Veterinary Immunology and Immunopathology , 12 (1986) 1 0 7 - - 1 1 6 107 Elsevier Science Publishers B.V., A m s t e r d a m - - Printed in The Netherlands

ONTOGENY OF GUT-ASSOCIATED LYMPHOID TISSUE (GALT) IN THE DOGFISH SCYLIORHINUS CANICULA L.

S. HART, A.B. WRATHMELL and J.E. HARRIS

Dept. Biol. Sci., Plymouth Polytechnic, Drake Circus, Plymouth PL4 8AA, UK

ABSTRACT

Hart, S., Wrathmell, A.B. and Harris, J.E., 1986. Ontogeny of gut-associated lymphoid tissue (GALT) in the dogfish Scyliorhinus canicula L. Vet. Immunol. Immunopathol., 12: I07-I16.

Cartilaginous fish occupy a fundamental position in vertebrate phylogeny and it is likely that this group has retained some of the ancestral immune

mechanisms. The ontogeny of GALT has received little attention in elasmobranchs and this study correlates this development with morphological differentiation, development of other lymphoid organs, exposure to seawater and transition

from yolk dependence to exogenous food as a source of nutrient. GALT was first represented by individual lymphocyte-like and macrophage-like cells

in the lamina propria. In later stages accumulations and intraepithelial leucocytes were recorded prior to hatching. The size of accumulations and the number of lymphocyte and macrophage-like cells infiltrating the lamina propria and epithelium increased in fish as they became dependent upon an exogenous diet. Although GAL T developed after the thymus and lymphoid- like tissue in the kidney and at approximately the same time as the epigonal,

Leydig and spleen, the source of cells populating the gut is unknown. Plasma cells and granulocytes were not observed in the developing fish until 6 months post-hatch after which the fish had a similar GALT distribution and content to the adult fish.

INTRODUCTION

Scyliorhinus canicula L., order Elasmobranchii, class Chondricythes,

is a member of a primitive group of fishes that occupy a fundamental position

in vertebrate phylogeny. Chondricythes are the most primitive vertebrate

group with a clearly defined thymus (Zapata, 1983), and it is considered

that within this group of animals a form of immunity exists which is not

different in principle from that of mammals (Marchalonis, 1977).

Studies have been undertaken on the nature of elasmobranch immunoglobulin

(Kobayashi et al., 1984); on lymphocyte function (Warr, 1981); phagocytic

activity of blood leucocytes (Parish, 1981); general immune function (Zapata,

1983) and on the morphology of the lymphomyeloid tissues (Fange, 1984).

There is, however, a paucity of information on mueosal immunology in the

cartilaginous fishes. Some work has been undertaken on gut-associated lymphoid

tissue (Dreszwina, 1905; Jacobshagen, 1915; Kondesa, 1956; Fichtelius et

al., 1968; Zapata, 1977; Tomonaga et al., 1984b and Hart et al., 1985).

0 1 6 5 - 2 4 2 7 / 8 6 / $ 0 3 . 5 0 © 1986 Elsevier Science Publishers B.V.

108

In this study, the ontogenesis of gut-associated lymphoid tissue in fish

1-2 months post-fertilization to 6 months post-hatch has been investigated

and confined to studying changes in the spiral intestine, for in the adult

this part of the gut harbours the majority of this tissue (Hart et al.,

1985).

MATERIALS AND METHODS

Eggs were collected from pregnant adult fish (600-1000g). The egg cases

were kept in circulating seawater at 12-14°C under a diurnal light regime

from December 1984 to July 1985. Fish were removed from the egg cases or

collected after hatching and fixed in neutralised 10% formal-saline, in

elasmobranch buffer (Hayle, 1965), at 4°C for 48 hours. Twenty fish between

stages i-4 (see Fig. 1); four at 30 days and four at 6 months were analysed.

Whole fish or specific tissue samples were dehydrated, infiltrated with

alcohol and embedded in methacrylate resin (TAAB). Semi-thin (I um) sections

were cut and material stained with methylene blue, Giemsa, Periodic acid

Schiffs (PAS) and methyl green pyronine.

The infiltration of leucocytes into the gut epithelium was quantified

by a stereological technique using a Werbel 2 graticule (Graticules Ltd).

The degree of infiltration was expressed as a percentage of the epithelial

volume occupied by the leucocytes.

RESULTS

The development of GALT in the spiral intestine was correlated with the

development of the gut and major lymphoid organs, the ventilation of the

eggs by seawater and transition from a dependence upon the yolk sac to an

exogenous diet (Figs. 1 and 2). The four major developmental stages, their

approximate age, and size and morphological appearance are illustrated in

Fig. 1.

At stage 1 (Fig. i) the tissue destined to become the spiral valve was

present as an outpushing of the gut (Figs. 2 and 3) and the anterior gut

was totally occluded. Partial occlusion is demonstrated in Fig. 4. Development

of the thymus and lymphoid-like tissue around the kidney had begun by mid-

Stage 1 (Fig. 1). Stage 2 was reached when the fish had developed external

gills (Fig. I). Ventilation of the eggcase by seawater did not occur until

approximately one third of the way through stage 2. The sprial valve had

begun to develop (Fig. 5) but, in the early part of stage 2 GALT was absent

and the anterior of the gut was still occluded. At the end of Stage 2 (Fig. l)

the spiral valve had undergone considerable development (Figs. 2 and 6)

and was compatible with the adult spiral valve (Fig. 7). It could be differ-

109

STAGE GUT DEVELOPMENT (NOT TO SCALE)

STAGE I

STAGE 2

STAGE______33

GALT

ABSEN'I

./

S,AGE 4 ~ V

LYMPIIOID TISSUE AND ORGANS

THYMUS KIDNEY SPt,EI.:N EPIGONALILEYDIG

%/ %/ ABSENT] ABSENT ABSENT

%/ %/ ,, ,,

%/ ./ ,/ .I

~/ ,/ ./ .i ./

./ ! ./ ./ ~/ ./

~/ ./ J J ./

Fig, i. The major developmental stages off the dogfish.

STAGE AND DEVELOPMENTAL SCHEMATIC REPRESENTATION

APPROXIMATE AGE FEATURES OF STAGES (NOT TO SCALE)

STAGE I 1-2MONTHS ALBUMIN COATED STAGE.

STAGE 2 2-5 MONTHS

STAGE 3 5-7 MONTHS

STAGE 4 POST-HATCH

UP TO 3-4 WEEKS

i - 2 CM LONG

EXTERNAL GILL STAGE.

2 - 5 CM LONG

INTERNAL YOLK SAC STAGE.

5 - 10 CM LONG

POST-HATCH STAGE.

16 CM LONG

Fig. 2. Initial development of gut-associated lymphoid tissue (GALT) in

the dogfish.

ii0

Fig. 3. Early developmental stage of the spiral valve in a Stage 1 fish.

Note the yolk platelets. Bar = i00 pm. I = intestine; S = spiral

valve; Y = yolk platelets; E = epithelium; Li = liver; B = body cavitz

Fig. 4. A partial gut occlusion. Bar = i00 pm.

0 = occluded tissue.

111

Fig. 5. Spiral valve showing the first coil development. Bar = I00 ~m.

L = lamina propria.

Fig. 6. Spiral valve showing the epithelium thrown into folds at Stage 3.

Bar = i00 ~m. F = intestinal fold; B = blood vessel;

IL = intralaminal leucocyte.

112

Fig. 7. Fully differentiated spiral valve tissue of a 1-year old fish.

Bar = I00 pm. C = connective tissue; L = lumen.

Fig. 8. A fold in the intestine showing vacuolar epithelial cells. Bar = 80 pm.

V = vacuolated epithelial cell; N = nucleus; Ci = cilia.

113

entiated from the adult, however, as the intestinal folds were small and

the epithelial cells vacuolated in appearance (Fig. 8). The gut is no longer

occluded at this stage and lymphocyte-like and PAS positive macrophage-

like cells were present in the lamina propria (Fig. 9). The leydig, epigonal

and spleen had also begun to develop by this stage (Fig. 2). Stage 3 was

characterised by the absence of external gills and the development of the

internal yolk sac. Stage 4 was free swimming but still possessed an internal

yolk sac. Stages 3 and 4 differed from previous stages as intraepithelial

lymphocyte-like and macrophage-like cells were recognised (Fig. i0), and

accumulations of predominately lymphocyte-like and macrophage-like cells

were observed in the lamina propria of the central core of the spiral (Fig.

ii). These accumulations increased in size as the fish aged. The development of

the intraepithelial leucocyte population is recorded in Fig. 12. This depicts a

relatively uniform increase in the volume of epithelium occupied by leucocytes

from the stages 3 and 4 to the adult. Granular cells and plasma cells were not

observed in the mucosae of fish examined approximately one month after hatch.

However, by six months granulocytes were observed in the mucosa and plasma

cells in the lamina propria.

DISCUSSION

Gut-associated lymphoid tissue was present in the dogfish in the latter

part off stage 2. First, as individual lymphocyte-like and macrophage-like

cells in the lamina propria and later in stages 3 and 4 as accumulations

in the lamina propria and as intraepithelial leucocytes. By six months

all the cell types described in adult GALT (Hart et ak, 1985) were present.

Precursors to GALT appeared after the thymus had differentiated and lymphoid-

like cells had appeared in the developing kidney. The spleen, Leydig organ

and epigonal tissue differentiated at approximately the same time. It is

impossible to decide from histological studies from which organ or cells,

developing GALT originated. The origin of most cells in the intraepithelial

tissue of mammals is also unclear. Some appear to be derived from precursors

in Peyers patches which migrate via the mesenterie lymph nodes to the lamina

propria and intraepithelial compartment. Thymeetomy reduces the intra-

epithelial cell population by 40-50%. Most granulated intraepithelial leuco-

cytes are, however thymus-independent (Ernst et al., 1985).

While the development of GALT has been mapped from embryonic, through

neonatal, to mature stages in S. canicula, the role of antigens in this

process is unknown. In mammals, the location and distribution of leucocytes

is independent of antigen (Ferguson and Parrot, 1972; Husband and Gowans,

114

Fig. 9. Intestinal fold surrounded by yolk platelets. Bar = i0 pm.

Fig. I0. Base of an intestinal fold infiltrated by leucocytes in a stage 3

fish. Bar = I0 ~m.

IEL = intraepithelial leucocytes.

115

Fig. Ii. Intralaminal accumulation of macrophage-like and lymphocyte-like

cells at the core of the spiral valve in a stage 3 fish. Bar = I00 pm. ILA = intralaminal accumulations.

o

I STAGE 3 STAGE 4 4 WEEKS 6 MONTHS 2 YEARS

Fig. 12. The percentage of epithelial volume occupied by leucoeytes.

116

1978) but the numbers of cells will increase in the presence of antigen

(Ferguson, 1977). The role of antigens in the development of GALT in elasmo-

branchs is further complicated as the developing embryo is exposed to seawater,

which is likely to contain antigens, from the middle of stage 2, whereas

the murine model, for example, will develop in a sterile womb until parturition.

The epithelial cells of the spiral valve appear to have a particular

vacuolar-like appearance when the fish is dependent upon the yolk sac for

nutrients. A preliminary experiment has indicated that the epithelial cells

at this stage will absorb carbon particles. This mechanism was not evident

in the adult gut. We are currently investigating when this mechanism is

lost, and whether it correlates with a transition to an exogenous diet.

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