Formation and Nature of the Operoular Chaetaeof Sabellaria alveolata.

By

F. J. Ebling, M.Sc.Department of Zoology, University of Bristol.

With 13 Text-figures.

CONTENTS.PAGE

1. INTRODUCTION 153

2. M E T H O D S 154

3. STRUCTURE OF THE OPERCULUM 154

4. CHAETIGEROUS SACS 159

5. M O D E OF FORMATION O F THE CHAETAE 162

6. STRUCTURE OF THE CHAETAE . . . . . . 166

7. CHEMICAL N A T U R E OF T H E CHAETAE 167

8. DISCUSSION 169

9. SUMMARY 175

10. R E F E R E N C E S . . . . . . . . . 175

1. INTRODUCTION.

THE nature and arrangement of the opercular chaetae ofS a b e l l a r i a a l v e o l a t a L. have been described by Watson(1910) and by Mclntosh (1923) who also figured the individualchaetae. Fauvel (1927) gives a brief description of the oper-culum and few sketches. Watson also gives a short account ofthe chaetigerous sacs ('nests') in a whole specimen 'renderedtransparent'. Wilson (1929) describes the 'chaeta-sacs' in thelate larval stages (see section 8 of this paper).

The author is indebted to Mr. D. P. Wilson for help andcriticism and also for the use of a number of pencilled notes onS a b e l l a r i a made by the late Mr. Arnold T. Watson. Thesenotes contain several references to the chaetigerous ' nests' andinclude some sketches.

The research was carried out under the supervision ofProfessor C. M. Yonge.

154 F. J. EBLING

2. METHODS.

The specimens of S a b e l l a r i a a l v e o l a t a were obtainedfrom the Bristol Channel either at Weston-super-Mare or Kilve.The animals were narcotized for several hours in calcium-freesea-water (formula of Allen (1913), p. 424, with magnesiumchloride substituted for calcium chloride), and fixed in Dubosq-Brazil.

Mounts of whole animals and of portions were made fromunstained specimens cleared in cedarwood or clove oil. Canadabalsam was found to be a more satisfactory mounting mediumfor showing the chaetae than Euparol or Hyrax.

Sections were cut at 8 or 10 fi and stained in Delafield'shaemetoxylin and eosin, or in Weigert's haematoxylin andMasson's stain. In the latter case several counter-stains areused; the first contains a mixture of acid fuehsin and orange G,then after immersing in 5 per cent, phosphotungstic acid thesections are again counterstained in light green. The methodwas found invaluable for differentiating the different types ofchaetal material.

3. THE STRUCTUBE OF THE OPERCULUM.

The operculum is situated at the anterior ends of the twoso-called ' peristomial' or 'opercular' lobes. A left side-viewof the anterior region of the animal is shown in Text-fig. 1.Each ' peristomial lobe' bears rows of tentacular filaments (TF)on its ventral face. Posteriorly the lobe is bounded by a some-what reduced segment, followed by three more chaetigeroussegments, each bearing a dorsal branchia (B), and a notopodiumin the form of a lateral chaetigerous lamella, which becomesprogressively larger in the posterior segments of this group, andhas about six oar-shaped chaetae (DC). Ventrally the para-podium forms a neuropodial ridge which also bears a group ofchaetae (vc). The preceding (i.e. the first post-peristomial)segment bears a dorsal branchia, and a lamella relatively moreventral than those of the other segments and bearing a fewminute chaetae. Ventrally the segment merges with the peri-stomial lobes in the mouth region. The lower lips of the mouthare surrounded by a horseshoe-shaped building organ, which is

OPEROULAR CHAETAE OF SABELLARIA 155

TEXT-FIG. 1.

Left side-view of anterior region of Sabel lar ia a lveo la t a .B, branchia of segment II (second post-peristomial segment); BO,building organ; c, ventral cirrus; DC, dorsal chaeta of segment I I ;i, inner fan of opercular chaetae; L, 'peristomial lobes'; o, outerfan of opercular chaetae; P, papillae forming a ring around thebase of the operculum; TF, tentacular filaments; u, uncinigerouslobe; vc, ventral chaetae of segment II. Scale on this and otherfigures in mm.

NO. 338,339 " M

156 P. J. BBLING

seen in side view in the figure (BO). It bears a small chaeti-gerous lobe on each side. Mclntosh (1923) says that the lamellaof the reduced first segment appears to belong to the ventralseries, and Fauvel (1927) opines that both this and the chaeti-

M

TEXT-ITG. 2.

Surface view of operculum. This and the following figures refer toSabe l la r ia a lveo la t a . D, dorsal edge; v, ventral edge;LC, loose chaetae about to drop out; M, middle fan of chaetae;other lettering as before.

gerous lobe of the building organ are neuropodial in origin. Thefifth post-peristomial segment and the succeeding segmentshave conspicuous branchiae9 and the notopodium is representedby a ridge (u) which extends over the greater part of the lateralsurface of each segment and bears a row of numerous toothedhooks or uncini. Ventral to each ridge the neuropodium is

OPERCULAR CHAETAE OP 8ABEI1LARIA 157

represented by a small chaetigerous tuft overhung on its ex-ternal side by a cirrus (c).

The operculum is surrounded at its base by a ring of papillae

O M

TEXT-HG. 3.Diagrammatic longitudinal section of opercular lobes, is, inner

chaetigerous sac; OS, outer chaetigerous sac; other lettering asbefore. The chaetae are shown stippled, with overlapping por-tions black.

(Text-fig. 1, P). It consists of two semilunar disks, each com-prising three crescent-shaped fans of chaetae (Text-figs. 2, 3).

158 F . JT. EBLING

Each chaeta consists of a blade set at an angle to a long taperingshaft, giving the chaeta a boomerang-like appearance in sideview (Text-fig. 3). The disks are formed from the blades, thoseof the outer (o) and middle (M) fans point outwards; those of

H

TEXT-FIG. 4.

Plan of transverse section of the lobes showing embedding of chaetalshafts. Inner fan is shown black. Lettering as before.

the inner row (i) inwards (Text-fig. 3). The shafts of the outerrow are set separately, but those of the inner and middle rowsinterdigitate at their bases, the chaetae sloping away from oneanother anteriorly (Text-figs. 3, 4). In the outer fan the chaetaehave flat blades with notched ends; each blade overlaps theone ventral to it forming a continuous serrated margin to theopercular crown (Text-fig. 2). The blades of the inner and middlerows are stockier in build and pointed; in the middle row eachblade slightly overlaps the one dorsal to it; those of the inner

OPBECULAR OHAETAB OF SABELLARIA 159

row do not overlap. At the ventral end of each fan chaetaebecome loose and drop out (Text-fig. 2, LO). After considerationof the position of formation of the chaetae, it is clear that chaetaegradually move from the dorsal to the ventral ends of the fans,where they drop off from time to time.

TEXT-FIG. 5.

Plan of transverse section of lobes in region of chaetigerous sacs.Lettering as before.

4. THE CHAETIGEROUS SACS.

In each opercular lobe are two chaetigerous sacs or 'nests'running longitudinally; chaetae of the outer fan arise in theouter sacs (Text-figs. 3, 5, os); those of the middle and innerrows from the inner sacs (is).

In the outer sac (Text-fig. 6) the smallest and youngestchaetae are in the bottom or posterior portion of the sac; they

160 F. J. EBLING

lie towards its dorsal side. Ventrally and forward the chaetaebecome progressively larger, each chaeta overlapping thesmaller one adjacent to it dorsally. It is clear that the chaetaeare formed in the base of the sac and pass forwards and ven-trally as they increase in size, as indicated in Text-fig. 6.

TEXT-BTO. 6.

Outer sac left lobe viewed from the dorsal side, slightly to the left,a little distorted in the preparation, s, sac; sc, sac cells investinga chaeta in an advanced stage of formation. The direction ofmovement of the chaetae is shown by a dotted arrow.

The blades are first formed; only the chaetae near the anterioror open end of the sac have shafts. Chaetae in an advancedstage of formation with complete blades and long shafts move

OPEROULAR OHAETAE OF SABELLARIA 161

from the anterior end of the sac to the dorsal end of the outerfan. There is a continual movement of chaetae from the dorsalto the ventral end of the fan, where they become loose andeventually drop out.

The inner sac (Text-fig. 7) lies a little nearer the dorsal surface

TEXT-FIG. 7.

Inner sac of left lobe viewed from its side, somewhat distorted.Lettering as before. The chaetae of the middle fan (M) in courseof formation are shown stippled. The direction of movement of thechaetae is shown by means of dotted arrows.

of the animal than the outer (Text-fig. 5), and arises a littleanterior to it (Text-fig. 3). The chaetae form in a similarmanner. The two sets of these (i.e. those supplying the innerand middle fans) can be clearly distinguished from each other

162 F. J. EBLING

at all stages in their formation; they are arranged alternately(Text-fig. 7). At the anterior end of the sac the chaetae passalternately into the dorsal ends of the inner and middle fans,the blade of each chaeta turning in the appropriate direction.

5. THE MODE OF FOKMATION OF THE CHAETAE.

Sections were cut of the sacs in order to determine the mode

TEXT-FIG. 8.Sagittal section of base of outer sac. CFC, ehaeta-forming cells;

ST, striations; D, dorsal edge; v, ventral edge.

of formation of the chaetae. In the outer sac the regular flatblades cut evenly in sagittal section and it is thus comparativelyeasy to trace the development of the chaeta from the base(posterior) to the open (anterior) end of the sac.

The chaetae lie with their bases towards the ventral wall ofthe sac, each chaeta arising from a cell having a conspicuousnucleus with two nucleoli (Text-fig. 8, CFC). The base of the

OPERCULAR CHAETAE OF SABELLARIA 163

chaeta is characterized by many tapering parallel longitudinalstriations (ST), running from the formative cell. In some cases

scv

D

ST

CFCBS

TEXT-ITO. 9.

Sagittal section of part of outer sac. BS, base of sac; F, fine striationsin distal part of cone of chaeta; PC, primary core; sc, sac-cellssecreting secondary layer; scv, secondary layer cells vacuolatedafter secretion; SL, secondary layer; other lettering as before.

less conspicuous fine striations were observed in the centralportion of the chaeta (Text-fig. 9, F), but these were not alwaysevident in the sections. These chaeta-forming cells appear to bein the process of moving round the periphery of the sac from

164 F. J. EBLING

its dorsal border (D), giving rise to the chaetae as they moveround towards the ventral edge (Test-fig. 8). Cell boundarieswere not clear in the preparations. The whole mechanismappears to be a sort of moving belt of cells, arising near the tip

PC

TEST-ETG. 10.Successive transverse sections through a chaeta showing stages in

formation of secondary layer, A, before secretion of any material;B and c, stages in secretion; D, layer continuous; lettering asbefore. .

of the sac along the dorsal wall and passing round the basewhere the formation of chaetae commences.

The remainder of the sac has a vacuolated appearance whichis most marked at the anterior end; although there is a denseaggregation of smaller nuclei (Text-fig. 9).

From a study of sagittal and transverse sections stained byMasson's method, it appears that chaetal material is not onlyformed from the basal cell. Chaetae a little way up the sac havea second layer of material (Text-fig. 9, SL) investing the original

OPEBCULAB, CHAETAE OF SABELLAEIA 165

core which is secreted by the surrounding cells (Text-fig. 9, so).The secondary material stains red in contrast to the green of theprimary core.

The laying down of this secondary layer commences around

SFC ST PC

TEXT-FIG. 11.

Diagram illustrating formation of chaeta. I, in longitudinal section;II, III, and IV, transverse sections at different levels as indicatedby dotted lines; I I corresponds to stage A in Text-fig. 10, III tostages B and c, and IV to stage D. Lettering as before.

the tip of the chaeta and appears to spread basally as the chaetamoves towards the anterior end of the sac. The lower end of theshaft is the last part to be formed; the two layers here aredifficult to distinguish, although the base of the shaft appearsto consist mainly of the secondary material. The shaft is notcompleted until the chaeta is actually in position in a fan.Each chaeta moving into one of the fans from the main bodyof the sac carries with it a coat of cells (Text-fig. 6, sc) which areforming the secondary layer.

Successive transverse sections through a chaeta in formationthe secondary layer being layed down (Text-fig. 10).

166 F. J. EBLING

Towards the base of the chaeta the primary core (PC) is sur-rounded by a ring of columnar cells (sc) having fairly denseprotoplasm (Test-fig. 10 A). A little anterior to this the cellsappear more vacuolated, and the secondary layer (SL) may beseen as a ring of separate' bricks' (Text-figs. 10 B, 10 c). Finally,

\ \Is , mm, 1 ^ \

TEXT-FIG. 12.

Whole chaeta from outer fan. OL, outer layer; ic, inner core;DB, dark region.

these fuse together to form a complete layer; the formativecells (sc) now have little cytoplasmic content (Text-fig. 10 D).The process is illustrated diagrammatically in Text-fig. 11.

6. THE STRTJCTUBE or THE CHAETAE.

The fully grown chaeta appears to consist of two distinctregions, an inner core (Text-figs. 12, 13, ic), and an outerinvesting layer (OL). This description applies to chaetae of allthree rows. At the base of each chaeta the demarcation is notdistinct.

The inner core is striated longitudinally. At the junction the

OPERCULAR CHAETAE OF SABELLARIA 167

striations curve round from the shaft into the blade and theregion is darkly coloured (DR). A similar structure may beinferred from the examination of transverse sections, although

OL -DR

TEXT-FIG. 13.Part of chaeta from middle fan. Lettering as before.

it is not easy to interpret the actual nature of the striations.This is discussed in Section 8. The outer layer is clear and homo-geneous forming an even investiture round the shaft of aboutone-quarter its total diameter, of the same actual thickness butrelatively thinner in the blade region. The notched tips of theblades of chaetae from the outer row appear to consist entirelyof this investing layer. These layers presumably correspond tothe ' primary core' and ' secondary layer' observed in the pro-cess of formation.

7. THE CHEMICAL NATURE OF THE CHAETAE.

Experiments were carried out to determine the chemical

168 P. J. BBLING

nature of the chaetae and to distinguish between the two layers.Chaetae free from other material were obtained by allowingthe animals to rot in fresh water, followed by washing in dis-tilled water.

The chaetae were first of all tested for chitin, using a methoddue to Campbell (1929). It consists of heating the material insaturated caustic soda solution to 160° C, thus converting it tochitosan, which may be detected by several chemical tests.In this case the chaetae dissolved completely, although a fewspecks of a brownish residue were seen floating on top of thesolution. These were insoluble in 3 per cent, acetic acid, andare clearly not chitosan. No part of the chaeta is made of achitinous material.

Protein tests were then applied. Millon's reagent gave noreaction, but the xanthoproteic test gave strong positiveresults. Addition of concentrated nitric acid caused the dark-straw coloured partly opaque chaeta to become transparentand a pale yellow; this changed to a brilliant orange on additionof ammonia. The whole chaeta appeared to give this reaction.It was noted that other concentrated acids gave the 'clearing'effect.

Attempts were made to distinguish the chaetal materials byvirtue of their iso-electric points. In the case of the chaetaefrom the middle and inner fans, the demarcation cannot be soclearly seen in the whole chaeta, due to its stocky build. Chaetaefrom the outer row, however, are flatter in build (Text-fig. 12)and proved more suitable for experiment.

The method of iso-electric point determination was devisedby Loeb (1922) and used by various workers including Yonge(1932) from whose paper details of the technique were obtained.It consists of placing the material in solutions of differenthydrogen-ion concentrations, to which anions or cations havebeen added. After washing, the range of pH over which theions combme with the amphoteric substance is demonstratedby the appropriate reactions.

The macerated chaetae were soaked for some hours in buffersolutions over a wide range of pH; a few drops of 1 per cent,potassium ferrocyanide solution being added to each member of

OPBECULAR OHABTAE OP SABELLARIA 169

one series and a few drops of 1 per cent, copper sulphatesolution to a second series. After washing, the chaetae from thefirst series were treated with dilute ferric chloride solution whichreacted with the anion Fe(CN)e" which had combined withthe material on the acid side of its iso-electric point, givingthe typical Prussian blue colour. The other series was treatedwith dilute potassium ferrocyanide solution which reacted withthe Cu++ ions which had combined with the material on thebasic side of its iso-electric point to form the reddish-browncopper ferrocyanide.

The method has great practical difficulties in the case ofchaetae. Owing to the original straw colour it is difficult todistinguish the copper ferrocyanide coloration, especially in thecase of the inner core material, which appears a uniformbrownish colour throughout the whole range of pH. At a pHof 2*2 the inner core material takes up the negative ion (i.e.the K4Fe(CN)6-FeCl3 series shows deep blue). At pH 3-0 theinner core in this series appears faint blue. Thus the iso-electric point would seem to lie between pH 3-0 and 4-0. Inthe case of the outer layer, the first series is blue when the pHis less than 5-0, and colourless when it is more than 5-8. Thesecond series again did not give sharp results; the outer, how-ever, appeared darker in colour in the more alkaline solutions.The iso-electric point would appear to be at between pH 5-0 and6-0 for the outer material.

8. DISCUSSION.

The mechanism of chaeta-formation in Annelida has receivedattention from several workers. Schepotieff (1903, 1904)describes investigations into the structure of chaetae andchaetal follicles in oligochaetes and polychaetes. He says thatearthworm chaetae were first described as homogeneous, butthat Vejdovsky (quoted by him) and others described them asfibrillar. Schepotieff observes both longitudinal and transversestriations, but claims that these represent an alveolar structureconsisting of gas-filled cavities arranged in longitudinal andtransverse chains. Longitudinal merging may form tubes.He albo describes a narrow zone at the base of newly developing

170 F. J. EBLING

chaetae which stains more deeply with clearer striations extend-ing from the formative cell. Some sections showed, in additionto the alveolar layer, an outer strongly staining granular layer.Each chaeta is situated in a special pocket with a formative cellat the base; in connexion with this is a special 'substitutepocket' in which a new chaeta is formed. He states that only theformative cell plays any part in the formation of the chaeta.

Pruvot (1913) gives an account of the process in the bristlesof N e r e i s. He states that the proximal extremity of the bristleis enclosed in an 'appareil basilaire' which is formed from a massof multinucleate cytoplasm without cell boundaries. He addsthat the part which occupies the bottom, of the capsule, havinga large nucleus with one conspicuous nucleolus, alone producesthe whole of the substance of the acicule, and names it the'chetoblaste'. The acicule has a fibrillary appearance in longi-tudinal section, consisting of fine parallel columns of groundsubstance, which are more and more compressed at the peripheryalthough there is no distinction between a cortical and a medul-lary substance. Pruvot remarks that it is a common mistake ofauthors to regard the fibrules as of a uniform nature along theirwhole length. He claims to show by his triple staining techniquethat there are two regions. First, the distal part of the acicule ischaracterized by fine parallel striations; the ground substancebetween them being stained, although less heavily in the youngerpart of the bristle, by light green. Second, at the base, these giveway to slim rods ('batonnets'), each one more or less curved andcompressed against the others. These are embedded in the'chetoblaste'. They take up haematoxylin strongly, and theinterstitial substance in this region stains in eosin, distinguishingit clearly from the distal part of the bristle.

Pruvot considers that these are true cilia, not only on accountof their general form and staining, but also because they showclearly a bulbous swelling; moreover, they are embedded in asurface which appears to be traversed by lines which he saysrepresent the 'pieces intermediares'. Under the surface thereis a thin stainable layer which he thinks represents the fusionof the basal granules. He says that the diverse inflexions of the'cilia' prove that they are motile in the matrix in which they

OPERCULAR CHAETAE OP SABELLARIA 171

are dipping and which is secreted by them. As soon as the'cilia' have appeared at the surface of the ' chetoblaste' theysecrete between them a ground substance which is at first softand plastic while it is in contact with them, but later when ithas been carried beyond them by the pressure of new secretedmaterial, it solidifies. When newly secreted it stains with eoain,when solid with light green. The fine parallel striations areimpressions left behind by the 'cilia'.

Pruvot concludes by stressing that certain 'cilia' are respon-sible for secretion, being capable of movement in the matrixwhich they form. His opinion is that the chaeta-forming ' cilia'occupy in the series of described ciliary formations a placeintermediate between motile cilia and 'oils fixes des borduresen brosse'. Presumably he is here referring to the so-called'free cell-borders' described for the mid-gut cells of manyanimals.

Bobin (1937) has described the 'appareil ciliaire' of Mar-p h y s a s a n g u i n e a of which she claims to recognize fivekinds. Each shows a characteristic arrangement of the 'cilia'and gives rise to a particular type of chaeta. The arrangementchanges throughout the development of the chaeta.

Jagersten (1937) has devoted a paper to the nature andformation of the chaetae of M y z o s t o m u m , based partly onhis own work but including a discussion on work by Stummer-Traunfels (1903, 1927). The chaetae of Myzos tomum areof two sorts, the hook and.the supporting chaeta. Severaldeveloping reserve hooks may be seen in each case; theyresemble the chaetae in the chaetigerous sacs of S a b e l l a r i a .The supporting chaeta consists of two layers, an inner with astriated appearance and an outer, named by Stummer-Traunfelsthe 'Markschicht' and the ' Mantelschicht' respectively. In thehook chaeta, on the other hand, although a peripheral layer ofa more homogeneous nature may be distinguished, Jagerstenconsiders that the whole chaeta is equivalent to the 'Mark-schicht ' of the supporting chaeta and that there is thus no true' Mantelschicht'.

The ' Markschicht' of the supporting chaeta and the whole ofthe hook chaeta are formed exclusively from the basal cell and

NO. 338, 339 N

172 F. J. BBLING

consist of a series of parallel pipes, hexagonal in cross-section,which fit closely together. These he considers to be secretedby the basal 'ciliary' apparatus, which accounts for theirhollow nature. Jagersten quotes considerable evidence insupport of this interpretation of the structure, but admits thathe has no explanation as to why the tubes should be separate.The peripheric layer of the hook is formed by a hardening ofthe outer regions of the material.

The ' Mantelschicht' of the supporting chaeta has no hint ofchannels, the only structure being a concentric layering. Itis only slightly stainable and does not take up acid fuchsin.(This is not true of the secondary layer in the chaetae ofS a b e l l a r i a . ) With regard to its formation the author hasno original contribution to o'ffer. He quotes Stummer-Traunfels(1927) who thinks that it must be brought about by the con-solidation of blocks secreted at the point of the supportingchaeta. Fedotov (1914) describes a similar process in P r o t o -m y z o s t o m u m . Jagersten concludes by stating that thebasal cell has nothing to do with the origin of the 'Mantel'and therefore some other cells must be concerned. Althoughhe has no first-hand evidence to offer, he considers that thesecretion of the above-mentioned parts must be due to the cellsof the 'chaeta-surrounding follicle'.

In Oligochaeta, Bourne (1894) has investigated the embryonicdevelopment of the chaetigerous sacs, although he was moreconcerned with the origin of the chaeta-forming cells than withthe actual mode of formation of the chaetae. He figures chaetaeforming from single cells with large nuclei, which are comparablewith similar structures in Polychaeta and M y z o s t o m u m .

Vandebroek (1936) has described the development of thechaetigerous follicles of E i s e n i a f o e t i d a . According to hisaccount, the chaeta develops from a cell which becomes wedge-shaped, and in which appears a bundle of converging proto-plasmic filaments; these are obviously equivalent to the' appareil ciliare' of Pruvot and the similar structures observedby Jagersten and Stummer-Traunfels in M y z o s t o m u m andthe present author in S a b e l l a r i a . Later the dome becomescone-shaped and shows longitudinal striations which are

OPBRCULAR OHAETAB OP SABELLARIA 178

continuous with the protoplasmic filaments. Then a black zoneappears at the apex of the cone; originating as several blackdots, but afterwards extending farther back as a continuousdome, while a corresponding diminution takes place in thestriated zone. It seems probable that this dome may be thecommencement of secondary layer formation, and the gradualapparent diminution of the striated zone may be due to itsinvestment by the darker-staining secondary material. Vande-broek's chaetae were probably too small for him to put anyfunctional interpretation to his observations. He figures chaetaeof the order of 30/x in length, whereas in the case of S a b e l l a r i achaetae at a similar stage of development are about 0-3 mm.

The foregoing accounts all agree that the chaeta, or at leastthe central core of the chaeta, is secreted by a single cell, andthat the zone of secretion is characteristically striated. Thedistal part of the chaeta is also finely striated, and it seemsprobable that this corresponds to a cast moulded during secre-tion by the pattern of the basal formation. Jagersten claims tohave shown by experiment and dissection that the distalstriations represent hollow pipes, in which case the basalstructures are presumably rods. Moreover, he claims to haveobserved this rod structure in sections through the basal partof the chaeta. Although Pruvot's original description of thisas a ' ciliary formation' which occupies a place between ' motilecilia' and 'free cell-borders' has not been contradicted, itappears meaningless in the light of more recent work. Newelland Baxter (1936) have classified such non-motile cell bordersinto three types:

(a) A 'rod-border' where the elements are probably homo-logous with the basal segments of cilia, such as occur inthe gut of Mel inna and L u m b r i c u s .

(6) A 'striated free border', found in the intestinal epithe-lium of vertebrates, which is probably similar to (a),

(c) A 'brush border' proper, typically found in insects, inwhich the elements are simple filaments and bear norelation to cilia.

Baker (1942) claims, with considerable evidence, to show thatthe vertebrate free cell-border, i.e. (b) is not composed of rods

174 F. J. BBLING

but of spindle-shaped canals opening by pores. It is clear thatthere is no evidence for supposing that the basal striations ofchaetae have any affinities with motile cilia or indeed with anyof the structures described as ' free cell-borders'.

A secondary layer of material investing the primary corehas been described in the case of the supporting chaetae ofMyzos tomum, the opercular chaetae of Sabe l l a r i a , andprobably the chaetae of B i s e n i a. There is no doubt that thisis formed by the surrounding ' follicle' or sac cells.

Goodrich (1896) worked on the chemical nature of the chaetaeof the oligochaetes Vermicu lus (Khizodri lus) , E n c h y -t r a e u s , and L u m b r i c u s . He found that the chaetae ofall three genera were insoluble in caustic potash. In Vermi-culus and E n c h y t r a e u s the material was insoluble inhydrochloric acid, so he concluded that it was not chitinous,whereas in L u m b r i c u s it was soluble except for a thincolourless layer which formed a sort of cap over the chaeta,and he therefore concluded that it was probably chiefly com-posed of chitin or some allied substance. Chaetae from all threegenera, however, gave strong Millon's and xanthoproteicreactions.

Sehepotieff (1903) is in general agreement with Goodrich.He obtained reactions for protein but not for chitin, andshowed the presence of calcium phosphate. This may explain theclearing action of acids on the opercular chaetae of Sabel-l a r i a .

With regard to the origin of the operculum, Wilson (1929)has described the 'great chaeta sacs' in the larvae of Sabel-l a r i a a l v e o l a t a , which, he says, turn upwards and forwardsand eventually take up a position projecting forwards and ratherdorsally, and thus come to form the peduncular crown of theadult. These observations, says Wilson, confirm the view ofFauvel (1927) that 'les deux gros pedoncles operculaires repre-sentent tres probablement les rames dorsales fusionees des deuxpremiers pieds rejetes en avant du prostoroium'. The segmentalchaetae of the adult are embedded alternately in the mannerreminiscent of the inner and middle fans. It seems likely thatthe inner sac together with the inner and middle fans are

OPBRCULAR CHAETAE OP SABELLARIA 175

derived from one embryonic segment; the outer sac and fanfrom another.

9. SUMMARY.

1. The structure of the operculum of S a b e l l a r i a a l v e -ola t a L. is described. There are three fans of chaetae set ineach opercular lobe.

2. The chaetae are formed in two longitudinal sacs on eachside; the outer one supplying the outer fan, and the inner thetwo remaining fans. There is a continual movement of chaetaefrom the sacs into the dorsal ends of the fans; they move roundthe fans and eventually drop out at the ventral ends.

3. The chaetae are secreted by a continuous band of cellsmoving round the posterior end of the sac and forwards alongits ventral side. The nature of the striations at the base of theforming chaeta is discussed.

4. Around this primary core secreted by the basal cell, othercells of the sac secrete a secondary layer.

5. The structure of the fully formed chaeta is described andits two layers correlated with those observed in process offormation.

6. The chaetae are shown to be composed of protein and notchitin. The materials of the outer and inner layers appear tohave different iso-electric points.

10. EEFERBNCES.

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