Morphology of the Lateralis Canal System 10 theShark Genus Carcharhinus1

ALBERT 1. TESTER and JAMES I. KENDALL2

THE SENSORY CANAL SYSTEM of elasmobranchshas been studied by several early workers. Gar­man (1888) described the gross morphologyof the system in several species of sharks andrays. A detailed account of the morphology andinnervation of the sensory canals was given byEwart (1891) for the shark, LaemargttS, andby Ewart and Mitchell (1891) for the skate,Raia batis. Johnson (1917) described the struc­ture and development of the lateral canals ofthe sharks, Mttstelus canis and Squabts acan­thias, and Norris and Hughes (1920) provideddetailed information on the innervation of thehead and lateral canals in the same two species.

The literature does not contain a descriptionof the sensory canal system of Carcharhimts,which includes many species of epipelagic sharkscommonly encountered in both inshore andoffshore waters. The present study attempts toovercome this deficiency, providing informationon the gross morphology and microanatomy ofthe system. It complements a study of the re­lated free neuromast or pit organ system ofsharks by Tester and Nelson (1967) and Testerand Kendall (1967). It is hoped that the in­formation will be of value to biophysicists,neurophysiologists, and behaviorists in inter­preting the results of experiments designed toincrease our understanding of how the lateralissystem functions as a water displacement re­ceptor (Dijkgraaf, 1963; van Bergeijk, 1967)or other sensor.

MATERIALS AND METHODS

Specimens examined included the following:Carchal'hilllts menisorrah-several near-term

1 Contribution No. 315, Hawaii Marine Laboratory,University of Hawaii. This study was supported bythe Department of the Navy, Office of Naval Re­search, Contract 2756 (00). Manuscript received May23, 1968.

2 Department of Zoology, University of Hawaii,Honolulu, Hawaii 96822.

1

pups (33 to 35 cm total length) from EniwetokAtoll, 2 adults (132 and 133 cm) from Eni­wetok Atoll, and 2 adults (125 and 162 cm)taken off Kauai; C. falciform is-several near­term pups (averaging 50 cm) taken off Kauai;and C. melanoptertts-several heads from juve­niles (about 30 em) taken from the Line Islands.

The binocular microscope was used for locat­ing modified scales associated with tubule open­ings and for gross dissection of the canals. Thepaths of the canals and their tubules were tracedboth by dissection and by clearing half-headsand pealed skins in glycerine after the canalshad been injected with either latex or India ink.Some preparations were bleached in Cloroxbefore clearing to render the skins more trans­parent.

The structure of the neuromasts and theirassociated epithelia were studied in freehand,frozen, and paraffin sections using freshly killedmaterial fixed in formalin, or Bouin's, Susa's, orRegaud's fixatives. In some paraffin preparationsit was necessary to decalcify the scales withDecal before sectioning. Various stains wereused including Heidenhain's hematoxylin andtriosin, Mallory's triple, Giemsa, Siegel's tolui­dine blue 0 and napthol yellow S, and carmine.Altmann's method was used for staining mito­chondria.

Nerve fibers and their terminations werestudied in frozen sections of formalin-fixed ma­terial using silver impregnation and gold toningaccording to the method of Gilbert (1965).

GROSS MORPHOLOGY

The sensory canal system is similar in allspecies of Carcharhimts examined. The paths ofthe canals and tubules are shown in Figure 1for a C. falciformis pup. The names assignedto various parts of the system follow, for themost part, those of Ewart (1891) and subse­quent authors and are based on both positionand innervation.

IV

10·ld 10,10 lE10·4

SO·2

10·3

10-:{ 10.1/ 10·

M

10-lb HM

SOCk SO'lb SO·la ST

A em B

FIG. 1. The lateralis canal system of a Cal'charhinus falciformis pup. A, Dorsal view of head; B, ventral view of head; C, lateral view of body.

SO·\c SO·lb SO~la lE ST

......\0

$

~...................

~'Tl......()

CFJ()......tTl

~.tTl

~

.......ll'

gll'

-<

co I 2 3 4 I

HMM

------.0

S(r2 10'3'10'2 10-1

Lateralis Canal System in Sharks-TESTER AND KENDALL 3

The intercommunicating canal system includesfour primary divisions: supraorbital (SO-I, SO­2), infraorbital (10-1 to 10-4), hyomandibular(HM) and lateral (L). The infraorbital of eachside is joined ventrally at the juncture of 10-3and 10-4. The lateral of each side is joinedanterodorsally by the supratemporal commissure(ST). A short length of canal from the junctureof Land ST to that of SO-l and 10-1 has beentermed the lateral extension (LE) by the presentauthors. Isolated mandibular canals (M) occurbehind the lower jaw.

For the most part, the canals are embeddedin or closely associated with the compact layerof the dermis. An exception is that part of thesystem comprised of LE, SO-la and 10-la, be­hind the eye, which lies well below the dermisand is covered in part by subcutaneous connec­tive tissue, musculature, or ampullar tubes.10-la has a thick fibrous wall which is closelyassociated with the postorbital process. Thehead canals SO-lc, SO-2 and 10-4 are deeplyembedded in subcutaneous connective tissueadjacent to the dermis and follow fibrous septawhich separate groups of ampullae of Lorenzini.

Attention is called to certain peculiarities ofthe system which also occur to a greater or lesser

extent in other shark genera, namely the pro­nounced supraorbital loop (SO-lb) on thedorsal surface of the head, the pronouncedinfraorbital loop (10-2b) in front of the eye,the slight loop toward the nostril of 10-3, andthe peculiar S-shaped curve of the lateral (L)below the second dorsal fin. The loops concen­trate the tubule openings in these areas; thebiological significance of this is uncertain.

In cross section, the canals are generally ovalin shape. Measurements of the inner diametersof the fibrous sheath surrounding the epitheliumand lumen are given in Table 1 for a 35-cm pupand a 162-cm adult C. menis01·rah. The cross­sectional areas (n: r1r2), which include the epi­thelium, are somewhat greater than the areas ofthe lumen proper, particularly in the anteriorhead canals where the epithelium distal to theneuromast is thickened and convoluted. Al­though there is considerable variation, in generalthe more anterior head canals (SO-I, SO-2, and10-4) have the larger bores. That of the lateralextension is also relatively large. Those of boththe hyomandibular and the lateral decreasecaudad. The maximum bore of the isolated man­dibular, at its mid-point, is relatively small com­pared with that of the intercommunicating

TABLE 1

SHORT AND LONG DIAMETERS OF THE LUMEN (INCLUDING EPITHELIUM), AND THE BORE (CROSS­SECTIONAL AREA INCLUDING EPITHELIUM), FOR SENSORY CANALS OF A 35-CM PUP (PARAFFIN

SECTIONS) AND A 162-CM ADULT (FREEHAND OR FROZEN SECTIONS) OF Cal'chal·hinus menisorrah

PUP ADULT

DlAMETERS (mm) DIAMETERS (mm)BORE BORE

CANALS':' SHORT LONG (mm2) SHORT LONG (mm2)

SO-la 0.38 0.42 0.123 0.8 1.4 0.88SO-lb 0.30 0.42 0.099 0.9 1.4 0.99SO-Ie 0.30 0.42 0.099 0.9 1.0 0.71SO-2 0.41 0.57 0.184 0.9 1.4 0.99IO-la 0.28 0.38 0.083 0.7 0.9 0.491O-1b 0.26 0.30 0.061 0.6 0.7 0.3310-2 0.7 0.9 0.4910-3 0.26 0.33 0.067 0.6 1.3 0.6110-4 0.22 0.68 0.118 1.1 1.4 1.21HM (start) 0.26 0.30 0.061 0.7 0.9 0.49M (max) 0.5 0.5 0.20LE 0.22 0.66 0.114 0.8 1.2 0.75ST 0.18 0.26 0.037 0.5 0.9 0.35L (start) 0.27 0.30 0.064 0.7 0.9 0.49L (1st dorsal) 0.13 0.17 0.017 0.5 0.6 0.23

* For identification of the canals see Figure 1.

4

canals. The canal bores increase with size ofshark. When length of shark increases by afactor of 4.6, the average bore increases by afactor of 7.7. However, this is a smaller increasethan would be expected with isometric growth(factor 21.4). The comparison of canal boresbetween sizes of shark may err slightly becauseof the difference in method of preparation(paraffin vs. freehand or frozen sections).

The canal tubules present an unexpected com­plexity in relative frequency, length, and orien­tation. They are difficult to map even when thecanals are injected with opaque substances, forthere is no assurance that impregnation has beencomplete. However, we believe that their repre­sentation in Figure 1 for the C. falciformis pupis reasonably accurate and complete.

In front of the eye, both dorsally and ven­trally, most of the tubules of the canals aredirected outward and forward, either antero­laterally (SO-lc and SO-2) or anteromedially(10-4). However, some are directed straightoutward, following the shortest possible routeto the surface (posterior part of SO-2, 10-2,and 10-ld). A few tubules are directed poste­riorly (10-3) or posteromedially (SO-1 b). Ingeneral the bore of the tubules is about one­quarter that of the canal but increases to morethan half that of the canal in the most anteriortubules. A few tubules are branched.

In the vicinity of the eye, the tubules of thesupraorbital (SO-la) and part of the infra­orbital (lO-lc) are directed toward the eye topartially surround it with pores. The tubulesof 10-lc branch profusely. Noteworthy are thevery long tubules arising from the infraorbitalcanal back of the eye (IO-lb) which are di­rected backward, branch profusely, and end inpores scattered among those of the hyoman­dibular. These long tubules do not form com­missures with the hyomandibular but pass overit distally.

Behind the eyes, the tubules are generallydirected outward and backward. Those of thelateral extension and the supratemporal arerelatively long and their pores are distributedacross the back. Those of the isolated man­dibular are very short and are directed poste­riorly. The tubules of the laterals are initiallyrelatively short but increase in length to thefifth gill opening, and maintain this length over

PACIFIC SCIENCE, Vol. XXIII, January 1969

most of the body. Most of the lateral canaltubules are directed outward and posteroven­trally; a smaller number, irregularly spaced, aredirected outward and posterodorsally. On thecaudal fin the tubules are relatively long and aredirected outward and posteroventrally. Alongthe body, and particularly on the caudal fin, theposteroventrally directed tubules tend to varyslightly in length in an alternating fashion.Their bore averages about one-third that of thecanal, thus progressively decreasing as canal sizedecreases along the body. On the caudal fin, thetubule bore becomes relatively larger; althoughstill small, it approaches that of the canalcaudad.

There are short lengths of canal without tu­bules (lO-la and part of SO-la) where thecanals have thick fibrous walls and lie deep be­low the dermis. It may be noted here, however,that these canals contain neuromasts.

fIG. 2. Pore of head canal tubule (c) bounded bynotched scales in contrast with larger pore of ampullaof Lorenzini (a) in a Carcharhinus menisol'rah adult.

Lateralis Canal System in Sharks-TEsTER AND KENDALL 5

The tubule openings on the head are smalland inconspicuous compared with those of theampullae of Lorenzini (Fig. 2). They occurbetween the crowns of scales which are notchedto conform with the circular shape of the pore.The pores are not covered by overhanging scalecrowns. On the side of the body, the scalecrowns above the pore are also notched to pro­vide an opening for the tubule; they are nar­rower and thicker than those of normal scalesand often have a rosette-like arrangement. Fre­quently the pore is covered completely or inpart by an overhanging crown.

It has been noted that the tubules of the moreanterior head canals are of relatively large boreand generally are directed forward, whereasthose of the more posterior head canals and thelaterals are of progressively smaller bore andgenerally are directed backward. This arrange­ment should produce a slow flow of seawatercaudad along the system because of water pres­sure on the head during forward swimming. Wehave demonstrated this flow in some sharkgenera3 but not yet in Carcharhimls because ofthe lack of small live specimens. Doubtless itoccurs. Smith (1930, 1933) has shown a flowin the canal system of some but not all of theteleosts that he studied.

MICROANATOMY

Semory Epithelillm

As found by Johnson (1917) in Sqllabls andMllstelm, the epithelium of the canal in Car­charhi1Uls is basically two-layered. For discus­sion, it may be divided into sensory and non­sensory regions. Its structure is illustrateddiagrammatically in Figures 3 and 4.

Within the canal opposite the tubule openings

3 A juvenile Sphyrna lewini (58 cm) was forced toswim in a weak (about 0.05%) solution of trypanblue for 50 minutes. Sectioning showed dye penetra­tion in the head canals and along the laterals to theregion of the second dorsal fin, but not beyond thispoint. Similar results were obtained with two juvenileGinglymostoma cil'l'atum (42 and 48 cm), althoughthe rate of flow was very slow in these sluggishsharks. In one, the dye penetrated the head canals andalong the laterals to the gi II region in 2 hours; in theother, it penetrated the head canals and along thelaterals to the region of the second dorsal fin in 4hours.

(i.e., medial to the dermis) the epithelium ismodified to form the sense organ-the neuro­mast or macula (Figs. 3A and B). This consistsof sensory hair cells, supporting cells, cupula,and a peripheral layer of mantle cells. Theneuromast is attached to a definite basementmembrane, supported by a loose fibro-elasticconnective tissue incorporating vascular andneural elements. Nerve fibers form a fiber zonein the connective tissue region before pene­trating the basement membrane to innervate thesensory cells of the neuromast. The connectivetissue sheath of the incoming nerve spreadswithin the thick fibrous sheath of the canal toform the loose vascular connective tissue sup­porting the non-sensory epithelium. The base­ment membrane in this region is absent orincomplete.

The sensory hair cells, with a prominentround or oval nucleus, are flask- or barrel­shaped (Fig. 3C). They extend from the distalsurface to about two-thirds of the distance to thebasement membrane. However, in some sensorycells of the pup neuromast, a strand of cyto­plasm extends from the proximal end of thecell to the basement membrane, indicating anorigin from cells extending the entire height ofthe organ. The "hair," about 6 'f.t long andtapering from about 1 f.t wide at the base, is seenin some preparations (Fig. 5). Presumably it isa bundle including a kinocilium and severalstereocilia as shown in teleosts by the electronmicroscope (e.g., by Flock, 1967).

Slender columnar or fusiform supporting cellssurround the sensory cells and extend from thefree surface to the basement membrane. Theirnuclei are oval and are located in the proximalportion of the cells. The distal ends of thesensory cells and the surrounding supportingcells are joined by intercellular cement showingclearly as terminal bars with Heidenhain'shematoxylin. Giemsa staining shows metachro­matic granules in the distal cytoplasm of thesupporting cells and extracellularly at theirdistal surface, indicating that these cells areactively secreting mucopolysaccharides. Degener­ating or necrotic cells with pycnotic nuclei andshrunken cytoplasm are of frequent occurrence.Mitotic figures (Fig. 6) occur occasionally aboveor near the level of the sensory cell nuclei.

Using primarily the above information, Tester

6

and Kendall (1968) showed that in sharks thesupporting cells secrete columns of mucoid ma­terial comprising the cupula and producing itstypical vertically-striated appearance. They con-

PACIFIC SCIENCE, Vol. XXIII, January 1969

elude that cupula production is a continuingprocess, accompanied by discharge, discarding,and regeneration of supporting cells and by con­tinual loss of cupular material at the distal free

+---TUBULE

DENSE FIBROUSCONNECTIVE TISSUE

.........----PIT ORGAN NERVE

----CUPULA

.__SENSORY HAIR:...J,.~r'=,rd>.C"'l7:1.",c-r7'"'1"'"rr .

.>;,-.---SENSORY CELL

~~~---SUPPORTING CELL

--NERVE FIBRILLAE

NERVE FIBRIL

FIG. 3. Diagrams of longitudinal sections of canals in a Cal'charhinus meniS01'rah pup. A, Head canal(ca. X 75); B, lateral canal (ca. X 75); C, head canal (ca. X 600).

Lateralis Canal System in Sharks-TEsTER AND KENDALL 7

FIG. 4. Diagrams of cross sections of canals in aCal'charhinus meniso1-rah pup. A, Head canal (ca.X 75); B, lateral canal (ca. X 75).

cell by an intracellular transducing process notyet clearly understood.

FIG. 5. Sensory cells in a pocket of the epitheliumof the supratemporal canal in a Carcharhinus menisor­t'ah pup showing sensory hairs (h). The hairs areusually lost during preparation. (Bouin's fixative,Mallory's stain, oil immersion.)

FIG. 6. Cross section through the infraorbitalcanal neuromast of a Cal'chal'hinus menisorrah pupshowing a mitotic figure (m) in metaphase amongthe sensory cell nuclei. (Bouin's fixative, Mallory'sstain. )

VESSEL

MUCUS

CUPULA

'BLOOD VESSEL

RIDGE

NERVE SUBRAMULUS

;JL,I;'.;i+--FIBER ZONE

~~~C-FIBER ZONE

~-'--NEUROMAST

VESICULATEDEPITHELIUM

A

B

surface. The delicate cupula is often lost duringroutine fixation and slide preparation. Evenwhen retained, it shrinks considerably (Fig. 7).In fresh-frozen sections of carefully handled,freshly killed specimens it appears to reachnearly to the top of the canal.

The presumed function of the cupula insharks is similar to that in teleosts. It is believedto move or "glide" slightly over the surface ofthe sensory epithelium under the influence of adisplacement of the canal fluid, thereby bendingthe hairs embedded in it (Flock, 1967). Bend­ing of the hair triggers electrogenesis in the hair

8

FIG. 7. Longitudinal section through lateral canalneuromast of a Cafchafhinus melanoptefus pup show­ing shrunken, striated cupula (c) partially torn fromthe neuromast surface. (Bouin's fixative, toluidineblue stain.)

The sensory epithelium is surrounded by aperipheral layer of inwardly curving supportingcells called "mantle" cells (Dijkgraaf, 1963).Metachromatic staining reveals the presence ofmucus-producing cells which are contributing tothe cupula.

In the pup and juvenile, the neuromasts ofthe canals are generally butted end to end,forming an almost continuous sensory epithe­lium. In longitudinal section (Figs. 3C and 8)the point of division between neuromasts canbe identified by the opposing curvature of themantle cells, producing a V-shaped indentationin the epithelial surface. The thickness of theepithelium decreases slightly (SO and 10) orabruptly (L) at the point of division. In theposterior part of the hyomandibular and in themandibular, the neuromasts are separated by afew cuboidal or columnar cells, similar to thoseof the side walls, between the mantle cells. Thissituation also occurs in the posterior part of thelateral canals, particularly in the caudal regionwhere the neuromasts are spaced by a distanceequal to about half their length. In the largerspecimens also, the neuromasts form an almostcontinuous epithelium. However, the spacingswhich occur are somewhat greater than in thepup and juvenile.

PACIFIC SCIENCE, Vol. XXIII, January 1969

FIG. 8. Longitudinal section through lateral canalneuromasts of a Cafchafhinus menisoffah pup show­ing V-shaped division (d) between two neuromasts.(Bouin's fixative, hemotoxylin-triosin stain.)

The neuromast is oval in facial section withits long axis parallel to that of the canal (Fig.9). Measurements of the sensory epithelium ina 33-cm pup and a 125-cm "adult" (apparentlystill immature) C. menisorrah are included inTable 2. For each canal section there are aver­ages of 2 or 3 measurements in each of 1 to 3preparations. Length measurements are few be­cause of the difficulty of obtaining perfectlyoriented longitudinal sections. The measure­ments of the pup and adult are not strictly com­parable because of a possible difference inshrinkage between paraffin and frozen sections.

In general, in both pup and adult, there ap­pears a slight decrease in thickness, a somewhatlarger decrease in width, and a still largerdecrease in length of the neuromast caudadfrom the most anterior head canals (SO-l and10-4). There also appears to be an increase insize of neuromast with growth. With increase in

I.ateralis Canal System In Sharks-TEsTER AND KENDALL

TABLE 2

MAXIMUM WIDTH AND THICKNESS (IN CROSS SECTIONS) AND LENGTH OF CANAL NEUROMASTS OF A33-CM PUP (PARAFFIN SECTIONS) AND A 125-CM ADULT (FROZEN SECTIONS) Cafchafhinus meniSOYfah

(All measurements in microns;

9

PUP

CANAL WIDTH THICKNESS

SO-la 160 30SO-lb 163 30SO-lc 171 3050-2 217 3810-1 103 2710-3 171 3410-4 190 38LE 106 30HM 95 27M 76 27L (start) 84 30L (above gills) 76 30L (1st dorsal) 76 34L (2nd dorsal)L (caudal

peduncle) 76 27L (mid caudal

fin) 60 19

LENGTH

504450

900

300

300

length of shark by a factor of 3.8, the neuromastincreased slightly in thickness (average factor1.3), considerably in width (average factor2.5), and, from the meager data, extensively inlength (factor 3.6 for SO-Ie, only). It is un­likely that these differences are due entirely todifferential shrinkage between paraffin andfrozen sections. Since the average maximumdiameter of a sensory cell appears to be similar(about 7 to 8 Il) in both pup and adult andsince the ratio of sensory to supporting cellsseems to remain about the same (about 1 to 5),it may be inferred that the number of sensorycells increases with growth.

Rough calculations may be made of the num­ber of sensory cells per neuromast. The distance(d) between several sensory cells (n) in oneplane (in sharp focus) is determined. The areaoccupied by one sensory cell and its surroundingsupporting cells may be calculated as d2/n2 .

Based on 10 determinations (SO-Ie and L) the

FIG, 9. Facial view of lateral canal neuromasts(n) from caudal peduncle of an adult Cafcha,'hinusmenisofl'ah showing their oval shape. (Formalin fixedfrozen section, Giemsa stained.)

10

average is 39 f!2 (range 20 f!2 to 56 f!2) for theC. menisorrah pup. Dividing this average intothe area of the neuromast surface (calculatedfrom Table 2) provides the following roughestimates (rounded to the nearest hundred) ofthe number of sensory cells per neuromast:10-4-3,400; SO-2-2,000; SO-lc-l,400; HM­600; L (first dorsal fin)-500. These estimatesagree in order of magnitude with those obtainedby counting the number of sensory cell nucleiat all depths of focus in a 10-f! cross section andmultiplying by the number of sections in theneuromast length. In the adult, assuming thesame unit area, for SO-lc (the only canal forwhich data are available) the estimated numberof sensory cells per neuromast is about 10 timesgreater than in the pup (15,700 compared with1,400) .

Non-sensory Epithelium

In the head canals, the epithelial lining ad­jacent to the neuromast has a peculiar "vesicu­lated" appearance (Figs. 4A and 10). It consistsof columnar or wedge-shaped cells which arejoined by distinct cytoplasmic processes enclos­ing well defined intercellular spaces. The cellnuclei are elongated and generally lie closer tothe distal than to the proximal end of the cell.Staining with Giemsa stain shows mucus on thesurface but not within the interior of the cellsor in intercellular vesicles. Staining with Alt­mann's technique shows mitochrondria denselyaggregated near the distal end of the cells (Fig.11). The distal cell membrane has a striatedappearance. Staining with naphthol yellow S(Siegel's stain) shows a finely granular contentindicating the presence of residual proteinswithin the intercellular vesicles, in adjacentblood vessels, and in the lumen of the canal. Thestaining responses show that the cells are secre­tory in nature, but are not producing mucus. Theaccumulation of mitochondria in the distal por­tion of these cells in the absence of mucus se­cretion suggests a possible similarity to the"chloride cell" described by Copeland (1948)in the gills of Ftmdultts heteroclitus, particuarlyin those fish which he immersed in hypertonicseawater. Tests for chloride and other ions (Na,K) await the availability of living specimens.

In the more anterior head canals of the pup,

PACIFIC SCIENCE, Vol. XXIII, January 1969

FIG. 10. Cross section of infraorbital head canalof a Carcharhinus menisorrah pup showing, from topto bottom, tubule (t) entering the canal, ridge area(r), vesiculated epithelium (v), and neuromast (11).(Bouin's fixative, Giemsa stained.)

FIG. 11. Longitudinal section of the supraorbitalhead canal in an adult Carcharhinus menisorrah show­ing mitochondria (m) aggregated at distal end ofcells of the vesiculated epithelium. (Altmann's stain­ing method.)

Lateralis Canal System in Sharks-TESTER AND KENDALL 11

those with forward-directed tubules (SO-Ie, SO­2, and 10-4 in particular), the vesiculated area iselaborated into folds and finger-like projections(Figs. 4A and 10). In the more posterior headcanals only the folds are present. In the laterals,it is reduced anteriorly and disappears posteri­orly (Fig. 4B).

In juvenile and adult Carcharhinus the epi­thelium is similarly modified. In the head canalsthe elaboration into folds and finger-like pro­jections is even more pronounced than in thepup and may be seen under the binocular micro­scope in gross dissection of the canal.

The function of this vesiculated epitheliumwill be discussed elsewhere. In brief, it is postu­lated that it may serve to regulate the ionic con­tent (Na, K) of the canal fluid and tissuesagainst a slow incursion of seawater through theanteriorly-directed tubules to facilitate electro­genesis in the neuromast. By controlling pH itmay also play a role in polymerization of thepolysaccharides involved in the cupular mucus.

Between the lining layer of cells and vesiclesand the vascular connective tissue there is alayer of ganglion-like cells (Fig. 4A) whichmay be differentiated from smaller connectivetissue cells. They are thin and elongate in crosssections of the canal but flattened and stellate inshape in longitudinal sections. They have a rela­tively large, round nucleus containing peri­pherally organized chromatin material and aprominent nucleolus. Vacuoles and fine granulesand fibrils appear under the phase microscope;also cytoplasmic extensions link the cells into ananastomozing network. However, no neurofi­brillae or neural connections to the neuromastnerve supply have been seen. It is possible thatthese ganglion-like cells exert neurohumeralcontrol of the overlying secretory cells of thevesiculated area.

The vesiculated area extends from the mantlecells to the squamous or low cuboidal epitheliumwhich lines the remainder of the canal and thecanal tubules. However, just below the level ofthe tubule openings, both cells and intercellularvesicles become irregular in shape and arecovered by a lining layer of cuboidal or lowcolumnar cells to form a ridge (Figs. 4A and10). The ganglion-like cells adjacent to thebasement membrane are no longer present. Thisthickened ridge is supported by vascularized

connective tissue, including a prominent bloodvessel.

The epithelial cells of the ridge area have adense, finely granular cytoplasm with a promi­nent round nucleus. The Altmann stainingmethod shows mitochondria condensed below adistal vacuolation in which metachromaticgranules are seen following Giemsa staining.The presence of mucopolysaccharides, both onthe distal surface and as granules within thecells, shows that they are producing mucus.

The ridges are well developed in the moreanterior head canals and may form one or severalfolds. Sometimes they extend into the canaltubule openings, forming valve-like flaps incross section. The ridges are less well developedin the more posterior head canals, forming onlya bulge or thickening of the epithelium. In thelaterals they are progressively reduced caudad,persisting as a double row of cells adjacent tothe neuromast and replacing the area occupiedmore anteriorly by the lining layer of vesiculatedepithelium (Fig. 4B). They are thickened into aweak ridge only in the more anterior part of thelateral canal and only in the vicinity of the canaltubules. It seems likely that in the more ante­rior head canals with forward-directed tubulesthe flaps serve to impede or regulate a slow flowof seawater into the canal, and the contiguousridges serve to direct the flow along the surfaceof the canal distal to the neuromast, thus mini­mizing the effect of flow on the cupula. Theyalso secrete a mucous cover for the epithelialsurface from the tubule openings to the neuro­mast.

The epithelium between the ridges in thecanal and lining the canal tubules is also two­layered but is relatively thin and low-cuboidalor squamous. Caudad, with the retreat of theridge cells toward the neuromast there is an in­creased circumference lined with this thin epi­thelium; it continues along the tubules until thetransition to the many-layered stratified squa­mous epithelium of the epidermis (Fig. 4B).Secretion of mucus occurs in small patches ofcuboidal cells along the course of the tubules.

Cephalic Glands

In close approximation to the more anteriorhead canals and the sacs of the ampullae ofLorenzini, there are numerous vascularized

12

gland-like pouches (Daniels, 1967) which,like the canals, open to the exterior by one ormore tubules ending in pores among the scales(Fig. 12). The tubules are shorter than thoseof the canals and proceed directly to the sur­face. In one section, a small pouch opens intoa canal tubule. The pore openings vary in sizebut are generally smaller than those of the canaltubules.

The glands have neither a neuromast nor anerve supply. Otherwise their epithelium is verysimilar to that of the more anterior head canals,with folds of vesiculated epithelium resting on athin layer of stellate, ganglion-like cells, with asmaller, lower ridge of mucus-producing cu­boidal cells resting on irregularly shaped cellsand vesicles, and with a thin, two-layered squa­mous or low cuboidal epithelium lining theneck of the pouch and continuing into the

FIG. 12. Cross section through the infraorbitalhead canal (10-4) of a Cat'chal'hinus menisOl'rah pupshowing the canal (c) and isolated cephalic glands(g). (Bouin's fixative, Mallory's stain.)

PACIFIC SCIENCE, Vol. XXIII, January 1969

tubules until it joins the stratified squamousepithelium of the epidermis. The glands appearto be partially developed anlagen of head canalswhich have remained as isolated units withneither neuromast nor innervation. The lumenis filled with a secretion containing mucuswhich, in paraffin section, is much shrunken.This mucus resembles that which covers thevesiculated area of the canal and is presumablyderived from the mucus-secreting ridge cells.It differs in density and lacks the striationsfound in the cupular covering of the neuomast.Residual proteins, demonstrated by naphtholyellow S both in the lumen of the gland andin the intracellular vesicles and blood vessels,may have been added to the lumen by thecolumnar and wedge-shaped cells of the vesicu­lated area. As in the head canals, the vesiculatedarea increases in size and in elaboration of foldsfrom pup to juvenile and adult.

The function of the glands is unknown. Itmay be postulated that they serve to controlthe ionic concentration and polarity of the tis­sues adjacent to the sensory epithelia of theampullae of Lorenzini.

INNERVATION

The gross innervation of the head canals inCarcharhimls has not been studied, but presum­ably it is similar to that described by Norrisand Hughes (1920) in SqttalllS acanthias. Themain nerves supplying the various canals in thatspecies are summarized in Table 3.

It may be noted that some of the rami of thefacial (VII) also innervate ampullae of Loren­zini, that some are intimately associated withcorresponding branches of the trigeminal (V)which may contain general cutaneous fibers, andthat one (r.m.e. VII) has a slender branch in­nervating the mandibular row of pit organs.Also, the rami of the vagus (X) innervate bothcanal and pit organs.

On leaving their ganglia, the nerves supplyingthe head canals proceed to a position just belowthe dermis. On reaching a canal, they follow itscourse, giving off ramuli which frequently di­vide into subramuli. These enter the canal mid­way along the length of a neuromast (Figs. 3Aand 3B). As seen in silver-impregnated, gold­toned frozen sections, they consist of a relatively

Lateralis Canal System in Sharks-TESTER AND KENDALL 13

• Compiled from Norris and Hughes (1920).

TABLE 3

SUMMARY OF INNERVATION OF SENSORY CANALS

FOR Sqttalus acanthias*

FIG. 13. Longitudinal section of lateral canal of aCal'cha,'hinus menisor1'ah pup showing two subramuli(s), cut after leaving the canal, each innervating threeneuromasts (n). (Gilbert's silver method.)

of the dorsal fin, and fewer more posteriorly)and form a correspondingly thinner fiber zonein the connective tissue supporting the neuro­mast. One bundle of fibers proceeds anteriorlyand another posteriorly, instead of each fiberdividing into two at the point of entry, as inthe head canals. The fibers of one subramulususually supply two neuromasts-one immedi­ately adjacent to the subramulus, and one-halfof each neuromast on either side. Occasionallythe fibers of one subramulus will supply 3neuromasts-one opposite the point of entryand one on either side (Fig. 13).

In both head and lateral canals, the fibrils ofthe fiber zone at intervals give off branches onapproaching the basement membrane. Themyelinated fibers appear to lose their myelinsheath on penetrating the basement membrane.After penetrating the membrane the fibrils areseen to branch repeatedly into fine fibrillae

MAIN NERVE BRANCH

Ramus oticus (VII)Ramus supratemporal is (IX)Ramus supratemporalis (X)Ramus supratemporalis (X)Ramus dorsalis (X)Ramus lateralis (X)

Ramus ophthalmicussuperJicialis (VII)

Ramus buccalis (VII)

}Ramus mandibularis

externis (VII)

{

large number (ca. 25) of myelinated nervefibers. Most of the axis cylinders are relativelythick (about 2 to 5 fl) in the 33-cm pup butwith a few very thin fibers among them. Afterpenetrating the canal wall, at least some of thefibers divide once, each branch proceeding in anopposite direction along the canal, as also notedby Johnson (1917) in SqllalllS acanthias. Thefibrils form a thick fiber zone in the vascularconnective tissue below the sensory epithelium.Although for the most part one neuromastseems to be innervated by the fibrils of onesubramulus, the fibrils from adjacent subra­muli overlap each other at the ends of adjacentneuromasts.

The innervation of the laterals (Tester andKendall, 1967) is similar to that of the headcanals but differs in detail. Over most of itslength the ramus lateralis lies close to the verte­bral column, moving to a more superficialposition adjacent to the canal in the caudalpeduncle and caudal fin. Ramuli are given offsegmentally and proceed posterodorsally in theconnective tissue sheath between the musclesegments. On reaching the lateral canal, whichlies in the compact layer of dermis opposite thejuncture of the dorsal and ventral musclemasses, the ramuli divide into subramuli which,after giving off fine nerves to the pit organs,enter the canal. The subramuli contain fewerfibers than those of the head canals (about 15in the region of the gills, about 9 in the region

CANAL

Lateral extension(LE)

Infraorbital (10)Hyomandibular

(HM)Mandibular (M)

Supratemporal (ST)

Lateral (L)

Supraorbital (SO)

14

FIG. 14. Longitudinal section of the supraorbitalcanal of a Carcharhinus menisorrah pup showingnerve fibers dividing into fibrillae (I) among thesensory cells of the neuromast. (Gilbert's silvermethod.)

(Figs. 3C and 14). These end among the haircells, presumably in contact with the cell mem­branes, as has been shown in teleosts by electronmicroscopy (Hama, 1965; Flock, 1965; andothers) .

It may be pointed out that, by profuse branch­ing, one nerve fiber supplies a large number ofhair cells (possibly 50 or more in the Carcha­rhinus menisorrah pup and perhaps several timesthat number in the adult). Moreover, thereseems to be overlapping innervation betweenneuromasts, but we could not determine whetherthe overlap involves one fiber or separate fibersfrom one subramulus. Frequently several un­branched fibrils are seen passing from oneneuromast to another. Electrophysiologists maybe able to determine the significance of theseobservations.

PACIFIC SCIENCE, Vol. XXIII, January 1969

SUMMARY

The lateralis sensory canal system in Carcha­rhinus consists of intercommunicating supra­orbital, infraorbital, hyomandibular, lateralextension, and lateral canals, and isolatedmandibular canals. The bore is largest in themore anterior head canals and decreases caudad.The canal tubules of the more anterior headcanals generally are relatively large and aredirected forward, whereas those of the moreposterior canals are generally smaller and aredirected backward. This allows a slow perfusionof seawater caudad through the system as theshark swims forward.

The sensory epithelium consists of neuromastswith typical hair cells embedded among sup­porting cells, covered distally by a mucouscupula and surrounded peripherally by mantlecells. The neuromasts are oval in facial sectionand, butted end to end, form an almost con­tinuous sensory epithelium over most of thesystem. They are somewhat more discrete in themandibular canals and in the posterior part ofthe hyomandibular and lateral canals. Theneuromasts decrease in length and width caudad,and there is a corresponding decrease in thenumber of sensory cells per neuromast. Theneuromasts in the adult are larger than in thepup. Since size and arrangement of the sensoryand supporting cells appears to remain thesame, it is inferred that the number increaseswith growth of the shark.

Adjacent to the neuromast, the columnarepithelium of the head canals is vesiculated andelaborated into folds. It rests on a layer ofstellate "ganglion-like" cells. The vesiculatedareas are reduced caudad. There is evidence thatthey are secretory in function, possibly adjustingthe ionic potential of the canal fluid and tissuesagainst an influx of seawater. The ganglion-likecells may exert neurohumeral control of thesecretion.

Distal to this vesiculated area, the epitheliumof the head canals is further modified to form athickened ridge of cuboidal mucus-secretingcells resting on irregularly shaped cells sepa­rated by vesicles. The ridge may serve to directthe slow influx of seawater along the surface ofthe canal distal to the neuromast. It decreases in

Lateralis Canal System in Sharks-TESTER AND KENDALL 15

prominence and retreats toward the neuromastcaudad.

On the head, adjacent to the ampullar sacs,there are numerous cephalic glands which opento the exterior by one or more short tubules andwhich contain mucus. Their epithelium is sim­ilar to that of the adjacent head canals exceptthat they lack a neuromast and lateralis innerva­tion. Their primary function may be to adjusttissue osmotic pressure and potential.

The neuromasts of the canals are innervatedby various branches of the cranial nerves (VII,IX, and X). The nerve ramuli and subramulienter the canal opposite the canal tubules andadjacent to the center of a neuromast. Belowthe sensory epithelium the nerve fibers form amore or less continuous fiber zone, from whichfine branches arise, lose their myelin sheath,penetrate the basement membrane, and branchrepeatedly into fine fibrillae which end amongthe hair cells. One fiber may innervate 50 ormore hair cells. Fibers from one nerve innervatethe neuromast opposite to its point of entry andalso the proximal ends of adjacent neuromasts.Both the number of fibers and the thickness ofthe fiber zone decrease caudad, corresponding tothe decrease in size and number of sensorycells per neuromast.

It is hoped that the information included inthis report will be of use to biophysicists, elec­trophysiologists, and behaviorists in designingmodels and experiments to clarify the functionand mode of operation of the lateralis systemin sharks.

LITERATURE CITED

COPELAND, D. E. 1948. The cytological basis ofchloride transfer in the gills of Fundulusheteroclitus. Journal of Morphology, vol. 82,pp. 201-228.

DANIELS, C. I. 1967. The distribution mor­phology, and innervation of the ampuilae ofLorenzini in the hammerhead shark and otherspecies. M. S. thesis, University of Hawaii,January 1967.

DI]KGRAAF, S. 1963. The functioning and sig­nificance of the lateral-line organs. BiologicalReviews, vol. 38, pp. 51-105.

EWART, J. C. 1891. The lateral sense organs ofelasmobranchs. I. The sensory canals of Lae­margus. Transactions of the Royal Society ofEdinburgh, vol. 37, pp. 59-85.

EWART, J. c., and J. C. MITCHELL. 1891. Thelateral sense organs of elasmobranchs. II. Thesensory canals of the common skate (Raiabatis). Transactions of the Royal Society ofEdinburgh, vol. 37, pp. 87-105.

FLOCK, A. 1965. Electron microscopic and elec­trophysiological studies on the lateral linecanal organ. Acta Oto-Laryngologica (Stock­holm), Supplement 199, 90 pp.

--- 1967. Ultrastructure and function inthe lateral line organs. In: P. H. Cahn, ed.,Lateral line detectors. Bloomington, IndianaUniversity Press. Pp. 163-197.

GARMAN, S. 1888. On the lateral canal systemof the Selachia and Holocephala. Bulletin ofthe Museum of Comparative Zoology (Har­vard), vol. 17, no. 2, pp. 57-119.

GILBERT, B. 1965. Triple silver impregnationfor selective staining of avian nerves. StainTechnology, vol. 40, no. 5, pp. 301-304.

HAMA, K. 1965. Some observations of the finestructure of the lateral line organ of theJapanese sea eel Lyncozymba nystromi. Jour­nal of Cell Biology, vol. 24, no. 2, pp. 193­210.

JOHNSON, S. E. 1917. Structure and develop­ment of the sense organs of the lateral canalsystem of selachians (Mustelus canis andSqualus acanthias). Journal of ComparativeNeurology, vol. 28, no. 1, pp. 1-74.

NORRIS, H. W., and S. P. HUGHES. 1920. Thecranial, occipital and anterior spinal nervesof the dogfish (Squalus acanthias). Journalof Comparative Neurology, vol. 31, no. 5,pp. 293-402.

SMITH, G. W. 1930. A mechanism of intakeand expulsion of colored fluids by the lateralline canals as seen experimentally in the gold­fish (Carassius atlYatus). Biological Bulletin,vol. 59, pp. 313-321.

--- 1933. The mechanism of intake andoutflow of fluids in the lateral line canal offishes. Anatomical Record, vol. 56, no. 4, pp.365-371.

TESTER, A. L., and J. 1. KENDALL. 1967. In­nervation of free and canal neuromasts in the

16

sharks CarcharhinlJs menisorl'ah and Sph)'rnalewini. In: P. H. Cahn, ed., Lateral Line De­tectors. Bloomington, Indiana UniversityPress. Pp. 53-69.

--- 1968. Cupulae in shark neuromasts:composition, origin, generation. Science, vol.160, no. 3829, pp. 772-774.

TESTER, A. L., and G. J. NELSON. 1967. Free

PACIFIC SCIENCE, Vol. XXIII, January 1969

neuromasts (pit organs) in sharks. In: P. W.Gilbert, R. F. Mathewson and D. P. Rail,eds., Sharks, skates and rays. Baltimore, TheJohns Hopkins Press. Pp. 503-531.

VAN BERGEI]K, W. A. 1967. Introductory com­ments on lateral line function. In: P. H.Cahn, ed., Lateral line detectors. Blooming­ton, Indiana University Press. Pp. 73-81.