Secondary Folding of Olfactory Organ in Young and Adult Sea Trout Gunnar Bertmar Department of Biology, Section of Ecological Zoology, University of Ume5, Sweden (Received September 9, 197 1 )

Abstract The development of the olfactory rosette in parr, smolt and adult sea trout is investigated by means of different fixation and staining tech- niques. The rosette consists of primary laminae which are folded in four types of secondary laminae. These are here called initial, cuneiform, filiform and fungiform laminae. At least initially secondary folding seems to be an interplay between a blood capillary and the sensory epithelium external to it. The ecological adaptations of folded olfactory organs are discussed.

It is well known that the olfactory organs in fish and cyclostomes differ from those of tetrapods in several respects of which one is that they are folded into primary laminae (Bertmar 1969). But it was only recently (Pfeiffer 1963; Holl 1965) that it became certain that a t least some teleostean fish also have a secondary fold- ing of the primary laminae. The develop- ment of these laminae and their eventual ecological significance are still unknown.

Material and Methods Material came from all together 82 speci- mens of Baltic sea trout Salmo trutta trutta I!!.. (Mills 1971) of River Umealven. The total length of the trout varied from 12.1 to 55 cm, and the age between 1 112 (1 + ) to 4 1/2 (4 + ) years. The youngest were parr taken from a pond of the Norr. fors’ fish hatchery, whereas the adult fish were caught at Norrfors when they were homing. Smolt were taken in May-June, a little more than 2 years old.

The entire nose of the smaller animals was fixed in toto with Bouin, alcalic

Bouin, Susa or Helly. Different fixation times between 2 and 24 hrs were used. After decalcification and paraffin embed- ding sections 6 1 0 pm thick were cut serially in frontal (transverse) and sagital projection, and stained with Azan-Mallory (Heidenhain) or Heidenhain ironhaemat- oxylin. In larger animals only the ol- factory organs were dissected out from anaesthetized animals (MS 222 1 : 3000 was used) and processed quickly in the same kind of fixations as for the other material.

Results Parr

These young fish have 10-12 primary laminae (Fig. 1). The laminae are built from a dermal fold of lome areolar tissue with a few lymphoid wandering cells, scattered pigment cells, blood and lymph vessels and nerve bundles of the nervus olfactorius (Figs. 1-3). In the top of the fold, reticular tissue is about to form. This tissue is still the best support of the primary laminae.

Acta Zoologica 53: 113-120 (1972) 8 - Acta Zool. 1972: 1

114 Gunnar Bertmar

Most of the primary laminae is covered with sensory epithelium, but indifferent epithelium is present on top of them. The indifferent epithelium is stratified and has a loose basal zone of 2-3 cell layers and a tight surface zone of 3 4 cell layers. Goblet cells and ciliated nonsensory cells constitute the mucus-covered free surface towards the olfactory chamber. In the basal zone there are blastema cells and a type of supporting cells which are very similar to fibroblasts. The latters are at- tached to small processes of the basement membrane (basal lamina).

Secondary folding of the posterior and middle primary laminae begins in young parr (1-1 1/2 year). Some cells aggregate into a little depression in the sensory epithelium, bounded by two diminuitive secondary laminae (Fig. 2) . The latters I call initial secondary laminae. They still consist of only sensory epithelium, and the basement membrane underneath is un- folded. A few cuneiform laminae, that is, the next stage in the development of secondary laminae, may be formed already in these young fish.

The depressions are somewhat more differentiated than the rest of the still rather homogenous sensory epithelium in the respect that a surface zone 2-3 pm thick and a basal zone about 2 pm thick are about to form on each side of a nuclear zone which is 50-60 pm wide., Four main cell types are found: receptors, supporting cells, basal cells and goblet cqlls. Of these I have found two types of receptors and supporting cells and four types of basal cells. These cell populations and their dynamics are described else- where (Bertmar 1971 a) .

The development of the olfactory roset- te in old parr (1 1/2-2 years) is charac- terized by further morphogenesis of sec- ondary laminae. The initial laminae

change into cuneiform secondary laminae. The latter type consists of a thin dermal fold covered with indifferent epithelium (Fig. 4) . The morphogenesis of secondary laminae results in a change of the distribu- tion of the sensory epithelium in these parr. I t becomes restricted mainly to the depressions and the proximal 3/4 of the cuneiform secondary laminae.

Smolt

These animals have an olfactory rosette which has grown from behind and for- wards. The posterior and middle primary laminae are larger than the anterior ones. This difference is present already in parr and remains in the adult. The number of primary laminae is 12-14.

All primary laminae are now folded. The secondary laminae cover all but the tip of the primary laminae. As before this tip consists of indifferent epithelium with a tight surface zone and a loose basal zone. The dermal part inside the laminae has rather loose connective tissue to sup- port them and reticular tissue is still present in the top of the fold. There are blood vessels, lymphatics, pigment cells and fila olfactoria inside the dermal fold.

Rather large secondary laminae have been formed in the posterior and middle primary laminae. The more differentiated of them are no Ionger cuneiform but fili- f o r m fn cross section (Fig. 5 ) , and basally

Figs. 1-3. Left olfactory organ of young parr of sea trout in sagittal section. The part within square is shown in higher magnification in the next figure. bm, basement membrane; c, cilia of ciliated supporting cell; d, depression of sensory epithelium; df, dermal fold; ft, fat tissue; g, goblet cell; isl, initial secondary lamina; m, mucous; o, olfactory nerve; p, pig- ment cell; p l , primary lamina; T, receptor cell; rg, receptor group; se, sensory epithelium.

Olfactory Organ in Sea Trout 115

116 Gunnar Bertmar

Fig . 4. Right olfactory organ of old parr of sea trout in cross section, showing parts of a cuneiform secondary lamina and two depres- sions. bc, blood capillary; GS, ciliated supporting cell; d, depression of sensory epithelium; g, goblet cell; ie, indifferent epithelium; ns, non- ciliated supporting cell; ok, olfactory knob; 7,

rod receptor; s, spindle receptor.

they have a dermal fold with a blood and a lymph capillary and a few scattered mesenchymal and blastemal cells. The sensory epithelium is restricted to the proximal half of the filiform secondary laminae.

The sensory epithelium is equally well developed in all primary laminae, and on both sides of them. There is no difference between proximal and distal secondary laminae of the same type and the same

lnitlol .sec lam + Cuneiform set lam

lyrnphotc. usscl pr imry lamina

F i g . 5. Diagram of developmental stages of secondary laminae in olfactory organ of sea trout.

Olfactory Organ in Sea Trout 1 17

Figs. 6-7. Right olfactory organ of adult fis, filiform secondary lamina; fo, filum ol- sea trout (47 cm) in cross section, showing factorius; fus, fungiform secondary lamina; g, different types of secondary laminae. bc, blood goblet cell; ie, indifferent epithelium; I , lym- capillary; bz, basal zone; csl, cuneiform sec- phatic vessel; rn, mucuous; r, rod receptor; sz, ondary lamina; d, depression; dct, dense con- surface zone. nective tissue; d f , dermal fold; fb, basal cell;

118 Gunnar Bertmar

primary lamina. The sensory epithelium is thinner on the slopes of the filiform laminae than in the depressions between them. This is caused by the growth of the secondary laminae and results in a deepening of the depressions into valleys. Furthermore, the sensory epithelium is more zonated than in parr.

Adult

These fish have an olfactory rosette of 14-16 primary laminae when they return to spawn. These are now supported all through by reticular and collagenous con- nective tissue. This makes the laminae more rigid thin before (Fig. 6 ) .

There are about 10 secondary laminae on the anterior primary laminae, and 16 on the posterior (and larger) primary laminae. The most anterior primary laminae has about 3 secondary laminae on the anterior (medial) side and about 7 on the posterior (lateral) side of it, whereas the most posterior laminae has about 7 secondary laminae on the anterior side and about 9 on their posterior side. Sometimes a secondary lamina lies op- posite that of the next primary fold, same- times not. I t is therefore no regular zip- system in the arrangement of the laminae.

The filiform secondary laminae have become more fungiform in cross section in the adult (Figs. 5, 7) . The dermal fold (papilla) of this final stage in the devel- opment of secondary laminae is com- paratively voluminous and contains, in addition to the blood capillaries of the two preceeding stages, a lymphatic vessel or sac and large intercellular space. The turgor of this keeps the “hat” expanded. Basally there is often reticular tissue. This makes the neck of the fungiform laminae more rigid than the more flexible top of it.

About 75 % of the fungiform laminae

is covered with indifferent epithelium, compared with about 50 % of the filiform type. The sensory epithelium is pseudo- stratified on the slopes of the fungiform laminae and stratified in the valleys be- tween them. As in smolt there are four zones in the valley epithelium.

There is still secondary folding going on in adult fish, especially in the top and base of the primary laminae. Cells ag- gregate there and elongate to almost threadlike cells which eventually are push- ed out into the olfactory chamber. Es- pecially in the basal zone macrophages and lymphoid wandering cells are gathered.

Discussion

Primary Laminae

Different types of olfactory rosette in teleosts was described by Reinke (1937) and Holl (1965). In Salmo gairdneri there is a continuous formation of primary laminae, according to Holl (1965), but according to Teichmann (1954) the number is constant after 15 cm length (adult). In S. trutta trutta L. the number of primary laminae increases in young fish (parr and smolt) and is largest in adult fish that return to spawn. I have not had access to fish which return to spawp a second time. I t is therefore im- possible for the moment to decide if the number increases with age all the time. Probably it is a function of size, not of age.

The salmonid primary laminae are ar- ranged in a 90 degrees olfactory rosette (Holl 1965) with the first fold situated posteriorly in the prolongation of the central axis. The next four laminae radiate from the posterior end of the axis, two on each side of the first fold. The younger laminae are then formed per-

Olfactory Organ in Sea Trout 119

pendicularly on each side of the axis from behind and forwards. This oval type of rosette (‘Type I1 of Holl, 1965) is also found in cyprinids, silurids, gadids, Anguilla, Conger and Muraena. It is ac- cordingly present in both macrosmate and microsmate fish, and it should therefore be of no ecological significance.

Secondary Laminae

Secondary folding was first described in Acipenser (Dogie1 1887). And it was not until 1954 that it was found in teleosts. Teichmann reported that he suspected to have seen secondary laminae in Salmo gairdneri, but if so he thought they might be a fixation artefact. Pfeiffer (1963) confirmed on fresh material that this species (he also found them in Oncorhyn- chus sp.) does have secondary laminae, and he suggested from fixed material that they consist of indifferent epithelium only, However, judging from his figures no ’8 and 9 they also have some sensory epi: thelium. Holl (1965) also could confirm that S. gairdneri has secondary laminae, and he also found them in S. trutta fario, but not in the other 16 teleost species he studied.

The above survey shows that there has been no study of the development of the secondary laminae. In this paper four stages have been described (Fig. 5) . Ini- tially there may be an interplay between organizators in a blood capillary in the dermis and the sensory epithelium exter- nal to it. In the cuneiform lamina a triangle of indifferent epithelium is form- ed, and the capillary lies in a small dermal fold (papilla) below the acute angle of the triangle. It seems as if indifferent cells are breaking through the sensory epithelium and pushing it apart (Fig. 4).

Simultaneously, some cells of the sensory epithelium degenerate, and some of these are extruded into the olfactory chamber whereas others are phagocytized by lym- phatic wandering cells, macrophages and granulocytes (Bertmar 1971 a) .

Ecological Adaptations

It has been assumed that animals with large olfactory organs and small eyes (e.g. the olfactory epithelium and the retina) are macrosmatic “nose” animals, and those with small olfactory organs and large eyes are microsmatic “eye” animals. Teichmann (1954) also considered a group inbetween these, but he put Salmo together with the microsmates. However, this does not fit with recent information that olfactory cues are more important than visual cues for successful homing in Salmo trutta, a microsmate (Bertmar 1971 b) .

The folding of the olfactory organs is an adaptation in fish and cyclostomes for effective use of the space of the olfactory chamber. The primary laminae in sea trout are stiffened by dense connective tissue and the secondary laminae are sup- ported by reticular tissue and turgor. This keeps the laminae upright and free from each other. And this in turn means that olfactory water has free passage to the sensory epithelium.

The result of the secondary folding pro- cess certainly is an increase in the area of the primary laminae, but about 75 ”/o of this increase can be referred to the indif- ferent epithelium. Sensory epithelium certainly covers only the proximal part of the filiform and fungiform secondary laminae, but still the secondary folding to some extent does increase the area available for chemoreception. In general, the increase in sensory area seems to be

120 Gunnar Bertmar

correlated with an increase in olfactory capacity. The larger sensory area in spawning sea trout should therefore mean that they have a better olfactory capacity when they return as adults than when they leave the river as smolt.

According to Pfeiffer (1963) the sec- ondary folding of the primary laminae is a function of size, and is not directly related to a marine phase in the life cycle of Pacific salmon. This is also valid for Baltic sea trout. All primary laminae are certainly secondary folded when the smolt leaves the river but new primary laminae are formed later on, in the adults, and these folds also become secondary folded.

Acknowledgements I am most grateful to Miss Karin Ekstrom for technical assistance, Mr. Harald Jo- hansson and his staff of the Norrfors’ fish hatchery for fish material, and the Swed- ish Natural Science Research Council for financial support via grants No. 9021 B, 9567 B and 282 B.

References Bertmar, G . 1969. The vertebrate nose, remarks

on its structural and functional adaptation and evolution. Evolution 23: 131-152.

- 1971 a. Cell populations in a fish olfactory organ. In manuscript. \

- 1971 b. Sensory mechanisms of homing in Salmonid fish. 2. Experiments on Baltic brown trout (Salmo trutta trutta L.). In manuscript .

Dogiel, A . 1887. Uber den Bau des Geruchs- organes bei Ganoiden, Knochenfischen und Amphibien. Arch. M i k ~ . Anat. 29: 74-139.

Holl, A. 1965. Vergleichende morphologische und histologische Untersuchungen am Ge- ruchsorgan der Knochenfische. Z . Morph. Okol. Tiere 54: 707-782.

Mills, D. 1971. Salmon and Trout, Oliver & Boyd, Edinburgh.

Pfeiffer, W. 1963. The morphology of the ol- factory organ of the Pacific salmon (On- corhynchus). Can. J . Zool. 41: 1233-1236.

Reinke, W. 1937. Zur Ontogenie und Anatomie des Geruchsorgans der Knochenfische. Z . Anat. Entwicklungsgesch. 100: 600-624.

Teichmann, H . 1954. Vergleichenden Unter- suchungen an der Nase der Fische. Z . Morphol. Okol. Tiere 43: 171-212.

Dr. Gunnar Bertmar Department of Biology Section of Ecological Zoology University of Umeh S-901 87 Umei 6, Sweden