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Peptidergic neurons in barnacles: an immunohistochemical study using antisera raised against crustacean neuropeptides.


The morphology of the cirripede central nervous system is unique amongst arthropods in that it has undergone extensive modification associated with the sessile mode of life in the adult barnacle. Indeed, the Bauplan of the arthropod nervous system is scarcely discernible. The supra-esophageal ganglion is a simple bilobed structure with no external topographical features distinguishing a proto-, deutero-, or trito-cerebrum, and it appears to be principally involved in neural integration of information from the lateral and median photoreceptors. There is no trace of a sub-esophageal ganglion; the circumesophageal connectives join a ventral ganglionic mass that is sometimes referred to as the thoracic ganglion. There is no trace of an abdominal ganglion (Gwilliam and Cole, 1979).

Although much is known about the neurobiology of the barnacle CNS, particularly with regard to the photoreceptors and the neural networks involved in cirral movement (review by Gwilliam, 1987), very little is known about the nature or anatomy of peptidergic cells in the barnacle CNS. At the light microscopic level, Gomori-positive neurons have been observed in the supra-esophageal and ventral ganglion (Barnes and Gonor, 1958a,b; Van den Bosch de Aguilar, 1976, 1979). At the electron microscopic level, membrane-bound electron-dense vesicles reminiscent of neurosecretory granules have been observed within axons in the median ocellar nerve of Semibalanus cariosus (Fahrenbach, 1965) and Chirona (Balanus) hameri (Clare and Walker, 1989); these vesicles apparently constitute a neurohemal area. Recently, Gallus et al. (1997) demonstrated the presence of FMRFamide immunopositive neurons in the ventral ganglion of Balanus amphitrite.

Because the morphology of the barnacle nervous system is so unusual, and because peptidergic neurons in those systems are so little known, the methods of immunohistochemistry were used to define and map neurons that might be producing peptides homologous to those now identified in some decapod crustaceans. From an evolutionary viewpoint, it was also of interest to determine whether peptidergic neuronal networks structurally homologous to those known in higher crustaceans could be identified in the greatly modified CNS of barnacles.

Materials and Methods

Animals and tissue preparation

Specimens of Balanus balanus were collected from rocks at extreme low-tide level at Church Island, Menai Strait, UK, and Balanus perforatus from rocks at mid-tide level at Concarneau, Brittany, France. Chirona (Balanus) hameri specimens were obtained by dredge from Modiolus beds off Langness, Isle of Man. Nervous systems were microscopically dissected under ice-cold saline, and since non-nervous tissue produces unacceptably high background staining, all of it was removed. Complete nervous systems were pinned with cactus spines to small pieces of Sylgard. Tissues were fixed for 8 h at 4 [degrees] C in Stephanini's fixative (Stephanini et al., 1967), or in 4% paraformaldehyde, 1% EDC (1-ethyl-3,3[prime]-dimethylaminopropyl-carbodiimide) in 0.1 M phosphate buffered saline (PBS), pH 7.4, for 8 h at 0 [degrees] C, washed in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.5 M sucrose, and permeabilized by extensive (24 h) incubation in PBS containing 0.5% Triton X-100 containing 0.02% sodium azide (PTX).

Immunohistochemical techniques

Nervous systems were incubated in primary antibody, diluted in PTX, for 72 h. Antisera (and dilutions) used were (a) anti-molt-inhibiting hormone (MIH), Carcinus 1:500 (Dircksen et al, 1988); (b) anti-crustacean hyperglycemic hormone (CHH), Carcinus 1:500 (Dircksen et al., 1988); (c) anti-red-pigment-dispersing hormone (RPCH) 1:250 (Schooneveld and Veenstra, 1985); (d) anti-pigment-dispersing hormone ([Beta]-PDH) 1:1000 (Dircksen et al, 1987); and (e) anti-crustacean cardioactive peptide (CCAP), 1:250 (Dircksen and Keller, 1988). After extensive washing in PTX (24 h), nervous systems were incubated in goat-anti-rabbit fluorescein isothiocyanate (GAR-FITC) (Sigma) 1:50 in PTX for 24-48 h, washed extensively in PTX, mounted in glycerol:PTX 1:1, and viewed under a fluorescence microscope. Permanent preparations were subsequently made by reincubation of tissues in goat-anti-rabbit IgG (Sigma) 1:100 (in PTX without sodium azide) (24 h), washing for 24 h in the same buffer, incubation in peroxidase-anti-peroxidase (PAP) (Sigma) 1:200 (24 h), and visualization with 3-3[prime]-diaminobenzidine hydrochloride as detailed by Dircksen et al. (1991); this was followed by graded ethanol dehydration, clearing in methyl benzoate, and mounting in DPX.

Specificity tests (preabsorbtion controls) were performed for antisera that yielded positive results by incubating (24 h 4 [degrees] C) 1 [[micro]liter] of antiserum with 10 nmol of the appropriate (synthetic) peptide dissolved in 10 [[micro]liter] PBS. After dilution, the antibody was then used for immunohistochemistry as detailed above.


Despite extensive investigation, immunopositive structures were never observed using MIH, CHH, or RPCH antisera. Nevertheless, PDH- and CCAP-immunoreactive (IR) neurons were consistently seen in preparations of B. balanus. B. perforatus, and C. hameri. Essentially, all species exhibited broadly similar PDH- and CCAP-IR structures; species differences were insignificant. Preabsorption controls completely abolished immunoreactivity.

PDH-immunoreactive neurons

The most striking and consistently observed immunoreactive structures seen in all species were a pair of large (30-50 [[micro]meter] diameter) anterio-ventral perikarya on the surface of the neuropil of the ventral ganglion (VG) ([ILLUSTRATION FOR FIGURE 1D-F, H, I OMITTED]; [ILLUSTRATION FOR FIGURE 2 OMITTED]). These cells projected a large, conspicuous (often beaded) axon along the great splanchnic nerve (GSPN), branching extensively at the junction of this nerve with the segmental splanchnic nerve (SSN) [ILLUSTRATION FOR FIGURE 1C OMITTED]. Additional branches from the axons ran to the posterior of the VG, and also to the midline, terminating in an extensive plexus ([ILLUSTRATION FOR FIGURE 1D, F, H OMITTED]; [ILLUSTRATION FOR FIGURE 2 OMITTED]). Fine nerves from the SSN, each containing immunopositive dendrites, branched extensively over adjacent musculature; the attrahens and anterior prosomal muscles were particularly well innervated. When attrahens muscles were examined, a consistent pattern of immunopositive dendrites were seen [ILLUSTRATION FOR FIGURE 1J, K OMITTED].

In a few (ca. 5-10) preparations (particularly C. hameri), a group of three pairs of perikarya were seen on the anterio-dorsal surface of the VG. The anterior two pairs of perikarya projected fine axons ipsilaterally and contralaterally along the GSPN, whilst the posterior pair projected axons ipsilaterally towards the GSPN, with a fine, posteriorly directed branch [ILLUSTRATION FOR FIGURE 3 OMITTED]. It should be emphasized that these neurons were not often visible in B. perforatus or B. balanus, but occasionally four immunopositive axons were observed in the GSPN. The large axon could invariably be traced to the anterio-dorsal perikarya mentioned earlier, but the fine axons (which exhibited prominent varicosities), could not be traced to the presumed group of three paired perikarya. Within the neuropil of the VG of B. perforatus and B. balanus, six pairs of small (15-20 [[micro]meter] diameter), weakly immunopositive, lateral perikarya projecting fine axons to the midline were observed [ILLUSTRATION FOR FIGURE 1F OMITTED], and a further six pairs of very weakly immunoreactive perikarya were located centrally [ILLUSTRATION FOR FIGURE 1D, F OMITTED].

Apart from the above-mentioned structures, the only PDH-immunoreactive structures consistently seen were in the supra-esophageal ganglia of B. balanus, where at least two pairs of strongly immunoreactive and three pairs of weakly immunoreactive neurons were seen, associated with extensive dendritic fields with prominent varicosities on the surface and within the neuropil [ILLUSTRATION FOR FIGURE 1A, B OMITTED]. Several contralaterally projecting fine axons were also observed [ILLUSTRATION FOR FIGURE 1B OMITTED]. Occasionally, prominent beaded axons originating from the supra-esophageal ganglion were seen in the circumesophageal connectives [ILLUSTRATION FOR FIGURE

1G OMITTED], but these could never be traced to the ventral ganglion [ILLUSTRATION FOR FIGURE 1E OMITTED].

CCAP-immunoreactive neurons

CCAP-immunoreactivity was observed in all species of barnacles examined, although immunoreactivity was weak, and background staining was high, even when nervous systems were fixed in carbodiimide fixative (the fixative of choice for this peptide in whole mounts of arthropod nervous tissue (Dircksen et al., 1991). For both B. balanus and C. hameri, similar morphologies of CCAP-immunoreactive neurons were observed [ILLUSTRATION FOR FIGURE 1,1-N OMITTED]. The anterior pair of perikarya (ca. 50 [Mu] diam.) are dorsal, the posterior two pairs (ca. 30 [Mu] diam.) are ventral. All perikarya project a single axon contralaterally which descends to the posterior margin of the ventral ganglion with fine branches projecting along each cirral nerve. A detailed camera lucida reconstruction of these neurons [ILLUSTRATION FOR FIGURE 4 OMITTED] shows the branching pattern of fine dendrites. In some preparations a pair of small (10-15 [[micro]meter]) neurons were seen in the supra-esophageal ganglia (the fine structure of these neurons was not investigated further).

For references, Figure 5 shows a schematic of the PDH- and CCAP-immunoreactive neurons observed.


In the present study, several antisera raised to native peptides of decapod crustaceans were used to determine the neuroanatomy of peptidergic neurons in barnacles. Although a limited array of antisera were used, a notable finding was that CHH- and MIH-immunoreactive neurons were never observed in barnacle nervous systems. This might be explained by the considerable group- or even species-specificity of MIH and CHH (Keller, 1992). However, CHH-immunoreactive neurons have been identified in isopods (Nussbaum and Dircksen, 1995) and mapped in the cladoceran Daphnia magna and in an anostracan, Artemia salina (Zhang et al., 1997). Thus, since CHH-like molecules seem to be a phylogenetically ancient group, the failure to observe CHH-like immunoreactivity in this study may well have been due to unsuitability of the fixatives used, although the same fixative (Stephanini) was used in all studies mentioned here.

The absence of RPCH immunoreactivity is surprising. This peptide is a member of the ever-expanding adipokinetic hormone (AKH) group. Although many different AKHs have been identified in insects (See Gade, 1997, for list), it appears likely that only one member, red-pigment-concentrating hormone (RPCH) occurs in crustaceans (Gaus et al., 1990). It might be argued that the absence of RPCH (as a circulating neurohormone) would be expected since chromatophores are present only in malacostracans; however, immunocytochemical studies (Mangerich et al., 1986; Nussbaum and Dircksen, 1995) using the same antiserum that recognizes the N-terminal tetrapeptide sequence common to many AKHs (Schooneveld and Veenstra, 1985) have shown that RPCH-immunoreactive interneurons are very widely distributed in the brain, thoracic and stomatogastric ganglia, and ventral nerve cord in malacostracans. Although RPCH from the central nervous system of decapods shows red-pigment-concentrating activity in bioassays (Fingerman and Couch, 1967), extracts of CNS from several species of barnacle result in pigment dispersion when injected into Uca pugilator (Sandeen and Costlow, 1961). This could indicate an absence of RPCH-like peptides in the CNS of barnacles, but it is more likely that the relative abundance of PDH-like material demonstrated in this study would override any pigment-concentrating effect of RPCH.

Although a pigment-dispersing hormone was first identified in crustaceans (Fernlund, 1976), immunocytochemical studies have not only verified a neurohormonal role for PDH, but have also documented a widespread occurrence in the CNS of crustaceans (Dircksen et al., 1987; Mortin and Marder, 1991) and insects (Homberg et al., 1991; Nassel et al., 1991; Stengl and Homberg, 1994); it seems likely that PDH-like peptides, or more precisely, pigment-dispersing factors (PDFs) have a widespread, perhaps universal, occurrence in arthropods (review by Rao and Riehm, 1993). Apart from the established role of PDH in controlling pigment migration in malacostracan crustaceans, the physiological significance of PDH-like peptides in arthropods has remained obscure. However, a role for PDH in control of circadian rhythmicity, proposed on the basis of the morphology of the immunoreactive neurons in the brain of orthopterans (Homberg et al., 1991), has been confirmed (Stengl and Homberg, 1994); furthermore, some neurons that express the period (per) gene in the Drosophila brain also display PDH- immunoreactivity (Helfrich-Forster, 1995). In the present study, PDH immunoreactivity was seen in several types of neuron in the ventral and supra-esophageal ganglia of barnacles. The neuronal system, consisting of a single pair of large cells projecting axons along the GSPN, was extremely prominent. Apart from these PDH-immunopositive structures, the only invariably immunoreactive PDH cells appeared to be interneurons: the perikarya in the supra-esophageal ganglia projected axons along the circumesophageal connectives towards the ventral ganglion. Small perikarya in the VG appeared to be segmentally arranged - although the VG is fused, six pairs of perikarya correspond with the number of thoracic limbs; thus these cells may delineate neuromers. Additionally, the dendritic areas associated with the posteriorly directed branches of the two large PDH-IR perikarya appeared to show some arrangement reminiscent of a segmentally iterated pattern [ILLUSTRATION FOR FIGURE 4D, H OMITTED].

With respect to the possible functions of PDH-IR material in barnacles, the presence of PDH-IR in neurons terminating on the attrahens and anterior prosomal muscles is of interest. Cotransmission of peptides and neurotransmitters is a widespread, if not universal, phenomenon (review by Kupfermann, 1991). In crustaceans, a thoroughly investigated cotransmitter system is that of the crayfish tonic flexor muscle, which is innervated by five motoneurons, three containing proctolin, that potentiate tonic contraction of these muscles (Bishop et al., 1987). It therefore seems possible that the two PDH-IR neurons innervating the somatic protractor may also have neuromodulatory roles in somatic extension in barnacles.

Crustacean cardioactive peptide (CCAP), originally isolated from pericardial organs of Carcinus maenas (Stangier et al., 1987), is another example of a neuropeptide with a wide, if not universal, occurrence in arthropods. The conserved neuronal networks of this peptide in arthropods have recently been reviewed by Dircksen (1998). Although the best known action of this peptide is its cardioacceleratory action, related (myotropic) actions in crustaceans include acceleration of scaphognathite rate and increased hindgut motility (Stangier, 1991). An important recent finding is that CCAP is a potent modulator of the pyloric rhythm of the stomatogastric ganglion of the crab Cancer borealis (Weimann et al., 1997). Immunocytochemical studies on the isopod Oniscus asellus (Nussbaum et al., 1995) suggest that CCAP might be involved in the biphasic exuviation pattern seen in isopods. For insects, broadly comparable myotropic roles have been observed, and it is expected (from morphology of CCAP-IR neurons that a multiplicity of functions have yet to be determined (Dircksen, 1998). In the present study, three pairs of CCAP-IR neurons observed in the ventral ganglion were notable. The perikarya essentially projected descending contralateral axons towards the cirri, but were too fine to allow the complete neural pathway to be traced. Although backfills have not demonstrated that barnacles have a neuronal architecture equivalent to that of higher crustaceans (Gwilliam and Cole, 1979; Gwilliam, 1987), the arrangement of the CCAPIR neurons in the barnacle ventral ganglion is broadly reminiscent of the arrangement of the cdn-type-2 neurons in the thoracic ganglion of decapod crustaceans (Dircksen, 1998) in the sense that they project contralateral descending axons. Further speculation with regard to homology or function would, however, be premature.


This work was supported by a Royal Society research grant. I am indebted to Dr. Heinrich Dircksen, University of Bonn, for providing antisera, and especially for his helpful advice and discussion. This paper is dedicated to the memory of the late Professor Dennis Crisp, F.R.S., who initiated and stimulated my interest in barnacle biology with his enthusiasm.

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Author:Webster, S.G.
Publication:The Biological Bulletin
Date:Dec 1, 1998
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