiles. (The terms used for the differ- ent parts of the protocerebrum cortex are those given by Walker (1935) for Oniscus (Isopoda).) No clear fluorescence is seen in the eyestalks or in the sinus glands. Two zones in the anterior part of the protocere- brum have immunoreactive perikarya: ‘ ‘ the lateral superior cells” and “the posterior superior cells” (PSC). Both groups are re- spectively divided into two symmetric cell groups. Within these four cell groups five to eight small polyhedrally arranged perikarya (about 10 pm/23 pm) (Fig. 1) present a clear fluorescence. Control tests with antisera saturated with cyclic SRIF proved totally inactive (Fig. 2). At the back of the PSC cortical area, at a short distance lateral to the median sagittal plane of symmetry of the protocerebrum, pear-shaped perikarya show a strong fluorescence (Fig. 3). The

FIG. superic

lateral

SOMATOSTATIN IN THE CRUSTACEAN Porcellio dilatatus 127

FIG. 2. Contiguous Arrows, artifacts.

serial section, control test: antiserum saturated with cyclic SRIF. (x 1500).

most striking structure in the protocere- brum is the very bright fluorescence of the central body (CB); at this structure axons from various parts of the brain converge and the neuropilar structure suggests an as- sociative function (Bullock and Horridge, 1965). In P. difatatus the CB is a quarter circle-shaped uniform mass of neuropile which runs transversely across the center of the protocerebrum (Fig. 3). The CB ta- pers into two fluorescent fiber tracts which extend to the tritocerebrum (Fig. 4). Ac- cording to Sharma (1977) it could be a part of the “fibrous connections between the tritoglobular tract and the central body.” These tracts consist of axons with relatively large diameters (Fig. 4). Above the CB, the upper part of the neuropile of the pro- tocerebrum is studded with fluorescent tiny fibers.

2. Immunoreactive Cells and Fibers of the Tritocerebrum

Under the antennary lobe, in the cir- cumesophageal connectives lie two sym- metric neuronal groups in which a perikary- on (about 25 pm/15 pm) probably mono- polar, with angular shape, shows a clear

128 MARTIN AND DUBOIS

rows, immunofluorescent material.

fluorescence (Fig. 5). In the contiguous neuropile some fibers are immunoreactive.

3. Immunoreuctive Cells and Fibers of the Subesophageal Ganglion

The subesophageal ganglion lies between the ventral nerve cord and the tritocere- brum, it gives rise to the usual four pairs of nerves of the mouth appendages. Briefly, this ganglion is built of four cortical areas and four neuropiles related to the mouth appendages. Each cortex contains few small perikarya showing a positive immu- nofluorescent reaction. Fine immunoreac- tive axonal processes run throughout the neuropiles of the subesophageal ganglion.

4. Immunoreuctive Cells and Fibers of the Ventrul Nerve Cord

The ventral cortex of each ganglion pre- sents small perikarya with a bright fluores- cence (Fig. 5). The contiguous neuropile contains small fibers with fluorescent var- icosities. All these fluorescent perikarya and fibers are Gomori-and PF-negative and, consequently, seem different from the neurosecretory cells of this species (Martin, 1972).

ve. Ar-

DISCUSSION

In the entire CNS immunocytological staining reveals precisely located discrete perikarya and fibers containing a SRIF-like substance; these data from P. dilututus are the first to suggest that a SRIF-like peptide occurs in a crustacean. Earlier, ‘the pres- ence of SRIF-like antigen was demon- strated in the neurosecretory cells of the pars intercerebralis of adult Locustu (Doerr-Schott et al., 1978; RCmy and Dubois, submitted for publication). Among the invertebrates, SRIF-like substance, es- pecially in insects, is mainly stored in perikarya; in the isopod P. difututus CNS there are few SRIF-positive cells but a huge number of varicosities of the CB fibers containing a SRIF-like substance. This dis- crepancy may indicate that the CB is a stor- age center for this peptide. Our results are preliminary and without experimental con- ditions we cannot decide about the physio- logical role of the peptide. Although the function of the SRIF-like peptide in this as- sociative structure (CB) is unknown, it may be suggested that this substance might serve as a neurotransmitter or modulator of neuronal activity.

SOMATOSTATIN IN THE CRUSTACEAN Porcellio dilututus 129

P

FIG. 5. Localization of immunoreactive perikarya in Porcellio CNS anterior part: a ventral view. Proto, protocerebrum; E, eyestalk; LSC, lateral superior cells; PSC, posterior superior cells; SG, sinus gland. Deuto, deutocerebrum. Trito, tritocerebrum; AL, antennary lobe; OA, esophageal area. SOG, sub- esophageal ganglion; MD, MX,, MX?, PMX, cortical areas of mouth appendages nerves. VNC, ventral nerve cord; TG,, first thoracic ganglion. Dashed lines set limits of cortical areas (Walker, 1935) Hachures. central body.

ACKNOWLEDGMENT

The authors wish to thank Mr. Lebourg and Mr. Terriot for technical assistance and Mme Nasreddine- Royer for typing the manuscript.

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