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Antennular sensory organs in cyprids of balanomorphan cirripedes:standardizing terminology using Megabalanus rosaJan Bielecki a; Benny K. K. Chan b; Jens T. Hoeg a; Alireza Sari c

a Department of Biology, University of Copenhagen, Copenhagen, Denmark b Biodiversity Research Center,Academia Sinica, Taiwan c Zoological Museum, School of Biology, College of Science, University of Tehran,Tehran, Iran

First Published on: 23 January 2009

To cite this Article Bielecki, Jan, Chan, Benny K. K., Hoeg, Jens T. and Sari, Alireza(2009)'Antennular sensory organs in cyprids ofbalanomorphan cirripedes: standardizing terminology using Megabalanus rosa',Biofouling,25:3,203 — 214

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Antennular sensory organs in cyprids of balanomorphan cirripedes: standardizing terminology

using Megabalanus rosa

Jan Bieleckia, Benny K.K. Chanb, Jens T. Hoega* and Alireza Saric

aDepartment of Biology, University of Copenhagen, Universitetsparken 15, DK-2100, Copenhagen, Denmark; bBiodiversityResearch Center, Academia Sinica, Taipei 115, Taiwan; cZoological Museum, School of Biology, College of Science,University of Tehran, Tehran, Iran

(Received 30 September 2008; final version received 11 December 2008)

Cirripedes are one of the major groups of fouling organism in the marine environment. The cyprid can, before apermanent attachment, actively explore and walk on the substratum using its antennules in a bipedal fashionwithout leaving the surface. Studying the structure of the cyprid antennule is therefore important for understandingthe events that culminate in biofouling by barnacles. There are at present no complete, standardised accounts of thestructure of the cyprid antennules in thoracican barnacles, and moreover, the existing accounts vary in their use ofterminology. This article describes the cyprid antennule of the barnacle Megabalanus rosa. This barnacle species iscommon in E Asia, and the cyprids have previously been used in several biofouling studies. All externally visiblesetae on the antennules have been mapped; these comprise both chemosensors with a terminal pore, a putativeaesthetasc-like seta and mechano-sensory setae. More setae were found on the attachment disc than in previousscanning electron microscope-based studies, but not all structures that can be seen with transmission electronmicroscopy were visible. The disc itself seems to have a variable surface area, which could assist in exploring roughsurfaces. The various lengths of the antennular setae, coupled with the disposition of the segments, enable the cypridto cover a wide swath of substratum during exploratory walking. A new terminology is proposed for cypridantennular setae, which will form a basis for future comparative and functional studies of cirripede settlement.

Keywords: biofouling; settlement; sensory organs; larval biology; barnacle

Introduction

The Cirripedia are pre-eminent in the fouling of man-made objects in the sea, and this problem enhancesdrag and consequently incurs large economic costs(Schultz 2007). Numerous studies have focussed on theprocess of cirripede settlement, recently using some ofthe most advanced biological and nano-physicaltechniques (eg Thompson and Nagabhushanam 1999;Berntsson and Jonsson 2003; Phang et al. 2006, 2008;Aldred and Clare 2008; Thiyagarajan and Qian 2008).

Among sessile marine invertebrates, cirripedesexcel in possessing a larva that is uniquely specializedfor initiating the attached phase of the life cycle(Walker 1992; Glenner 1999; Høeg et al. 2004). Thecirripede cyprid can, before a permanent attachment,actively explore the substratum by walking on itsantennules in a bipedal fashion (Crisp 1976; Walkeret al. 1987; Lagersson and Høeg 2002; Prendergastet al. 2008). This ability to explore a large surface areawithout leaving the substratum incurs obvious advan-tages during the settlement process and goes a longway towards explaining why barnacles are among thedominant sessile organisms in marine systems.

Before settlement, the fusiform cyprid swims in thewater column using its six pairs of natatory thoracicappendages. Little is known about its behaviourduring this phase, but it is possible that it can usechemosensory lattice organs exposed on the headshield (carapace) in navigating towards suitable areasfor settlement (Jensen et al. 1994; Høeg et al. 1998).When actively exploring the substratum, the cypridwalks on the antennules and the highly complexmorphology of these appendages enables an impressiveenvelope of motions, including a 1808 turn around onthe spot. During this exploratory walking, cyprids arewell known to react to a variety of physical, chemicaland biological properties of the substratum and usethese to make a decision if and where to attachpermanently (Crisp 1976, 1979; Clare 1995; Walker1995; Aldred and Clare 2008). The lattice organs,located on the shield and thus turned away from thesubstratum are unlikely to play a major role duringsurface exploration. Therefore, testing of the substra-tum must predominantly rely on the multitude ofantennular setae, although the frontal filaments andsetae on the caudal rami may also play a role

*Corresponding author. Email: [email protected] arranged alphabetically.

Biofouling

Vol. 25, No. 3, April 2009, 203–214

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(Chan and Leung 2007). The seminal papers of Nott(1969) and Nott and Foster (1969) on Semibalanusbalanoides (L. 1767) were the first to use ultrastructuraltools (scanning electron microscope (SEM) andtransmission electron microscopy (TEM) to describethe impressive array of sensory setae located on theantennules, but in contrast with the vast number ofpapers on settlement factors employed by balanomor-phan cypris larvae (Aldred and Clare 2008) there havebeen almost no further studies on cypris antennules inspecies from this taxon. This is unfortunate because thecomparative study by Moyse et al. (1995) revealed thatthere are very considerable morphological differencesamong cirripede cyprids in terms of both attachmentorgans and antennular setation, indicating that thismight correlate with differences in habitat selectionbetween the species.

Classification of the multitude of antennularsetae has also impeded both the comparison ofspecies and understanding the role of the differentsensory elements. Terminology has been somewhatconfusing using ‘seta’, ‘sensilla’, ‘organ’ or ‘sac’without further explanations. The classification byNott and Foster (1969) relied exclusively on TEM,which, although superior for understanding thesensory modality of setae, is very slow for comparingmany species. TEM was also used in the brief notesby Gibson and Nott (1971) and Lagersson et al.(2003). On the basis of SEM, Moyse et al. (1995)suggested a consistent classificatory scheme of thecypris antennular setae, but unfortunately theyomitted the fourth antennular segment, which Clareand Nott (1994) argued to be of primary importancein testing the substratum. The only comprehensivestudy of all external features in a cypris larva is byGlenner and Høeg (1995) for Amphibalanus amphi-trite (Darwin 1854), but the present authors’unpublished information shows that they also missedsome antennular features. The proper naming of thelatter species (Balanus amphitrite or A. amphitrite)has recently been debated; this paper adopts currenttaxonomy despite previously published reservations(see Clare and Høeg 2008; Carlton and Newman2009; Evans 2009).

In this article, SEM is used to map all externalfeatures in the cypris antennules of Megabalanusrosa (Pilsbry 1916). This species is common in farEast Asian waters, and its cyprids have previouslybeen used in studies on settlement and biofouling(Kamino et al. 1996; Okano et al. 1996, 1998;Khandeparker and Anil 2007). The purpose is topresent a consistent classificatory system of theantennular setae in line with current knowledge ofcrustacean sensory organs (Hallberg et al. 1992;Garm 2004).

Materials and methods

Larvae of M. rosa were reared to cyprids as describedby Okano et al. (1996) and were provided by Prof.Nobuhiro Fusetani (Hokkaido University, Japan). Thefixed cyprids were critical point dried from CO2,mounted for SEM and first examined in a JEOLJSM 840 SEM. Areas of particular interest were re-examined in a high performance JEOL JSM-6335FEG-gun SEM and photographed for the plates in thisarticle. Among the examined cyprids, a total of six hadboth antennules extended from the mantle cavity. Inthese, the individual segments of the right and leftantennules were systematically photographed in theJeol 840 in standard views (lateral, ventral, dorsal,frontal) to allow accurate measurements and compar-isons. Following that, details of setation and otherfeatures were photographed in the JSM-6335 atappropriate viewing angles to facilitate accuratemeasurements and comparisons. The photographsshown here are not the standard views used formeasurements, but those found most suitable toshow the features described.

Results

General shape and size of antennules

Previous accounts have described the complex cuticu-lar elements and musculature in the antennules ofbalanomorphan cyprids and combined this with videoobservations to model the biomechanics of surfaceexploration (Glenner and Høeg 1995; Glenner 1999;Lagersson and Høeg 2002). The following summaryserves as a background for describing the placement ofthe antennular setae and their possible orientationduring walking.

The two antennules are situated anteriorly in themantle cavity, whence they are extended duringsurface exploration (Figures 1 and 2). There are noappendages between the antennules and the thorax,but a pair of frontal filaments with unknownsensory function extends just behind them (Figure 1).Formally, an antennule consists of four segments(Figures 1–7), but the first one is composed of twoarticulating sclerites, so the entire appendage hasfive motile elements interconnected by four joints(Figure 2A). Basally, the proximal (first segmental)sclerites of the two antennules are interconnected bya pivot joint located in the roof of the mantlecavity.

For the surfaces of the antennules, the termsmedial, lateral, pre-axial and post-axial are used(Figure 2A). Medial and lateral refer to the situationat rest in the mantle cavity, but some of the antennularjoints allow considerable rotation so the antennules

204 J. Bielecki et al.


can be oriented very differently during walking(Lagersson and Høeg 2002). Pre- and post-axialcorrespond to dorsal and ventral when the antennulesare fully retracted into the mantle cavity, and the termsderive from comparison with the setation in thepreceding nauplius larva (see Nott and Foster 1969;Walossek et al. 1996). They are used here forconsistency with earlier literature, but have little

explanatory power in the cyprid. Firstly, the cypridantennules can assume many different stances duringwalking when compared with the nauplius, where theyalways point more or less straight ahead (Figure 2B–D). Secondly, in the cyprid, the attachment surfacebecomes the functionally distal end of the appendage,whereas the fourth segment, in principle the distal mostone, projects laterally from the third. In this article, anew system for naming the antennular setae is used(Figures 2A, 7). In this terminology, the proximal–distal axis of the third segment runs through the post-axial seta 3 (PS3), post-axial disc seta (PDS), axial discseta (ADS) and radial disc seta 0 (RDS0).

Figure 1. Cyprid of M. rosa. Lateral view with bothantennules extended. The distal part of the first antennularsegments and segments 2–4 are visible. Arrows indicate thejoint between the proximal sclerite (most often hidden in themantle cavity) and the distal sclerite of the first segment.AS1–4, antennular segments 1–4; FF, frontal filament; FP,frontal horn pore; PS2, post-axial seta 2; TS-A, terminal setaA; TS-B, terminal seta B.

Figure 2. General morphology of the antennule. A: Thecuticular elements and joints in the antennule (setae omittedfor clarity). The basal pivot joint is not articulating with thebody cuticle, but is suspended by muscles only. This confersa high degree of flexibility to movements of the twoappendages. The terminology used for orientation isindicated. B–D: Some of the stances assumed by theantennules during surface exploration (redrawn fromLagersson and Høeg 2002); AS-1–4, antennular segments1–4.

Figure 3. Distal part of the left antennule of the cyprid inFigure 1. The segments 2 and 3 are connected by a zone ofthin arthrodial cuticle. Segment 4 sits on the lateral side ofsegment 3. The attachment disc faces ventro–distally and isof concave shape in this specimen. The dotted line marks thepost-axial side (ventral midline, see text). Lower inset showsthe terminal pore in TS-D. Upper inset shows the insertionsof the thin setules on terminal setae A. AS2–4, antennularsegments 2–4; PRS2, pre-axial seta 2; PS2, post-axial seta 2;PS3, post-axial seta 3; RDS3–5, radial setae 3–5; STS1–4,subterminal setae 1–4; TS-A–D, terminal setae A–D.

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The segments

The first segment

Both the proximal sclerite and much of the distalsclerite of the first segment (Figure 1) are normallyhidden from view in SEM images. No arthrodial cuticlewas seen with the SEM between the first and secondsegments, but Lagersson and Høeg (2002) showed thatconsiderable motion can take place in this joint.

The second segment

The second segment (Figures 1 and 3) is cylindrical,somewhat laterally compressed and tapers towards the

distal end (Table 1). There is an extensive band of thinarthrodial cuticle in the joint to the third segment,enabling extensive flexure and rotation.

The third segment

The third segment (Figures 3 and 4) is short and bellshaped. Distally, it carries the attachment disc

Figure 4. Segment 3. A: Oblique view of the attachmentdisc (right antennule) densely covered by cuticular villi. Thedisc is virtually flat in this specimen and the centrally placedaxial disc seta (ADS) is raised above the level of the cuticularvilli. (Radial disc setae 0–2 not indicated, but some of themvisible in the original SEM photograph). B: Lateral view(right antennule). The dotted line marks the arthrodialmembrane between segments 2 and 3. Elongated velar flapsextend along the side of the segment towards the attachmentdisc. C: Detail of the pre-axial seta 2 (PRS2) in B; arrowdenotes terminal pore. ADS, axial disc seta; AS2–4,antennular segment 2–4; PDS, post-axial disc seta; PRS2,pre-axial seta 2; PS3, post-axial seta 3; RDS3–5, radial setae3–5; STS1–4, subterminal setae 1–4; TS-A–D, terminal setaeA–D.

Figure 5. Segments 3 and 4. A: Detail of Figure 3. Proximalpart of segment 3 with the post-axial seta 3 (PS3) insertingoutside the attachment disc and facing a narrow gap in theseries of velar flaps (arrow). PS3 has a conspicuous socket;near the terminal pore there is a narrow part (arrowhead) setoff from the rest of the seta. Note how the short radial discseta 6 (RDS6) could easily be obscured if viewed fromanother angle. B: The insertion of segment 4 laterally onsegment 3 showing its inverted cone shape, the subterminalledge and apical platform. Arrow marks where the apicalpart of TS-D can bend relative to the short, proximal part. *,the basal hump of terminal seta C (TS-C). Inset shows thespinules (arrow) and the terminal pore (arrowhead) on post-axial seta 3 (PS3). C: The relative position of terminal setaeA–D on segment 4. A low fold of cuticle surrounds the apicalplatform (arrows). Terminal seta E is (probably because ofspecimen processing) strongly curved rather than straight. *,the basal hump of terminal seta C. D: Detail of subterminalsetae 1–4, all with terminal pores. Arrows designate theconstriction near their apex. AM, arthrodial membrane;ADS, axial disc seta; PDS, post-axial disc seta; PS3, post-axial seta 3; RDS4–6, radial setae 4–6; STS1–4, subterminalsetae 1–4; TS-A–D, terminal setae A–D.



surrounded by a velum, consisting of thin, narrowpanels of cuticle. The segment is 24 mm high (long)when measured through the centre of the attachmentdisc, but the bell is asymmetrical, with a slightly longerdorsal than ventral side. This causes the attachmentdisc to be angled slightly ventrally from the antennularlong axis, even when the third segment points straightahead from the second (Figures 4B, 7A).

The fourth segment

The fourth segment (Figures 3, 5B–D) is small (30 mmlong) and rod-shaped. It is articulated to the lateral

side of the third segment, almost equidistant from thedorsal (pre-axial) and ventral (post-axial sides) andvery close to the joint to the second segment. In lateraland medial views, it has the shape of an inverted cone,with a 258 angle between the apically diverging sides.On the medial side, it has a narrow (4 mm wide) ledge

Figure 6. Details of selected features in the cypridantennule. A: The post-axial disc seta (PDS) and the axialdisc seta (ADS) sited deeply in a distinctly concaveattachment disc. Inset shows the narrow pore at the tip ofthe PDS. Dotted circles enclose radial disc seta 1 and 2,almost hidden in the carpet of cuticular villi. B: Rectangle inA showing the terminal pore of the axial disc seta and radialdisc seta 6, also with a terminal pore. C: The axial disc setaadorned basally with a few villi but surrounded by an area ofcompletely naked cuticle. D: Overview of an attachment disc.E: Rectangle in D, area near the distal rim with the smallradial disc setae 1 and 2 and the medially placed radial discseta 0 (enlarged in inset). F: Segment 3, lateral view of rightantennule. Several flaps of the velum are reflexed in thisspecimen, revealing how the carpet of cuticular villi proceedsdown the lateral side of the segment. The basal part of radialdisc seta 5 is situated in the carpet of villi a little inside thevelar flaps (dotted circle). ADS, axial disc seta; AS4,antennular segment 4; PDS, post-axial disc seta; PS3, post-axial seta 3; RDS1–6, radial setae 1–6.

Figure 7. Schematic diagram on the cypris antennules of M.rosa. Distal antennular segments in semi-diagrammaticrepresentation (traced from SEM micrographs with relativesize and position of structures approximately to scale, except inD). A: Lateral view of right antennule. Segment 4, hereextending ventrally, will normally be extended laterally; whenexploring a surface, the post-axially (ventrally) situated PS2and PS3 setae will probe area immediately behind theattachment disc. The small RDS 0, 1, 2 and 6 omitted;position of the medially sited PRS-2 indicated. B: Anterior(frontal) view with segment 4 extended straight laterally. Setaeon the attachment disc omitted. Thick black bars mark theareas probed by the setae on segments 3 and 4 during surfaceexploration; the staggered lengths of the setae on segment 4entail that the cyprid probes a wide swath lateral to the areabeneath the attachment disc itself; the aesthetasc-like TS-D canprobably sense stimuli along most of its length, the remainingchemosensory setae only at their terminal pores. The curvedshapes of STS 1–4 ensures probing of the surface adjacent tothe attachment disc; the short TS-C will probe the areaimmediately lateral to STS 1–4, when the apex of the segment istouching the substratum. TS-AþB sense water currents. C:Face on view of the attachment disc showing relative positionsof all setae on segments 2 and 3. Dotted circles mark theposition of radial disc setae found only by TEM in S.balanoides. D: Apical end of segment 4. Highly schematicdrawing of the relative positions of the subterminal andterminal setae. AD, attachment disc; ADS, axial disc seta;AS1–4, antennular segments 1–4; PDS, post-axial disc seta;PRS2, pre-axial seta 2; PS2, post-axial seta 2; PS3, post-axialseta 3; RDS4–6, radial setae 4–6; STS1–4, subterminal setae 1–4; TS-A–E, terminal setae A–E.

Biofouling 207


situated subterminally and very close to the apex. Theledge extends across the entire medial face of thesegment. The fourth segment terminates with a ca.11 mm wide apical platform, which has slanting surfaceso its lowermost part lies almost at level with thesubterminal ledge. The apical platform is (in top view)almost circular, ca. 15 mm in diameter and surroundedby a low rim of cuticle (Figure 5C).

The velum

The velum consists of a single row of thin and veryflexible cuticular flaps encircling the attachment disc(Figures 4B and 6F). Individual flaps have a semi-rectangular shape, being much longer (ca. 12 mm) thanwide. The flaps originate on the side of the segmentmore or less at the same level and are applied rathertightly to the side of the segment until they splitapically into small fringes.

Attachment disc

The large distal surface of the third segment, confinedby the velum, forms the attachment disc. It has adistinctly oval shape (ca. 30 6 16 mm), with theshortest axis at right angles to the antennular longaxis (Figure 3). The midline (long axis) passes throughthe PDS and ADS. (Figures 4A and 7). In the SEM,the apparent shape of the disc depends extensivelyon the viewing angle and can only be measuredaccurately in perfect face on views. A carpet ofcuticular villi covers the entire disc and obscures thetrue surface in the SEM. The length of the denselycrowded villi is difficult to measure. They can be atleast 5 mm long but never more than 10 mm. Because of

the flexibility of its thin cuticle, the profile of the discsurface is very dynamic. It can vary from being slightlydepressed to distinctly bowl-shaped with a centraldepression up to 5 mm deep (Figures 3, 4 and 6). Asseen where the flaps of the velum are accidentally rolledback, the villus covered disc extends almost halfwaydown the outer side of the third segment (Figure 6). Thecomplex shape of the disc entails that the effectivesurface area may vary, and it is in most situations largerthan estimated from perfect ‘face on’ views.

Position of the antennular setae

Tables 1 and 2 summarize the characteristics of theantennular setae (Figure 7). The first segment carriesno setae. The second segment carries two setae, thepost-axial seta 2 (PS2) and the pre-axial seta 2 (PRS2),both situated near the distal end close to the joint tothe third segment.

On the third segment, only the PS3 sit outside thevelum, whereas the remaining setae are located withinthe confines of the attachment disc. These disc setaecomprise the PDS, ADS and at least seven RDS 0–6.The PDS and ADS are both situated well inside theattachment disc. In distal (face on) views of the disc, allthe RDS seem to sit near the rim of the disc, but theTEM study of Nott and Foster (1969) shows that at leastsome of them continue for a considerable distance downinside the velum and they are therefore longer thanappears from most SEM pictures. This is also evident inthe SEM, when some of the velar flaps are accidentallyrolled back (RDS5 in Figure 6F). On the second andthird segments, four setae (PS2, PS3, PDS, ADS) sitalong a straight line that runs along the post-axial (mid-ventral) side of the second segment and continues as thelong axis of the oval shaped attachment disc.

The setae of the fourth segment sit in two groups.The ledge carries four subterminal setae (STS1–4),whereas the apical platform carries five terminal setae(TS A–E).

Pre-axial seta

This tiny, 10 mm long seta (Figure 4C), sits distally andpre-axially on the second segment, but slightly downthe medial side and almost at the arthrodial cuticle. Ittapers distally towards a distinct terminal pore.

Post-axial seta 2

This prominent, ca. 40 mm long seta (Figure 3) sitsdistally on the ventral (post-axial) side of the secondsegment, close to the joint to the third (Figures 1and 3). It has a simple, curved shape with few, smallscattered spinules and tapers towards a small terminal

Table 1. Lengths of selected antennular features and setaein cyprids of M. rosa.

Mean (mm) SD N

AS2 Length AS2 134 15.3 5AS2 Proximal height (longest) 69 8.3 5PS2 length 38.7 2 5PS3 length 32.61 4.3 6RDS5 length 9.6 1.69 5AS3 height 24.06 3.69 6Attachment disc – long axis 27.79 2.19 6Attachment disc – short axis 12.68 2.51 6AS4 length 24.79 2.69 6STS setae length 23.4 3.3 5TS-A length 80.87 12 5TS-B length 79.91 20 4TS-D length 61 5.3 5TS-E length 46 3 5

The basal parts of RDS5 are hidden from view; the length is the freepart above the cuticular villi. Lengths of TS-A,-B are the centralshaft, not including setules extending more apically.



pore. Near the base is has a distinct and slightly wider(ca. 1.5 mm) socket part. It resembles PS3 hence detailsonly of that seta are shown.

Post-axial seta 3

This 33 mm long seta sits on ventral (post-axial) side ofthe third segment, close to the base of the velum, whereit protrudes from between the base of two velar lappets(Figures 1 and 5A). It is simple, distinctly curved,tapering distally and ornamented with a few apicallydirected spinules. It has a distinct socket part(Figure 5A) and near the apex a distinctly narrowerpart with a terminal pore (Figure 5B).

Post-axial disc seta

This seta has previously been called the post-axial setaor even the post-axial sense organ. It sits somewhatinto the attachment disc, at the edge of the centraldepression when the disc has a concave shape(Figure 4A). It is slender, simple, and distinctlynarrower and shorter (ca. 8–9 mm) than the PS2 andPS3 setae (Figure 6A,D). It is normally straight or onlyslightly curved and just long enough to extend a littlebeyond the rim of the disc in perfect lateral views of thethird segment. Near its origin, where emerging from thevilli, it is ca. 0.5 mm wide. Distally, it has a small pore.

Axial disc seta

This structure was previously called the ‘axial organ’ orthe ‘axial sensory organ’, but the present authors revert

to calling it a seta as by Nott and Foster (1969). Theaxial disc seta (ADS) is part of a prominent featurenear centre of the attachment disc (Figure 4A). Theseta itself is placed on top of a small hump, and theentire structure is encircled by an area devoid of thecuticular villi that covers all other areas of the disc.The hump can, unlike the surrounding area, carry afew villi at least on one of its sides. From the SEMalone it is unclear whether the hump is actually anintegral part of the seta. Near the axial seta sit ca. threevillus-like filaments that are longer than normal villi,but they are not true setae. The axial seta proper has ashort, ca. 2 mm long and stumpy shape, with a widebase and a conspicuous terminal pore with small knobsaround its periphery. The seta extends only slightlyabove the top of the villi so it can easily be partly orfully obscured at some viewing angles.

Radial disc setae (RDS 0–6)

These comprise seven setae (radial disc setae (RDS 0–6)). Except for the medially placed RDS0, they areplaced pair-wise around the perimeter of the discrelative to its long axis (Figure 7). Although apparentlysited at the disc perimeter (Figure 4A, RDS 1, 2), closeup examination reveals that a stretch of villi alwaysseparates them from the velum (Figure 6D, E). Theirapparent size varies considerably. Three are veryprominent (RDS3, RDS4, RDS5), whereas the remain-ing ones are small and easily missed in the SEM. Someif not all of them continue down the side of the segmentcovered by the velum (RDS5 in Figure 6F). This entailsthat SEM examination only shows their distal most

Table 2. Classification of antennular setae in cyprids of M. rosa.

Abb. Name Segment Terminal pore Setules Modality

PS2 Post-axial seta 2 2 Yes No Chemo (& mechano?)PRS2 Pre-axial seta 2 2 Yes No Chemo (& mechano?)PS3 Post-axial seta 3 3 Yes No Chemo (& mechano?)PDS Post-axial disc seta 3 Yes No Chemo (& mechano?)ADS Axial disc seta 3 Yes No Chemo (& mechano?)RDS0 Radial disc seta 0 3 Yes No Chemo (& mechano?)RDS1 Radial disc seta 1 3 Yes No Chemo (& mechano?)RDS2 Radial disc seta 2 3 Yes No Chemo (& mechano?)RDS3 Radial disc seta 3 3 Yes No Chemo (& mechano?)RDS4 Radial disc seta 4 3 Yes No Chemo (& mechano?)RDS5 Radial disc seta 5 3 Yes No Chemo (& mechano?)RDS6 Radial disc seta 6 3 Yes No Chemo (& mechano?)STS 1–4 Subterminal setae 1–4 4 Yes No Chemo (& mechano?)TS-A,B Terminal setae A,B 4 No Yes Mechano onlyTS-C Terminal seta C 4 Yes No Chemo (& mechano?)TS-D Terminal seta D 4 Yes No Chemo (& mechano?)TS-E Terminal seta E 4 Yes No Chemo & mechano

RDS setae 7 and 8 were found by TEM in S. balanoides by Nott and Foster (1969) and may be present, but hidden from SEM view, in M. rosa.The alleged functions are based on external morphology and the TEM study of similar setae in A. amphitrite by Lagersson et al. (2003). All setae,except TS-A and B, are surmised to have a mechano-receptive function and therefore be bimodal sensory structures.

Biofouling 209


part, hence their true lengths cannot be accuratelymeasured. All RDS setae have a terminal pore.

RDS0

This seta sits in the midline at the distal end of theattachment disc and is normally obscured by the carpetof villi from which it barely projects at all. It appearstiny in the SEM but the TEM analysis of Nott andFoster (1969, their seta 1) shows that, in S. balanoides,it starts far down the side of the segment but is coveredby the velum for almost its entire length.

RDS1 and 2

This is a pair of setae that are placed symmetricallynear the distal rim of the disc (Figure 6D,E). They arestumpy with about the same diameter as the PDS.They easily escape detection even if projecting slightlyhigher from the villi than the RDS0 seta.

RDS3 and 4

These two slender setae sit slightly more proximallythan the RDS 1–2 pair (Figures 3 and 4A). With a freelength of ca. 10 mm they always project conspicuouslybeyond both villi and velum in a latero–distal direction.

RDS5

This conspicuous seta (which could also be called themedial disc seta, or MDS) sits near the medial rim ofdisc, about at level with the ADS in the centre(Figure 4). It stands apart from the other radial setae,because its partner on the other side (RDS6) is muchsmaller (Figure 6A,B). A specimen where the velum wasaccidentally rolled back revealed that RDS5 continuesfar down the side of the segment, but its base could stillnot be seen (Figure 6F). It is almost twice as long asRDS3 and 4 and hence the longest (ca. 30 mm) of theRDS. It is slender, 0.8 mm wide and simple and in SEMimages often distinctly flexed (Figure 6A, F), perhaps asan artefact because of its narrow width.

RDS6

This small seta is placed near the lateral rim,symmetrically with RDS5. It resembles RDS 1 and 2and normally it barely projects above the villi.

Subterminal setae 1–4

On the fourth segment these four similar, 33 mm longsetae sit in a single row on the narrow andsubterminally placed ledge. They are simple setae,

with occasional spinules and a terminal pore. From abasal width of 2 mm, they taper very gradually towardsa short, narrower tip set off from the rest of the seta(Figure 5B). The subterminal setae (STS) curve inmedial direction away from the fourth segment, andtheir tips can sometimes point directly down towardsthe attachment disc. This curvature is quite evident,also on live cyprids, and is therefore not an artefact.

Terminal setae

This group comprises five setae (TS A–E). Setae A, B,C and D are all long, whereas seta C is diminutive. Allfour setae sit close together on the slightly slopingapical platform of the segment (Figures 5C and 7D).Setae A and B sit along the outer rim and apparently alittle removed from the insertion of seta D. Seta C sitsin the near centre of the platform. Seta D sits justoutside seta E, more lateral than the centre of theplatform. Seta E sits close to the medial rim of theapex, just facing a subterminal seta at the end of therow of STS 1–4.

TS A and B

These two setae are identical in external morphology(Figure 3). They consist of a 35 mm long and nakedbasal part and a 50 mm long apical part adorned withlong setules to the very tip. The diameter may beslightly smaller in the setulated part than in the basalpart. The very fine setules are up to 100 mm long andsit pair wise in two rows along the shaft, spaced 7–8 mm apart. Both setae lack a terminal pore.

TS-C

This tiny seta sits on a small hump that may be part ofthe seta itself. Without the hump it is 7 mm long and itterminates in a distinct pore (Figure 5B,C).

Seta D

This 61 mm long seta is a 2–4 mm wide sac, with athin wall reinforced by conspicuous cuticular ridges(Figure 3). It consists of a 7 mm long proximal part and amuch longer apical part with distinctly different ridgepatterns (Figure 5B). In the proximal part, the reinfor-cement is a rather regular series of circumferential ridgesthat stabilizes the attachment to the fourth segmentwhile enabling the long apical part to bend (at the arrowin Figure 5B). In the long apical part, the closely spacedreinforcement ridges have a complicated and wavypattern (Figure 3, inset). TS-D can be up to 4 mm wideand it remains almost isodiametrical until close to theapex, where it has a distinct pore (Figure 3, inset).



TS-E

This is a simple smooth seta, isodiametrical for most ofits length and with a very small terminal pore. In thedistal part, it is somewhat laterally compressed into aband shape (Figure 5B). Although this seta seems to besomewhat curved in live cyprids of A. amphitrite (Clare1995), the extreme curvature in seen here in the SEMimages of M. rosa is undoubtedly an artefact caused byspecimen processing.

Discussion

Until now, only a few studies have mapped the sensorysetae in cyprids of balanomorphan cirripeds in anycomprehensive manner. For S. balanoides, Nott (1969)gave the first SEM-based account. The follow-upbenchmark paper of Nott and Foster (1969) usedTEM to study all setae on the third segment, whileGibson and Nott (1971) used TEM to study the setaeof the fourth segment. Since then, A. amphitrite hasbecome a model barnacle in studies on settlementfactors and fouling problems, but only three studieshave used ultrastructural methods to study theantennules of this species (Clare and Nott 1994;Glenner and Høeg 1995; Lagersson et al. 2003) andnone of these dealt with all setae. This study of anothermodel barnacle, M. rosa, provides the first compre-hensive account of the entire antennular setation in abalanomorphan cyprid. All setae visible with the SEMare described and a consistent classificatory system foruse in future morphological, behavioural and physio-logical investigations is presented.

Moyse et al. (1995) found very considerablevariation in the morphology of the third segment andits attachment organ, both across all cirripedes ingeneral and between the few thoracican speciesincluded in their study. To these results, Prof. A.S.Clare, Newcastle University, UK, remarked (para-phrased): ‘How shall we ever understand cyprid sensorybiology when the setae vary to this extent!’ (AmericanSociety of Zoologists Annual Meeting, WashingtonDC, 1995). The recent paper by Blomsterberg et al.(2003) further emphasized this by showing that cypridsof the Lepadidae differ in several important ways fromthose of the Balanomorpha. The generality of thedescription and classificatory system given here wouldsuffer if such variation prevails also within theBalanomorpha. A few differences were found betweenthe antennules of M. rosa, A. amphitrite andS. balanoides (Nott and Foster 1969; Gibson and Nott1971; Clare and Nott 1994; Glenner and Hoeg 1995;Lagersson et al. 2003). However, the general similaritybetween the cyprids of these balanomorphan barnaclessuggests that the classificatory system for antennular

setae given here forM. rosa can easily be extended to themajority of balanomorphan species and serve as ageneral background for studies on their settlement.

Classification and function

On the cyprid antennule, all sensory structures can beclearly classified as setae, and their morphologyprovides some hints to their function (Table 2). Apartfrom eyes, most sensory structures in crustaceans aresetae (sensilla), although sometimes highly modifiedones. The lattice organs, peculiar to cypris larvae, areputative chemoreceptors that have in most species lostany similarity to a seta (Jensen et al. 1994; Høeg et al.1998; Yan and Chan 2001; Høeg and Kolbasov 2002).

The current classification reflects the externalmorphology and the position of the setae on theantennules, using a simple numbering system. It istherefore not a functional one per se. As an example,Nott and Foster (1969) showed that the RDS differsomewhat in their TEM level structure, probablyreflecting functional differences. This article appearedbefore the TEM level structure of chemo- andmechano-receptors were well established for crusta-cean setae, impeding comparison with modern studies,but the few subsequent TEM studies on cypridantennular setae serve to emphasize the importanceof this technique for further functional characteriza-tion (Lagersson et al. 2003; Pasternak et al. 2004).

The terminal pore seen in most of the setaeindicates a chemoreceptive function. Setae with thismorphology have their sensory cilia (the outerdendritic segments) shielded along their entire lengthby thick enveloping cells (dendritic sheath), beingexposed to the environment only at the terminal pore(Garm 2004). This strongly suggests that they arecontact chemoreceptors that can sense stimuli only atthe tip. Aesthetascs are sac-shaped and thin-walledsetae that can detect water borne compounds (Hallberget al. 1992; Derby et al. 1997). Their thin cuticle allowsthe compound to enter over the entire surface of thesespecialized chemoreceptors. Van der Ham and Felgen-hauer (2008) described a novel type of seta, where theterminal pore had a secretive function, but in cypridsnone of the antennular glands terminate on setae (Nottand Foster 1969), so the interpretation of a terminalpore is taken as indicative of a chemoreception.Wherever investigated, plumose setae with two oppo-site rows of long setules, as in TS-A and -B, detecthydrodynamic forces (Garm 2004). In other types ofsetae, a mechano-receptive function is more difficult todemonstrate from external morphology alone. More-over, TEM investigations have revealed that allchemoreceptive setae in Crustacea, except the aesthe-tasc type, are bimodal and therefore also have

Biofouling 211


mechano-receptive properties (Schmidt and Gnatzy1984). In M. rosa cyprids, it is speculated that allantennular setae, except terminal setae A and B, arebimodal. The following interpretation is based on theSEM results presented here and the TEM data inLagersson et al. (2003) from the very similar setae of A.amphitrite, although it cannot be excluded thatdifferences could exists between the species.

For the setae on segments 2 and 3, there is no TEMdata other than the early study of Nott and Foster(1969). The terminal pores on post-axial setae 2 and 3and the setae on the attachment disc suggest that theyare contact chemoreceptors. RDS 3, 4 and 5 aresignificantly longer than the remaining setae on thedisc. They could therefore test the surface during atentative step even before the disc itself is in firmcontact with the substratum. This could assist indeciding whether to complete the step or turn awayand proceed in a different direction as described byLagersson and Høeg (2002). The ADS has previouslybeen described as a complex structure (eg Moyse et al.1995), but the present micrographs clearly show that itis just a single, short seta situated on a characteristichump. There is accordingly no reason to classify it asan ‘organ’ as has been usual until now (eg Glenneret al. 1989; Moyse et al. 1995; Chan 2003; Høeg andRybakov 2007). The few cuticular villi (setules) sittingon the hump are, like those elsewhere on theattachment disc, purely structural (Nott and Foster1969).

On segment 4, the interpretation is that the fourSTS 1–4 are contact chemoreceptors. Having identicalexternal shapes there are, at least in A. amphitrite,slight differences in their internal structure (Lagerssonet al. 2003), indicating that their functions might differ.The plumose terminal setae A and B function ascurrent detectors, either when exploring the substra-tum, while swimming or both. The simple, straightterminal seta E can probably test the physical proper-ties of the substratum, although the pore indicates thatit also is chemoreceptive. The tiny terminal seta C mustbe a contact chemoreceptor, although TEM data arelacking. The conspicuous and sac-shaped terminal setaD (TS-D) has an external morphology consistent withan aesthetasc, except that Lagersson et al. (2003) foundthat it also has the internal characteristics of amechano receptor. A terminal pore, as seen in TS-Dof M. rosa, is also absent from true aesthetascs. It istherefore preferable to call TS-D an ‘aesthetasc-like’seta. Its surmised chemoreceptive function was pre-viously suggested by Clare and Nott (1994) and Clare(1995). Its complex surface pattern may serve toreinforce the otherwise thin cuticle of this seta, and itis suggested that flexure can take place in the distinctneck in the basal part (arrow in Figure 5B).

Detailed video analysis of cyprids during theirexploratory walking could assist in interpreting thefunction of the setae (Lagersson and Høeg 2002;Prendergast et al. 2008). The short setae on theattachment disc (PDS, ADS, RDS 1–6) can only probethe small area within its perimeter, but as indicated inFigure 7B, the staggered lengths of the setae onsegment 4 could enable the cyprid to test a ca. 100 mmwide swath on either side of the exploring larva.

The short and always inwardly curving STS setaecover the area immediately lateral to segment 3,whereas the minute TS-C seta and the longer TS-Dseta cover more laterally placed stretches. The long,medially placed PS-2 and 3 setae will normally coverthe area immediately below the appendage and behindthe attachment disc, but might sweep to either side alsowhen their segments rotate. The aesthetasc (TS-D) canprobably detect stimuli all along its length, but mayprimarily serve in detecting waterborne chemicals,including those in the boundary layer, rather thandirect surface stimuli. Terminal seta E (TS-E) issurmised to be bimodal and test both the physicaland chemical surface properties far away from theattachment disc. The mechano-receptive terminal setae(TS-AþB) sense water currents. The constant back andfourth sweeping of the entire fourth segment will assistits setae in testing the surface.

The attachment disc

The morphology of the attachment disc is important inunderstanding both the temporary adhesion duringsurface exploration and final cementation. Althoughstatic views, the SEM images suggest that the shapeand therefore the active surface area of the disc is verydynamic. It is further suggested that the villi covereddisc area extending down the side of the third segmentmay be of functional significance when the cypridexplores topographically rough surfaces. If so, it mightbe expected that there would be a morphologicaldifference between the attachment discs in speciessettling in the rocky intertidal and those normallyinhabiting smooth surfaces. The arrangement of theADS on a hump in the centre of the disc is of interest inrelation to the mechanism of adhesion during surfaceexploration, because Phang et al. (2008) noted that thefootprints left by the cyprid have little adhesive depositin this area. Clearly, sensory setae, antennular glandsand the disc cuticle form a complex attachment systemthat is yet but little understood, especially in dynamicterms. The cuticular villi impede the detection of thesmall setae on the disc. Using TEM, Nott and Foster(1969) found a pair of radial setae (their setae 7 and 8)located symmetrically in the proximal part of the disc,and their surmised position is tentatively indicated in



Figure 7. But, using SEM, this study found no trace ofthem in M. rosa, and their apparent absence empha-sizes the need for more detailed comparative studies onantennular setae.

Conclusions

All externally visible setae on the antennules of abalanomorphan cyprid have been mapped. The setaecomprise both chemosensors with a terminal pore, aputative aesthetasc seta and two obviously mechano-sensory setae. On the attachment disc, more setae werefound than in previous SEM-based studies, but therewere not as many as can be seen with TEM. Thevarious lengths of the setae coupled with the disposi-tion of the segments enables the cypris to cover a wideswath of substratum during exploratory walking. Anew terminology for cypris antennular setae isproposed as a basis for future comparative andfunctional studies on cirripede settlement.

Acknowledgements

This study was completed with the financial support of theCarlsberg Foundation (2007-01-0095) and also receivedsupport from the Danish National Science Research Council(FNU 272-07-0260) and Irma Kaffe. Support from theSYNTHESYS Project http://www.synthesys.info/, financedby European Community Research Infrastructure Actionunder the FP6 ‘Structuring the European Research Area’Programme is acknowledged. Finally, the authors are indebtedto Dr Anders Garm, University of Copenhagen, and Dr JohnMoyse, Swansea University, UK for advice and support andto Prof CG Satuito and Prof Nobuhiro Fusetani, HokkaidoUniversity, Japan for providing the cyprids.

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