The morphology and histology of the cirripede pemis

J. exp. mar. Biol Ecol., 1972, Vol. 10, pp. 243-265; @ North-Holland

Publishing Company

THE MORPHOLOGY AND HISTOLOGY OF THE CIRRIPEDE PENIS WALTRAUD KLEPAL Lehrkanzel

fiir Meeresbiologie,

I. Zoologisches

Institut,

Universitiit

Wien, &terreich

H. BARNES The Dunstqfnage

Marine

Research Laboratory,

Oban, Argyll,

Scotland

and E. Agricultural

Research

Council Institute

A. MUNN

of Animal Physiology,

Babraham,

Cambridge,

England

Abstract: The general morphology and detailed histology of the penis of two common boreo-arctic cirripedes, Balanus balanoides (L.) and B. balanus (L.) have been investigated. The penis is a highly extensible, annulated organ beset with four rows of sensory setae. The paired vesiculae seminales unite within the pedicel of the penis to give the single ductus. Distally, the exoskeleton is invaginated into this ductus. Circular muscles are present in the vesiculae seminales but do not continue into the penis. The histology of the ductus epithelium indicates a secretory nature as does that of a specialized group of cells, termed the ‘cushion’, towards the distal end: this group of cells is surrounded by circular muscle bands. Longitudinal muscles extend virtually the whole length of the penis; they give off fibres which are inserted at the junctions of the annulations. The muscles of the pedicel are described. Paired nerves in the pedicel give rise to four in the penis. The sensory innervation of the setae is described. The possible functional relations of the structure to the activities of the penis at copulation and during the emission of semen is discussed. Zusammenfassung: Die allgemeine Morphologie und die Histologie des Penis im einzelnen wurde an zwei boreo-arktischen Cirripediern B. balanoides (L.) und B. balanus (L.) untersucht. Der Penis ist ein 5uBerst dehnungsfahiges, “annuliertes” Organ, mit vier Borstenreihen besetzt. Die paarigen Vesiculae seminales vereinigen sich innerhalb des Penis-Pedicel zum unpaaren Ductus ejaculatorius. Am distalen Ende des Penis ist das Exoskelett ein Stuck in den Ductus eingezogen. Urn die Vesiculae seminales sind Ringmuskel vorhanden, die sich jedoch nicht in den Penis erstrecken. Die Histologie des Ductus-Epithels l&Bt Sekretion vermuten, genauso wie eine Gruppe spezialisierter Zellen, genannt ‘cushion’ am distalen Ende des Penis. Diese Gruppe von Zellen ist von ringformig angeordneten Muskelblndern umgeben. fiber die gesamte Lange des Penis erstrecken sich Langsmuskel. Von diesen abgehende Fasern inserieren an den Verbindungsstellen der Annuli. Die Muskel im Pedicel werden beschrieben. Paarige Nerven im Pedicel spalten sich auf zu vier im Penis. Die Innervation der Borsten ist beschrieben. Eine mbgliche funktionelle Beziehung zwischen der Penis-Struktur und der PenisAktivitlt wlhrend der Kopulation und wlhrend des Samen-AusstoBens wird diskutiert. INTRODUCTION

The general morphology of the cirripede penis and some of the more marked specific differences were described by Darwin (1854): the basal pedicel from which, what Darwin termed the articulated part, arises, has recently been shown to have a complex structure (Barnes & Klepal, 1971). Relatively little attention has, however, been given to the detailed structure and histology of the penis. It is now well known that at the time of copulation the penis, much extended by turgor pressure, makes searching movements before it enters a functional female and that once the female has been located and apparently recognized there follows a whole series of responses and behav243

244

ioural

WALTRAUDKLEPAL,H.BARNESAND

patterns

associated

with copulation

E. A.MUNN

(Barnes

well provided with setae, and it is reasonable of these are sensory and probably concerned

& Barnes.

1956). The penis is

to suppose that at least sortie, if not all, in the stimuli associated with these re-

sponses. Furthermore, recent investigations on the motility of the spermatozoa of Balmus balunoides have indicated that in this species passage of the semen through the penis has an activating action on the motility of the spermatozoa and it has been suggested that this may be due to the secretory activity of certain cells in the penis (Barnes, Klepal & Munn. 1971). Walley, White & Brander (1971) have shown that the clear, fluid secretion of the oviducal gland is a powerful activating agent inducing motility in the spermatozoa of B. bahnoidrs after the semen has been transferred to the functional female but they found that spermatozoa from the vesiculae seminales could not be activated in the same way: taken together, these observations suggest that passage through the penis followed by further activation from the oviducal gland secretion may be essential for the attainment of full motility in this species. The following account refers particularly to the mature penis of two common boreo-arctic species namely, B. balunoides (L.) and B. bahus (L.), the former taken in October, the latter in January-February, at Millport and Oban, Scotland. THE MATERIAL

AND

METHODS

Whole penes, before and after treatment with dilute sodium hydroxide as well as exuviae were examined by light and phase contrast microscopy. For ordinary histological work the whole ‘body’ or the penis after separation was fixed in sea-water Bouin: the material was embedded in paraffin wax; sections were cut at 7 and IO pm and stained with Haematoxylin-Eosin and Masson’sTrichrome stain. In addition, material was embedded in Araldite by a standard procedure after fixing with glutaraldehyde (followed in some cases by osmic acid) and dehydrating in acetone; after sectioning at I -2 pm on an ultramicrotome it was stained with Toluidine Blue. Staining of whole penes or parts with either silver nitrate or methylene blue was not very successful. Some general dissections of the penis were made on large B. bulanus but the gross musculature of the penis and pedicel was largely elucidated from sections and from the dissection of several very large (3 - 8 cm basal diameter) B. nubilus Darwin. GENERAL

MORPHOLOGY

ot THIZPENIS

The penis arises from a basal pedicel situated between the sixth pair of cirri and lying, therefore, above the anus. In B. bafanoides the resting penis is more or less cylindrical in form whereas in B. bafunus it is distinctly conical: that of the latter species is more intensely coloured brown. It is a highly extensible, ‘articulated’ (perhaps annulated is a better description) organ’ (Figs 1. 2, 3, 4). ’ In the strictest sense of the word the penis cannot be said to be made up of a series of annuli but common biological usage of the word annulated -- “with ring-like construction” (Henderson & Henderson, 1967) makes it the most appropriate and convenient expression.

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Figs 1, 2. Bulunus balmus: stereoscan of surface of the penis: 1) freeze-dried preparation: 2) fixed in glutaraldehyde whilst under tension to show annulations (a.) which particularly in (2) have shrunk; seta, s.

Figs 3, 4. Bulanus balunoides: phase contrast (X 80): exuviae of penis: 3) distal part of penis with groups of setae (s.) in four lateral rows, variable number per group, arising at upper part of a nnulation; chitin lined tip extruded (see Fig. 6): 4) proximal part of penis, fewer setae per group, individual setae shorter and stronger.

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The penis is covered by a chitinous exoskeleton some I .3 pm thick: under the action of turgor pressure the articulated form allows of considerable extension (some 3 - 4 times the resting length) during searching activity and copulation. The exoskeleton between successive annulations is thrown into what, as seen under the scanning electron microscope, appears to be a series of smaller folds (Fig. 2) but which in section are seen to be small, apparently solid exoskeletal ridges, irregular and running round the penis (many are about 2.5 pm high and 1.3 mu wide, but others are higher and thinner (Figs 5,27). The number of these ridges increases in the more proximal depressions e.g., B. balanddes, extreme distal 7, extreme proximal 50; B. balunus, extreme distal 7, extreme proximal 27 (averages of counts on the same penis, at light microscope level). The number of these ridges appears to vary somewhat according to the species: their function is unknown; clearly they give a greatly increased surface area, but since they are solid they cannot contribute to the increase in length under turgor pressure: they may serve to give a certain rigidity to the extended penis. In B. balanoides and B. balanus the penis bears four longitudinal rows of setae with some 3 -7, (sometimes up to 12) individual setae in each group; the number per group is smaller proximally. Prior to copulation the mature penis of B. balanoides has sub-terminal setae somewhat longer (158 pm in a moderate-sized animal) than the terminal setae (105 pm in the same animal) (Fig. 3): at this stage the most proximal setae, each relatively thick and short ( % 40 pm), may only be present in groups of two or three (Fig. 4). -Combs

,/

Conneclwe

ionghdmoi muscles, inner group Longhdinol muscles, oufer group

‘\

Fig. 5. Bulanus

(Issue

bahus:

schematic drawing of a penis seen in longitudinal and in transverse showing the general disposition of tissues and setae.

sections

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247

Fig. 6. Balanus balunus: phase contrast (X 170): (ex. d.) extruded and its combs (comb.) evident; setae, s. Fig. 7.

Bulanus balunoides:

dium hydroxide;

phase contrast (x 170): exoskeleton after treatment of animal with sochitin-lined terminal part of the ductus, ex. d. and terminal setae, s.

Fig. 8. Bulunus buhnoides: transverse section of vesicula seminalis. H and E (X 530): Darwin’s inmer tunic (= epithelium), ep. and outer tunic (= circular muscle bands), c.m.b.; connective tissue, con I. t.; spermatozoa, sp.

248

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AND

E. A. MUNN

According to Darwin the paired vesiculae seminales unite at the base of the penis proper after passing through the pedicel; however, in both 3. baianaides and B. balams, they unite at the level of the girdle, i. e. within the pedicel. The extreme distal part of the penis beyond the terminal annulation is somewhat conical in outline and the exoskeleton passes some distance into the lumen of the ductus, there to form its inner lining: this recalls the fact that the terminal part of the oviduct (oviducal gland) is lined bv exoskeleton (Walley, 1965). Although this terminal part of the penis is not naturally everted the exoskeletal-covered part can easily be extruded by light pressure (Fig. 6) it is then seen to be ornamented with the comb-like structures common on the pedicel and other parts of the body exoskeleton but absent on the outer exoskeleton of the penis itself. The generaf morphology of the penis is illustrated in Fig. 5 to which reference may be made in what follows.

THE DUCTUS EJACULATORIUS

Darwin (1854) stated that the wall of each vesicula seminalis consists of a thin inner “tunic”, “which is strengthened by thicker reticulated lines” and an outer of “transverse muscular or elastic fibre”; only the first of these layers is continued into the penis. According to Gruvel (1905) the structure of the vesicula s~lllinalisvaries with age (and size) and differs from species to species; he states that in general, an epithelium is surrounded by a layer of smooth circular muscles covered by cellular connective tissue, whose cells have very flattened nuclei, and in which longitudinal muscles are embedded: he further states that the circular muscles are continued - after the junction of the two vesiculae - right to the end of the penis. In the species considered here, B. balanoides and B. balanus. the presence of circular, but not longitudinal, muscles in the vesiculae has been confirmed (Fig. 8) but as stated by Darwin, these circular muscles are not found in the penis: a group of circular muscles does, however,encircle the group of large innerepithelial cells (see p. 252) situated near the distal end of the ductus (Figs 13, 14, 15). Inter-specific variation may account for the discrepancy with Gruvel’s description. Except as already described for the terminal part, the central ductus through which the semen is ejaculated, is lined by an epithelium (Figs 9, 3 1, 12). Initially uniform, the height of the cells of this ductus epithelium increases from base to tip during growth or regeneration in B. balanoides. In the mature penis of this species and in B. balanus the height of the epithelial cells increases from 3 ,um in the pedicel to 6 /irn over much of the ductus and reaches 12 pm distalIy (Fig. 11) Immediately proximal to the terminal exoskeleton-lined part there is a further increase in cell height - here quite abruptly - the large cells being some 22 pm high and 5 - 6 ktrn wide, with nuclei 9 x 6 pm and having a single nucleolus about 1.5 pm in diameter: this region will be called the ‘cushion’ (Figs 13, 14, 15). In both species the cross-section of the lumen

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Figs 9-12. Bnlun~ balunus: transverse section of penisin mid-part: Araldite-Toluidine Blue: 9) showing general relation of tissues (X 202): four main nerve strands situated peripherally, well marked lacunae: 10) enlargement of peripheral part of 9 ( X 820): 11) enlargement of 9 ( x 790) showing high columnar epitheIia1 cells with vacuoles lining ductus: 12) ductus later in season, February ( x 530) with epitheIia1 cells more prismatic, some only lightly stained, and with abundant villi. Connective tissue, con. t. (cellular and non-cellular); ductus epitbelium, d.ep.; ductus, d.; epidermaI cells, e. c.; exoskeleton, ex.; lacunae, la.; longitudinal muscles, 1.m.; nerves, a.; pigment granules, p.g.; sheath cell nucleus, s.c.nu.; spermatozoa, sp.; vacuoles, v; vilii, vi.

250

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AND

E. A. MUNN

Figs 13, 14. Balunus bulunus: longitudinal section of penis near tip: Araldite-Toluidine bands of circular muscles around the cushion and combs on exoskeleton, lining ductus enlargement of 13 ( i 1109). Fig. IS. Balunus balanus: Araldite-Toiuidine Circular muscle bands, c.m.b.;

Blue ( 1: 908): transverse combs, comb.: exoskeleton

Blue: ( y 630):

13) 14)

section rn region ofcushion, (cu. I. lining the ductus, ex.d.

MORPHOLOGY

AND HISTOLOGY

Fig. 16. Bulunus balanus: longitudinal section through cells of the cushion: Araldite-Toluidine Blue (x540): balloon-like secretory cells; circuiar muscle bands (c.m.b.) around the cushion (cu.).

Fig. 18. Baianus balanoides: longitudinal section of ductus in mid-part of penis, after copulation: AraIdite-Toluidine Blue (X 360): ductusepithelium (d.ep.) with vacuoles (v.) and spermatozoa (sp.).

OF CIRRIPEDE

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251

Fig. 17. Balanus balanoides: Iongitudinal section of ductus in the region of the pedicel: Araldite-Toluidine Bfue f x 360): globular epithelial cells (gIb. ep. c.) on the basement membrane &me.).

Fig. 19. Balanus balanus: transverse section of penis near base, Araldite-Toluidine Blue (X 370): ductus, d.; lacuna, la.; oil droplets (o.dr.) in cells amongst epidermal cells.

at the level of the cushion is much reduced (Fig. 15). The cells of the cushion appear to be secretory in nature; they can be seen at different stages of development or secretion, as indicated by their contents; how far this activity is cyclical is unknown. They may be elongate or grossly enlarged distally, when the terminal globular part (11 pm in diameter) stains only lightly with Toluidine Blue while the basal part stains quite

152

strongly,

WALTRAUD

In B. bahus

KLEPAL,

H. BARNES

the cells of this cushion

AND

E. A. MUNN

(Fig. 16) closely resemble on occasions

those epithelial cells lining the mesenteron - most commonly seen in the hepatic caeca which have been described by TGrnHva (1948) as giving off “balloon” secretions however. in spite of the ;such ‘b a 11oon’ secretions are known in other Crustacea): resemblance to these mesenteric cells, separate vesicles have not been seen free in the ductus, If there is secretory activity it recalls the statement of Gruvel that in Polficipes sp. the epithelial cells of the vesiculae seminales are secretory in character. It is possible that this secretion is partly concerned in the activation of the spermatozoa: it seems extremely unlikely that, like those of the mesenteron, the cells can be concerned in any digestive processes. So far, the columnar cushion cells in Balanus balanoides have only been seen to differ from the other columnar epithelial cells in their greater height (perhaps they have not yet been seen during an active secretory cycle since on occasions the cushion of B. balanus may only have large columnar cells). In the pedicel and at its junction with the penis proper the epithelial cells of B. balunoides are, however, large and globular; they are 12 pm high and 9 pm wide over much of theirlength, tapering to 5 pm at their basal ends (Fig. 17): the granular nucleus and the nucleolus of these cells are similar to those of the ‘balloon’-like cells already described for the cushion of B. balanus and, like the balloon portion of the latter stain lightly with Toluidine Blue: the condition of these cells may vary according to season. It is possible that the circular muscle bands present in both species around the cushion serve to expel any secretory material contained in it along with the semen. Ling (1971) has pointed out that in Lineus ruber secretory material is pressed out of the glandular cells lining the proboscis on stretching. In Balanus balanus, the cellsof the inner epidermis lining the ductus -- except for those making up the cushion - appear to have villi (Fig. 12) on their inner border around the ductus. It is possible that the appearance is due to adherent spermatozoa, but it seems more likely that it represents residual secretory material (perhaps the remains of ‘balloon’ secretions) preserved on fixation since these epithelial cells are markedly vacuolated (Figs 12, 21) and in B. balanoides vacuoles occupy most of the cell after copulation (Fig. 18) and the inner wall of the cell may appear broken. OUTER EPIDERMIS AND CONNECTIVE

TISSUE

The singlelayer of epidermal cells underlying the exoskeleton is made up of prismatic cells (in B. bafunoides 6- 12 pm high and 3 -6 pm wide; in B. bafanus 5-6 pm high and 3 -. 12 pm wide): the large, centrally placed granular nucleus more elongate (6 ,um x 3 pm) in B. balanoides than in B. bulunus (Figs 10, 26) (7 pm x 6 pm), has a single nucleolus (1.5 - 2.0 pm diameter). In B. balanus these epidermal cells contain a considerable number of densely staining granules probably of pigment material: such granules are far less abundant in B. balanoides. Cellular connective tissue surrounds the ductus and strands run to the periphery (Fig. 9). Typical parenchymatous tissue such as is found elsewhere in the body although present between the muscles is Iargely absent from the penis. Nearthe base of the penis, however, these parenchymatous cells

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253

Figs 20, 21. Longitudinal

section of penis: Araldite-Toiuidine BIue: 20) Balunu~ ~lanoj~es ( x 200); 21) Balanlrs balanus ( x 230). Ductus, d.; basement membrane, b.me.; ductus epithelium (d.ep.) with vacuoles (v.); connective tissue (con.t.) closely opposed to ductus epithelium and forming a network in lacunae (la.); longitudinal muscles 1.m.; nerves, n.

amongst the epidermal cells are, at certain times (e.g., October), full of oil droplets (Fig. 19). In B, ~alano~~~ the four penis nerves lie in pairs in the convective tissue surrounding the ductus and, together with the longitudinal muscles, may occupy most

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AND

E. A. MUNN

of the space between the ductus and epidermis (Fig. 20) (see also below). In B. bafanuJ the four nerves are situated peripherally (Fig. 9) and are also surrounded by connective tissue. In B. balanus what appear to be multicellular glands have been seen lying under the epidermis and opening to the exterior at about the level of the cushion (Fig. 22); they may have 2, 3 or 4 cells (18 pm long, 5.5 ,um wide distally and with a neck 3.0 /ml in diameter) in each group; the nucleus is 4.5 - 6.2 by 3.0 pm with a nucleolus 1.5 pm in diameter; granules are present in the cytoplasm and nucleus. Dr BocquetVtdrine (pers. comm.) has pointed out that their small size, the small nucleus, the absence of a characteristic orifice, and their irregular occurrence all indicate these glands are not comparable with cirral glands (Walley, 1967) the activity of which is related to the reproductive cycle. They seem to resemble much more closely the simple epiderrnal glands of the nauplius and cypris stages (Walley, 1969). Their very infrequent occurrence is difficult to explain and the hypothesis that they are parasites does not appear

satisfactory.

TIIE MUSCLES

Longitudinal muscles are a conspicuous feature of the penis. In a small but mature B. halanoides there are, basally, four groups somewhat triangular in cross-section and

Fig. 22. Bulanus balmus:

transverse section of penis: Araldite-toluidine cells, (e.gl.) at level of the cushion.

blue

( I 300): unidentified

Figs. 23. 24. Ba/unus balmus: transverse section of penis: Araldite-Toluidine Blue ( ’ 146): showing varying disposition of tissues and lacunae in penis: 23) tip, 24) near base, ductus d.; lacunae, la.

MORPHOLOGY AND HISTOLOGY OF CIRRIPEDE PENIS

255

surrounded by connective tissue; these occupy much of the penis so that the lacunae are small. They give way to eight groups further distally (Fig. 33) whilst near the tip the muscles are arranged peripherally. The same disposition of the muscles is found during regeneration of the penis of older animals but subsequently, as maturity is reached, the muscles are all peripheral from base to tip - and the lacunae, therefore, relatively larger. In a young B. balanus the muscles are still in groups but these are evenly spaced around the ductus from base to tip leaving only small lacunae: in older animals, however, the muscle groups become distinctly peripheral and larger lacunae develop between this ring of muscles and the ductus (Figs 23,24). An examination of serial sections indicates that the lacunae are ramifying channels rather than, as would sometimes appear from a limited number of sections, direct longitudinal channels running from base to tip of the penis (Fig. 21). The extreme distal part, beyond the annulations has, in both species, about five well-defined large lacunae separated by strands of connective tissue (Fig. 28). Gruvel (1905) refers to several distinct lacunae lying between the ductus and peripherally arranged muscles and in particular to what he calls afferent and ventral canals; there is no evidence for any such designations as was pointed out by Cannon (1947) for Lithotrya. Contrary to the appearance in many sections, staining with Heidenhain’s Iron Haematoxylin and with Toluidine Blue shows that the longitudinal penis muscles are not striated; those of the body and the pedicel are striated. The striated appearance often seen in some preparations may be due to the fact that in any one section some of the fibres are cut obliquely - an effect enhanced in the contracted muscle on preservation, particularly since there will be very considerable shortening as seen in sections of the main nerves (see IIanson & Lowy, 1960). Proximally, the longitudinal muscles of the penis are attached within the pedicel (Fig. 25) which is variously thickened so giving some rigidity to the points of attachment, and distally to the exoskeleton at the bottom of the annulations (see below). Within the pedicel they insert as follows: the upper outer layer of muscles runs along the upper, inner surface of the pedicel to become attached in B. balanoides at the median, carinal part of the girdle and in B. balsas at the upward projecting process which arises from the median part of the girdle: the inner layer of longitu~nal musdes attaches at the uppermost part of the carinal processes where the latter leave the girdle. The remainder of the longitudinal muscles are attached to the paired lateral processes and to that part of the girdle lying between these processes, i. e. above the shield. Distally, the outer longitudinal muscles, each 12 - 19 pm diameter, give off bundles of about five fibres opposite each annulation (Figs 26, 27): these fibres are attached to the exoskeleton at the junction between two annulations. In many sections the inner part of the exoskeleton appears to be ridged at this point of attachment, but this is probably an artifact and may depend upon the stage in the moulting cycle. Clearly, any given muscle must, therefore, decrease in size distally. Beyond the point at which a given muscle eventually terminates, a muscle internal to it becomes similarly attach-

256

WALTRAUD

ed to the exoskeleton;

KLEPAL,

the number

H. BARNES

AND

E. A. MUNN

of muscles and the size of each both decrease there-

fore. on proceeding distally along the penis (Figs 5, 23, 24). On occasions, an inner muscle may give off fibres to the exoskeleton before a bundle peripheral to it has terminated (Fig. 26). The innermost muscles, namely, those attached to the paired lateral processes and that part of the girdle lying between them, as well as the inner, upper longitudinal muscles, pass beyond the last annulation into the terminal part of the penis to be inserted at the smooth exoskeleton (Figs 5. 28). The pedicel itself is also supplied with a series of paired muscles (Figs 25, 29). One band (I) of muscles connects each lateral process with each lateral part of the girdle. Muscle II arises from the girdle at the point where it joins the shield and, curving over muscles III and IV (see below), becomes inserted at the thickened exoskeleton at the base of the 6th cirrus: muscle III arises underneath muscle II and inserts at the 6th cirrus opposite (further carinally) to the insertion of II. Muscles IV and V originate together largely on the lateral edge of the shield, V lying over IV: the latter muscle passes between III and V and, passing over V runs together with II to its point of insertion also at the base of the 6th cirrus; similarly muscle V inserts near muscle III. Paired muscles VI also run from the carinal processes downwards into the body. Each of the muscles I - V gives a few fibres to the connective tissue surrounding the ductus (Fig. 29). Except during the activities associated with copulation, the mature, relaxed penis, ,

Horn

/

Fig. 25. Schematic

drawing

of the pedicel showing muscles and proximal muscles of the penis proper.

insertion

of the longitudinal

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251

Figs 26, 27. Balunus balunus penis: attachment of longitudinal muscles: 26) transverse section: Araldite-Toluidine Blue ( x 545): giving off flbres to the exoskeleton; 27) longitudinal section H and E ( x 380). Attachment of longitudinal muscles, m.a.; longitudinal muscles, 1.m.; pigment granules, p.g.; exoskeletal ridges, e.r.

normally lies, at first, rostrally in front of the cirri of one side and then, curling carinally behind all the cirri, comes in B. balanoides to lie along the surface of the prosoma. Prior to copulation and with the body raised in the mantle cavity, the action of turgor pressure ‘unrolls’ the penis and leads to an increase in length: at the same time,

258

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section of penis near tip: H and E ( :, 530): showing terminaFig. 28. BU Imus b&anus: longitudinal tion s of lOI3 lgitud inal muscles (t.1.m.) large terminal Iacunae (la.) obliquely trave rsed by strands of connective tissue; a terminal seta, s.

Fig. 2Y. Bulunus balmus: pedicel, oblique section: Hand E ( -c 64): insertion of muscleson thickened areas of girdle (g.), lateral processes (lat. p.), and shield (sh.); note muscles crossing: arrows indicate attachment of muscle fibres on the ductus; longitudinal muscles, 1.m.: nerves. n.

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259

contraction of muscles I, II, IV and VI will rotate the penis carinally and together with the relaxation of the appropriate longitudinal muscles allow the penis to turn upon itself and pass between the 6th cirri to project carinally - a posture usually assumed before the initiation of searching movements. Muscles I, II, IV andV1 seem to be ‘opposed’ by muscles III and V which on contraction would lower and rotate the penis. That the longitudinal muscles are concerned in some of the searching movements is indicated by the fact that the exuviae of a penis (B. balanoides) shed after copulation and still containing much of the penis tissue has been seen to perform many of the bending movements associated with searching. It may be noted that the resting penis of B. balanus is, for a given size of animal, shorter than that of B. balanoides, and so only reaches part way round the cirri: this may be due to the greater contraction of each annulation - in turn related to the smaller number of exoskeletal, annular ridges in B. balanus. In addition, the annulations in B. balanus decrease more in diameter towards the tip so that the resting penis has a more conical, rather than cylindrical shape. Since turgor pressure is responsible for expansion of the cirri and since the penis is not expanded except at copulation there must be some nervous or other control over the distribution of hydrostatic pressure. To some extent the absence of lacunae in the young penis will resist the entry of body fluids but in the adult the penis has abundant lacunae: no valve-like mechanism has been found. It is perhaps significant in this connection that before the penis unrolls the cirri are in extreme extension and the muscles may be so arranged as then to faciliate entry of body fluids into the penis under further contraction of the body muscles. Nussbaum (1890) considered that two muscle bundles crossing one another in one sinus of Pollicipes polymerus were able to act as a sphincter. Such control must be exercised not only during normal activities but also repeatedly during copulatory activities since the penis may be partially contracted during each successive intromission while the cirri remain distended. The simplest mechanism would consist of a sphincter, under nervous control - the latter receiving feed-back from the reproductive organs either neurally or hormonally - and it could well be situated near the girdle: such has not, however, been found. The muscles around the ductus at the level of the cushion may also act as a sphincter (see however, p. 248 and p. 252) and togetherwith those joining the ductus to the exoskeleton of the pedicel (see p. 256) and those of the vesiculae seminales, serve to control the emission of semen during the several intromissionary acts of the fully turgid penis which are required for fertilization: the primary contraction would seem to be that of the vesiculae muscles. NERVES AND

SENSORY

RECEPTORS

According to Darwin, the nerves to the 5th and 6th pairs of cirri run together from the thoracic ganglion and a branch from that to the 6th, and of the same size, goes to the penis opposite the 5th cirrus. In the pedicel there are paired nerves (Figs 29,30,31) which give rise to four (Fig. 32) in the penis proper and these correspond to the four groups of radially arranged setae (Figs 3, 4, 7). The setae arise at the highest point

260

Figs 30-32.

WALTRAUD

KLEPAL,

~~~/ff~us ~~~~~ui~e~:transverse

H. BARNES

AND

E. A. MU&N

section of penis pedicel: Araldite-l‘oltiirline

30, 31 f paired nerves (n.) in the pedicel: 32) further

distally,

Blue

( A 350):

four ncrvec;.

of an annulation (Figs 3, 4): in a freshly dissected penis individual setae of a group have sometimes been seen to twitch but they do not seem to be supplied with muscles so that this may b:: due to extremely Jocal movements of the exoskeleton for which the fine fibres of the Iongitud~~aJ muscfes are responsible. la transverse section the seta is seen to have a thin and thick-walled cavity, the latter being excentric at some Jevefs.

MORPHOLOGY

AND HISTOLOGY

OF CIRRIPEDE

PENIS

261

Figs 33, 34. Bulanus balunoides: transverse section of penis: H and E: 33) general dispositic m of tissu es and setae ( x 235): 34) detail of 33 ( x 950). Basement membrane, b.me.; specialized ‘epsiderma1 cells, etc.; dendrites (de.) passing through the exoskeleton (ex.); epidermis, e.; ductus, d.; longitudinal muscles, 1.m.; nerves, n.; nucleus of neuron, ne.nu.; setae, s.

261

WALTRAUD

A small ( 5 pm diameter)

KLEPAL,

eosinophilic

H. BARNES

AND

E. A. MUNN

body can often be seen at the base of a seta.

The dendrites from a seta pass through the exoskeleton and lead to bipolar neurons, fusiform in shape 25 pm x 9 pm in B. bafanoides and 27 pm x 12 /OTTin B. balanur (Figs 5, 33, 34) with a single large, somewhat ovoid nucleus (6-9 pm in diameter) and a single nucleolus (I .2 pm diameter) in both species. The neurons are surrounded by sheath cells (Figs 35, 36) 12.5 pm long and 2.5 /Lrn wide in I?. balanoides and

Figs 35, 36. Balanus balanus: transverse

Ftg. 37. Balanus balanus: longitudinal

section section

of penis: of penis:

Araldite-Toluidine Araldite-Toluidine

Blue Blue

(

5SO).

( 460).

Fig. 38. Balanus bahoides: transverse section of penis: H and t ( 540). Specialized epidermal ceils (ct.c.) and their nuclei (nu.ct.c.); ductus epithelium, d.ep.: longitudinal muscles, 1.m.; nerve, n.; neuron (ne.) with sheath cell (SC.): exoskeletal ridges fc.r.) on annulations.

MORPHOLOGY

AND HISTOLOGY

10.5 ,um long 6.0 pm wide in B. balanus; a count of the number consecutive

sets of setae indicates

263

OF CIRRIPEDE PENIS

that 3 - 4 dendrites

of neurons

between

enter each seta. The length

of

the post-neuron section of the nerve depends upon the position of the seta; in the terminal setae of B. balanoides they are 50 - 60 pm in length, but further proximally when the base of the seta is nearer the main nerve they are 38 - 44 pm, and even further to the base only 11 pm. Situated near to the neurons are a number of cells which in their staining characteristics have all the properties of epidermal cells (Figs 33, 37,38): the number of these cells seems to correspond to the number of setae. They are very similar to the cuticulotubal cells described by Hayes (1966, 1971) in Limulus where they lie near the epidermis and serve to secrete the cuticular tube surrounding the dendrites. In both Balanus balanoides and B. balanus no such cuticular tube is present, the exoskeleton only being slightly invaginated at its tip as in the post axial sense organ of the attachment disk of the cyprid described by Nott & Foster (1969). Possibly these cells are associated with the development of the seta itself, being somewhat analogous to the specialized epidermal cells giving rise to the neck of the cirral gland in Chthamalus stellatus (BocquetVedrine & Ovechko, 1959). DISCUSSION

In many animals, invertebrate and vertebrate, in which semen is introduced into the female tract via a penis, the latter simply - or largely - acts as a ductus for the transfer of semen often together with the secretions of auxilliary glands, during copulation. The copulatory act may be, and usually is, preceeded by a complex series of events and interactions - behavioural, physiological, and biochemical - between the male and female. In a sessile animal pre-copulatory activity which involves movement of either partner must of necessity be absent: the presence of receptors linked to such pre-copulatory activity as searching and attending would, therefore, not be expected. Characteristically, in balanid barnacles immediate pre-copulatory activity is confined to an interaction between the penis of the functional male in locating the functional female and its acceptance by the latter prior to the transfer of semen. The mature resting penis is extremely long relative to body-size and its length is further increased by turgor pressure as it is extruded; the annulated nature of the penis allows such extension while the ridges still confer on it some rigidity. The complex musculature of the pedicel and the longitudinal muscles of the penis proper allow the extended penis to explore a considerable area around the functional male. Whilst initially the exploratory movements seem to be random, once a female has been located they become more directed and may be presumed to be dependent upon a chemosensory response: the setae described above are clearly suited to this purpose. By the time of copulation the penis is well provided with such setae; richly endowed terminally, an animal of 1 cm basal diameter has a total of some 200 annulations and with four rows of grouped setae will have perhaps a thousand such receptors each containing a num-

264

ber of dendrites:

WALTRAUD

KLEPAL,

H. BARNES AND E. A. MlJNl\i

the sensory input could clearly be considerable.

That

the longitu-

dinal muscles, together withthe nerves (whichprobablyalsocontain motoraxonsinnervating the muscles) come to make up the bulk of the penis tissue is not surprising, In many animals the testis itself subserves important functions .- glandularand hormonal - other than the production of spermatozoa and the male reproductive organs are associated with one or more accessory sex organs contributing a variety of secretions to the semen, the function of some of which is still only partly known (Mann, 1964): no such accessory organs are known in cirripedes although the seminal plasma is still a complex mixture which in some respects resembles that of animals with accessory reproductive organs (Barnes, 1962, 1963). The apparently secretory nature of the inner epithelium lining the ductus and the specialized glandular cushion perhaps subserve some of these functions. It has already been indicated that in B. balurzaides passage of the semen through the penis has an activating effect on motility of the spermatozoa (p. 244): these glandular secretions may play some part in this. On the other hand, in B. balanus the cushion is also very well developed and yet at copulation many spermatozoa even from the vesicula seminalis are motile before their passage through the penis. Walley, White & Brander (1971) have suggested that the epidermal cells lining the ductus may secrete material which 15 associated with the coagulation of the semen and there is abundant evidence in the present observations for such secretory activity. The small quantity of parenchymatous tissue, abundant elsewhere in the body, IS striking-its place being to some extent taken up by connective tissue. In B. balanoides the penis re-develops each season after the period when the parenchymatous tissue of the body is full of reserve material. Some regeneration also takes place in B. bahus: storage in the penis is not, therefore. necessary and indeed its most rapid redevelopment takes place when stored material in the body is being utilized.

ACKNOWLEDGEMENTS

We would like to thank Professor M. S. Laverack and Professor W. Stewart for providing facilities for cutting Araldite sections and Miss Evelyn Davidson and the Cambridge Scientific Instrument Company for assistance with the Stereoscan photographs. REFERENCES BAKMS, H.,

1962. The composition of the seminal plasma of Bdunus hulonrr.~.J. ~‘~17.Bid., Vol. 39, pp. 345-351. BARNES, H., 1963. Organic constituents of the seminal plasma of Bdunrrs holu,ws. J. rxp. Bid., Vol. 40, pp. 587-594. BARNES, H. & M. BARNES, 1956.The formation of the egg mass in Balmus htrlunoidrs (L.). Arch. SIN. C’nnam~~, Vol. 1I, pp. 1I-16.

BARNES, H. & W. Klepal, 1971. The structure

to other taxonomic

characters.

of the pedicel of the penis in clrripedes J. E.XP. mnr. Bid. Ecol., Vol. 7, pp. 71-94.

and its relation

MORPHOLOGY

AND HISTOLOGY

OF CIRRIPEDE

PENIS

265

BARNES,H., W. Klepal & E. A. Munn, 1971. Observations on the form and changes in the accessory droplet and motility of the spermatozoa of some cirripedes. J. exp. mar. Biol. Ecol., Vol. 7, pp. 173-196. BOCQUET-V~DRINE, J. & N. OVECHKO,1959. Contribution g l’&ude des ‘glandes salivaires’ de Chrhamalus stellatus. Bull. Sot. [inn. Normandie, T. 10, pp. 106-l 18. CANNON,H. G., 1947. On the anatomy of the pedunculate barnacle Lithotrya. Phil. Trans. R. Sot. Ser. B, Vol. 233, pp. 89-136. DARWIN, C., 1854. A monograph of the subclass Cirripedia. Vol. 2. The Balanidae, The Verrucidae etc. Ray Sot., London, 684 pp. GRUVEL,A., 1905. Monographie de Cirrhipddes ou ThPocostracPs. Masson et CiB, Paris, 472 pp. HANSON,J. & J. Lowy, 1960. Structure and function of the contractile apparatus in the muscles of invertebrate animals. In, The structure andfunction of muscle, Vol. I. Structure, edited by H. Bourne, Academic Press Inc., New York, pp. 265-335. HAYES,W. F., 1966. Chemoreceptor sensillum structure in Limulus. J. Morph., Vol. 119, pp. 121-141. HAYES,W. F., 1971. Fine structure of chemoreceptor sensillum in Limulus. J. Morph., Vol. 133, pp. 205-239. HENDERSON, I. F. & W. D. HENDERSON,1967. A dictionary ofbiological terms. Oliver and Boyd, Edinburgh, 640 pp. LING, E. A., 1971. The proboscis apparatus of the nemertine Lineus ruber. Phil. Trans. R. Sot. Ser. B, Vol. 262, pp. l-22. MANN, T., 1964. The biochemistry of semen and the male reproductive tract. Methuen and Co. Ltd., London, 493 pp. NOTT, J. A. & B. A. Foster, 1969. On the structure of the antennular attachment organ of the cypris larvae of Balanus balanoides (L.). Phil. Trans. R. Sot. Ser. B., Vol. 256, pp. 115-134. NUSSBAUM,M., 1890. Anatomische Studien an Californischen Cirripedien. Bonn, 97s. TGRNKvX, S. R., 1948. The alimentary canal of Balanus improvisus Darwin. Acta zool. fenn. No. 52, 52 PP. WALLEY,L. J., 1965. The development and function of the oviducal gland in Balanus balanoides. J. mar. biol. Ass. U. K., Vol. 45, pp. 115-128. WALLEY,L. J., 1967. The cirral glands: a new type of epidermal gland in cirripedes. Crustaceana, Vol. 12, pp. 151-158. WALLEY,L. J., 1969. Studies on the larval structure and metamorphosis of Balanus balanoides (L.). Phil. Trans. R. Sot. Ser. B, Vol. 256, pp. 237-280. WALLEY,L. J., F. White & K. M. Brander, 1971. Sperm activation and fertilization in Balanus balanoides. J. mar. biol. Ass. U. K., Vol. 51, pp. 489-494.