Blood reactions in vitro in crayfish against a fungal parasite, Aphanomyces astaci

JOUBNAL

OF INVEBTEBRATE

Blood

PATHOLOQY

Reactions Fungal

19,

94-106

in Vitro

Parasite,

of Physiological

in Crayfish

Aphanornyces

TORGNY UNESTAM Institute

(1972)

AND JAN-ERIK

Botany, University

against

a

astaci N~LUND

of Uppsala, Swedm

Received June 18, 1971

The immediate and subsequent reactions in crayfish blood upon making contact with hyphae of the crayfish plague fungus were observed in vitro. Blood cells agglomerated and clumped rapidly on the hyphae in streaming blood. Very brief contact by chance with a hypha, or with cell extensions from cells or a cell clump on the hypha, caused flowing cells to stop and adhere. Chemotaxis was not believed to be of any importance. Particles originating from hemocyte granules were specifically attached to, and “encapsulated” the distal portion of, the hypha (even without the presence of the cells themselves), where their content of polyphenol oxidase became activated. These processes may be connected with the defense mechanism in crayfish. Within a few hours the process of melanization could be observed on the hyphal surface. Both the enzyme(s) and the substrate(s) for the melanization reaction seem to originate from the blood cells. Dihydroxyphenylalanine (DOPA) could replace the natural substrate and greatly enhanced the process. The melanization in whole blood was much heavier in a crayfish resistant to the disease than in a susceptible one.

blood cells on the parasite and melanieation were two easily observed reactions in both groups of crayfish after injection of zoospores. Bacteriocidal principles as well as encapsulation, agglutination, and melanixation have often been described in arthropods as defenses against invading parasites, but the total significance of these mechanisms is little known (Bang, 1967; Salt 1963, 1970; Chadwick, 1967). Very little is known about the events after fungal attack in arthropods (MacLeod and Loughheed, 1965). The present investigation was conducted in order to follow the frrst series of events after contact between the fungal hyphae and the crayfish blood and to draw some conclusions about their interrelatiomhipe.

In an earlier investigation (Unestam and Weiss, 1970) the degree of resistance to the crayfish plague fungus, Aphuncmyces astuci, was tested in two closely related species of crayfish. One of these, Pacijastacus Zeniuscu-

lus, showed very high resistance to external attack from spores added to the aquarium water, while the other species, Astacus a.stams, was very susceptible. The difference in resistance to external attack was believed to be located in the integument of the animals rather than in the hemolymph. This is supported by the fact that hyphae are seldom found in the hemolymph before the final stage of disease, but almost entirely in the inner layers of the exoskeleton, especially the ventral intersegmental membranes of the abdomen. Further, when spores were injected into the hemocoel, resistance was high in the susceptible species and even higher in the resistant one. Melanization of hyphae was heavier in resistant (Unestam

MAT-

AND

METHODS

The strains of Aphamyces astaci used in this investigation were JI and D1. They were kept in stock culture on peptone-glucase agar (Unestam, 1965) and were precultured (incubated for 24 hr) for the experi-

crayikh than in susceptible ones and Weiss, 1970). Clumping of 94

BLOOD REACTIONS IN CRAYFISH

ments in drops of peptone-glucose medium (PGl) in plastic petri dishes (Unestam, 1969) at room t.emperature. Small pieces (
The events following the exposure of hyphae of Aphanomyces a-staci to crayfish

95

blood in vitro as well aa in vivo were: Trapping of hemocytes on the hyphae, “explosion” or disintegrat.ion of certain cells in the capsule, formation of a light refracting zone on the hyphal surface, and melanization of this surface. Blood Cell Reactions to Hyphae

When a drop of crayfish blood is placed on glass or plastic, the hemocytes settle quickly and uniformly on the surface. After a few m inutes, clumps of cells are formed. Apparently, by some mechanism the cells are attached to each other and pulled together in groups (see Florkin, 1960). Inside the crayfish, blood cells were always found to attach and clump onto hyphae of A. a&xi growing from zoospores introduced in the blood by injection (Unestam and Weiss, 1970). In an attempt to arrange an artificial blood stream around hyphae of A. a&xi in vitro, a mycelium (strain I&) in serum under a coverglaSs was exposed to fresh streaming blood (see Materials and Methods). The velocity of the flow was about the same as in craylish (as observed through the intersegmental membrane of the abdomen). Some of the blood cells hitting the hyphae (by chance) immediately adhered to them, more or less tightly clinging to the hyphae (Fig. 1). Only very seldom were the attached cells seen to spread out over a hypha in an attempt to phagocytize (?) it. Often a blood cell would make contact with a hypha but, pass it and become “hanging” 20-50 p beyond it (downstream), pulled by the streaming blood. But. as t,he stream slowed down, the cell in a few seconds pulled itself back to the hypha and becamefast.ened to it. Evidently, some kind of connection must have existed between the cell and the hypha during these events. It also happened that a cell passing the first cell within some distance (without hitting either the cell or the hypha) became attached to the first cell by some mechanism and was pulled to the hypha together with the first cell.

96

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FIG. 1. Time lapse photographs of the trapping in vitro of crayf%h blood cells on hyphae of Aphanomyces aslaci at 30 set (a), 10 min (b), and 60 min (c) after addition of fresh blood. X ZOO. FIG. 2. Blood cells of Astacus astacua adhering to a hypha by means of pseudopodia. Five minutes after blood addition. Phase contrast. X 480. FIG. 3. Electron micrograph of crayfish blood cells adhering to a hypha. Citrate, 0.04 M was added with the blood. Fixed in glutaraldehyde-osmium 1 hr after addition of blood. X 4,000.

After the first cell (or pair of cells) was attached to the hypha, other cells often joined the first one(s) (Fig. 1) giving rise to localized clumps of cells around the hypha. Evidently the site of the Crst cell became a focus for clumping. Often stellate cells connected other cells and hyphae via their pseudopodia (Fig. 2). In such a case chemotaxis did not appear to play an important role. When the blood stream had ceased, after 1 or 2 min, the clumps were observed for several hours. During the first hour it was quite evident that the clump became more densely packed (Fig. l), apparently the f!.rst stage in capsule formation.

As cells became attached to a hypha or a cell clump, many soon disintegrated, each leaving only its nucleus and a surrounding, transparent cell plasma. Poinar et al. (1968) observed a Eimilar bursting of insect blood cells upon contact with a parasitic nematode in vivo. Apparently, some kind of local coagulation, possibly “cell coagulation” (Florkin, 1960) soon took place around many hyphae, since many cells m-ere trapped at some distance from it and could not be removed by the streaming blood. Strings visible under the phase-contrast microscope were sometimes seen connecting hyphae and blood cells. When blood was added on top of the

BLOOD

REACTIONS

mycelium (not in streaming blood) the clumping of cells on the hyphae n-as much less pronounced and no big clumps, as depicted in Fig. 1, were formed. Single cells, however, attached very closely to the hyphal surface (Fig. 3) in the same way m in the very initial stage of clump format,ion. Within minutes the first signs of disintegration or explosion of cells were seen. “Explosion”

of Gamlar

Hernocytes

The disintegrat.ion of many blood cells, particularly the granular ones (Toney, lg.%), was easily observed under the light microscope. But, the events in a single blood cell were too rapid for a more detailed, continuous study. Citrate-treated blood was more useful since citrate at, the concentration used delayed cell disruption. Fresh blood (0.1 ml) was added to 1 ml of 0.2 M sucrose solution containing 0.04 M citrate and 0.07 H phosphate buffer, pH 6.3, was immediately

IN

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97

cent rifugcd lightly and the cent:rifugate was studied under the light, microscope. Endrr t.hese conditions the changes in the granulocytes occurred slowly and could be photographed. Figure 4 shows that within a few minutes the granules began to be liberated from the blood cell. During the folloGng 40-50 min almost all the granules wry rrleased. During the “explosion,” transparent nonrefract.ile (under phase-conkast) blisl ers expanded out, from the cell surface (Fig. 4): apparently containing material (but usually no organelles) from the cell and taking up water, at least, from the surrounding cnvironment. The blister e.xpanded, st.ill in close connection with the cell, and often finally burst. Such a blister sometimes surrounded the whole cell, giving it the appcarante of a fried egg with the cell as the yolk in the center and the blister contents as the white of egg. Finally, n-hen none or wry few

FIG. 4. Release of granules and transparent blisters (arrow) (cf. Fig. 9) from granulocyt,es in phosphate buffer with 0.2 M sucrose and 0.04 M citrate. Time lapse: a 4 b, 2 min; b + c 8 min; c -a d. 40 min. X 1,200.

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AND

FIO. 5. Crayfish granulocyte with melanieed portion. Fixed upon bleeding in 1% formaldehyde in phosphate buffer (0.07 M, pH (3.8) containing 0.2 M sucrose. Incubated (after careful washing) for 6 hr in 2.5 m M DOPA in sucrose-phosphate buffer. X 1,200. FIG. 6. Melanized granules of a granulocyte. Treated as in Fig. 5. X 1,200.

granules were left in the cell, nothing visibly happened to the blister(s) and the rest of the cell for many hours. (The same kind of blisters could also be seen in coagulating blood and in blood cells on hyphae.) The plasmalemma and the nucleus seemed sometimes t,o be intact. The liberated granules somet,imes exploded and the contents disappeared, but frequenbly, while “floating” away from the mother cell, they budded off small particles. Apparently, the sugar-cit,rate treatment not only slowed down the explosion process of the cell but also conserved the granules when outside the cell. Cells in different stages during the explosion process were fixed in 1% formaldehyde for 2 hr, washed, and transferred to 2.5 mM L-dihydroxyphenylalanine (DOPA) in sucrose and phosphate as above. Now, many

NYLUND

of the cells showed brow-n pigment after a few hours, indicating the presence of polyphenol oxidase. The pigment appeared particularly in peripheral portions of the granulocytes, in pseudopodialike extensions of such cells, and in granules (often budding) or smaller particles still attached on the outside of t,he cell (Fig. 5). But only occasionally were the granules inside the cell stained (Fig. 6). Perhaps the enzyme becomes activated when outside the cell and/or DOPA might not. be able to penetrate easily into the cell, even after formalin treatment. Figure 7 shows an elect,ron micrograph of an intact blood cell with its osmiophilic granules. In electron micrographs granules leaving or having left the cell appeared grained and usually lacked the membrane envelope (Figs. 8, 9). Smaller particles were formed on the surface of the granule, probably the buds as observed with the light microscope. After treatment with DOPA these particles became more electron dense and so did the area in the cell inside the “erupt,ion” site (Fig. 10). The granules inside the cell were still homogeneous. Proper treatment wit,h osmium revealed a delicate, thumbprint-like structure inside both small and large granules in the cell (Fig. 11). This structure was composed of tubulelike filamcnts, considerably thinner than microtubules (Fig. 11). Similar tubules are knon n to be formed during the clotting process in the horseshoe crab (Dumont, et, al., 1966). In dried smear preparations of blood on a glass slide, the nuclei of many hemocytes stained brown after addition of DOPA, but not the granules. Nuclear phenol oxidase is probably not uncommon (Okun et al., 1970) but has no immediate connection with the melanization of the hyphal surface. Deposition the Hyphae

of the Light-Refraciing

Zones

on

When blood was added on top of the mycelium (grown in serum and washed in redistilled water or grown in peptoneglucose

BLOOD

REACTIONS

IN

CRAYFISH

Fl LG. 7. Electron micrograph of an intact crayfish blood cell with large osmiophilic fixation. X 5,COO. Tom :y, 1958) (g) and pseudopodia (ps). Glutaraldehyde-osmium

medium), blood cell attachemnt was observed as described above. Within a few hours, some portions of many hyphae became surrounded by small particles that were barely visible under the light microscope. Gradually during the first hour, a light-refracting zone appeared on these hyphae. This zone was heavily stained by methylene blue. Giitz (1969) found similar, darkening material on nematodes placed in midge hemolymph. After this stage in the investigation, myCelia grown in peptone-glucose medium were always used. Also, if not otherwise indicated, the mycelia were kept frozen until used. In this way factors such as hyphal growth, growth product formation, etc., could be omitted. All events were, however, identical with those on living hyphae so far as could be seen. The light-refracting zone of living hyphae exposed to blood for less than half an hour

99

granules (see

was studied in the electron microscope. It was found to be composed of small bodies (Fig. 12) which had the same structure as those liberated from released granules (Fig. 9). These bodies attached firmly to the cell wall (Figs. 12, 13, 14), became electron dense with DOPA (Fig. 13), and seem to be balls of thin tubules (Fig. 15) (as in the granules, Fig. 11). It is, therefore, plausible that these bodies were the same as those released by the blood cells granules (Figs. 9, 10) and that they contained the polyphenol oxidase. Citrate-treated and centrifuged blood was filtered through membrane filters (Sartorius) with different. pore sizes. The filt.rate passing through 0.2 p pores and added to mycelia gave refracting zones and melanin with DOPA on hyphae as did nonfiltered citrate blood. But the filtrate passed through 0.05 p pores did not. This suggested that particles bigger than 0.05 p in diameter but often

UNEBTAM

AND

NYLUND

Localization of Melanin Deposition About 3-15 hr after the addition of blood without DOPA, the light-refracting zones became brown due to a pigment (which was not soluble in organic solvents but was soluble in 0.5 M NaOH at Xl-6O”C), apparently melanin (Fig. 16). These melanin zones resembled those found on hyphae in vivo in muscular tissue (Fig. 17), in blood, and in the integument of infected crayfish (Unestam and Weiss, 1970). By adding DOPA (0.025 ml of a 2 mu solution to 0.1 ml blood) immediately before blood was added to the mycelium, the time for brown pigment formation of the hyphae was reduced to l-2 hr. Also, melanization was heavier with DOPA than without: more hyphae became visibly melanized, and the zones of brown pigment surrounding each hypha were usually much broader (Fig. 18). In peptoneglucose medium (PGl), with

FIG. 8. Electron micrograph of crayfish blood cell releasing its granules (g) which have became grained and electron dense after incubation for 6 hr with DOPA (as in Fig. 5) after 6xation in 1% formaldehyde. Fixation made upon bleeding from the hemocoel. Postfixation with glutaraldehydeamium. x 12,060. FIG. 9. Electron micrograph of a granule released from a crayfish blood cell. Blood added to 0.04 M citrate in 0.07 M phosphate, pH 6.8. Fixation after 3 hr in glutaraldehyde-osmium. Note the particles (p) being liberated from the granular surface. Part of the nonmembranous “wall” (arrow) of a simultaneously formed “blister” (cf. Fig. 4) is also seen. X 25,000.

smaller than 0.2 p were responsible for the melanization on the hyphal tips. This is in full agreement with the electron microscope observations where the bodies attaching to the hyphal wall and being the foci of pigment formation had a diameter of 0.1 /L or more.

FIG. 10. Electron micrograph of the surface of a crayfish blood cell treated in DOPA and fixed as in Fig. 8. A granule (g) has released particles (p) which have become electron dense after the DOPA treatment. X 25,000.

FIG. 11. Electron micrograph of granules in a crayfish blood cell. Immediately fixed in glmaraldehyde--osmium. Groups of parallel tubules cut at different angles (arrows) are seen in the granules. These tubules are thinner than microtubules (mt). X 35,COO;inset X 40,000.

FIG. 12. Electron micrograph of an obliquely sectioned hypha of Aphanomyces astaci in crayfish blood. Fixed in glutaraldehydeosmium 60 min after the addition of blood. Particulate materiai (p) is attached to the hyphal cell wall (w). X 7,000. FIG. 13. Same as Fig. 12, but 0.5 mdd DOPA was added together with the blood. The particulate material is electron dense. Melanized material (m) also surrounds the hypha (cf. Fig. 18). X 7,000.

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102

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FIGS. 14 and 15. Electron micrographs of particulate material (p) attaching to the hyphal cell wall (w). The material has become partly electron dense with DOPA. Treated and fixed as in Fig. 12. Fig. 14. Particle surface and center are electron dense. X 40,000. Fig. 15. FIigher magnification showing tubules cut at different angles (arrows). X 60,000.

or without DOPA, no melanin was ever formed on living or dead fungal hyphae. In preheated blood (55°C 30 min), with or without DOPA, or preheated plasma plus DOPA, no melanization or only very weak melanization occurred on the hyphae. Nonheated blood or plasma plus DOPA caused melanization, as usual. Piasma without DOPA did not cause melanization on the hyphae. Pigment formation did not occur in the presence of diiethyldithiocarbamate (Cu chelating) and was very weak under an atmosphere of nitrogen. Also, addition of catalase (1 mg/ml, Sigma C loo), to blood or plasma 15 min before use had no inhibitory effect on pigment formation with or without DOPA. This indicates that hydrogen peroxide and peroxidase did not play any major role (see Okun et al., 1970). As expected a polyphenol oxidase from the blood was therefore probably involved. In tests of the enzyme activity with DOPA as a substrate (unpublished) it was found that this activity was very much weaker if the plasma

was rapidly separated by centrifugation (within 30 set after bleeding) than if left for a few minutes in the glass tube before centrifugation. These data indicate that most, if not all, of the polyphenol oxidase was released rapidly from the cells while the normal substrate for melanization was slowly released by the hemocytes or liberated by some factor from these cells. In Cancer pagurus most blood tyrosinase was found in blood cells (Bhagvat and Richter, 1938). Both with and without DOPA this melanization was always much heavier in blood from Pacifastacus leniusculus than in blood from Mucus astams. The same was found in vivo by Unestam and Weiss (1970). In blood, melanin deposition occurred almost only on the distal portions of the hyphae, usually up to 100-300 p from the tip (Fig. 18). The localization of the melanization was exactly the same when DOPA was present as without DOPA or in plasma plus DOPA. Heavy melanization in the blood cells was seldom found in hemocytes

BLOOD

REACTIONS

Ih-

CRAYFISH

103

16. Melanired particles on a hypha to which crayfish blood and DOPA had been added 2 hr Note that hyphal portion formed after blood addition is not melanized. Phase contrast. X 1,200. FIG. 17. Melanired hypha in the abdominal muscle of a live crayfish into which zoospores had been injected 40 hr earlier. X 350. FIG. 18. Heavily melanized distal portion of a hypha 5 hr after the addiCon of crayfish blood and 0.4 mu DOPA. X 224. FIG.

earlier.

not, clumped on (or close t,o) the hyphae. Therefore, the involvement of t,he hypha as such in the induction of the process of heavy melanization was indicated. Furthermore, with DOPA the first signs of a brown pigment appeared on the hyphae in about 1 hr, while in blood cells or fragments of blood cells located elsewhere in the coagulated plasma no pigmentation was seen until 3 or 4 hr after the addition of blood. However, living hyphae were not required for melanin deposition on the surface. Even after the mycelium had been frozen or preheated (killed) in water, ethanol, or acetone (6O”C, 30 min) melanization in blood took place as

usual on the hyphae, again preferentially on the tips. Nor did weak hydrolysis of the mycelium, with 5% HCl in methanol for 3 hr at 6O”C, prevent. melanization. On the other hand, pure cellulose fibers (blood added to cotton fibers) did not induce melanization. Cellulose is one of the glucans in the cell wall of the Oomycetes, to which Aphunomyces belongs (Cooper and Aronson, 1967). It has to be stressed here that although blood cells were clumped on or attached to all hyphae extending from the inoculum, a small fraction of hyphal tips were not visibly melanized. The reason for t.his is not known. It should also be pointed out that if a live

104

UNESTAM

hypha succeeded in breaking through the melanin layer on and around the tip, no further melanization occurred on the new portion (Fig. 16). This indicates that the presence of fresh blood cells is necessary for the melanization of the hyphal surface. Melanincoated hyphal tips, however, often were not able to grow further, became misshaped, or showed other signs of disturbance. By adding 0.2 ml of 0.5 M sodium citrate, pH 6.8, per milliliter of blood, coagulation was prevented. When added to mycelia such citrated blood also produced the normal formation of refracting zones, and melanization of the hyphal tips but no brown pigment could be seen until after about 4 hr. Apparently the citrate delayed the process. DISCUSSION The involvement of hemocytes in arthropod defense mechanisms against internal parasites is well known, especially in insects (Salt, 1970). However, our knowledge of the mechanism of attachment to the parasite and of its inhibitory effect against the invader is extremely limited. In insects, weak chemotactic stimulation by the parasite (Salt, 1963) as well as chemotactic attraction among the hemocytes (Heimpel and Harsbarger, 1965) have been suggested. In the present investigation it was found that crayfish blood cells hitting the parasite hypha were immediately at,tached to it. The first cell might release some substance or use its pseudopodia to trap cells in the vicinity which were then pulled toward the site of the first cell. No chemotaxis is believed to be involved here, and the normal blood cell clotting mechanism (Florkin, 1960) may take part, if not sufficient in itself. Salt (1961) suggested that in insects, hemocyte encapsulation may occur only on surfaces not contributed to or laid down by hemocytes. This could be true also in crayfish. If so, there does not have to be any basic difference between blood clotting in wounds and on invading parasites in these

AND

NYLUND

animals. Levin (1967) found that in the horseshoe crab, Limuluus polyphemus, endotoxin or bacteria cause cell disruption and subsequent plasma coagulation with production of a gel which immobilizes bacteria. Encapsulation has not been observed to take place in vitro in insects (Salt, 1970); nor has it been established previously in vitro in other arthropods as far as known. The simple method used in our work gives initial cell clumping which appears identical to capsulelike structures on the Aphunomyces hyphae in vivo (&e&am and Weiss, 1970). Our method may be used in studies of encapsulation as well as phagocytosis in arthropod species which do not undergo immediate gelling of the liquid portion of the hemolymph. The apparent events following the beginning of encapsulation of the hyphae by blood cells were disruption of granulocytes, release of granules (during the first few minutes), and deposition of polyphenol oxidse (PPO) particles on the distal hyphal portions. These particles oxidize some substrate slowly released by the blood cells or liberated due to some substance from these cells. The evidence in the present work seem strong that most, if not all, of the tubule-containing PPO particles originated from the granules of the hemocytes. Interestingly, similar in vertegranular structures were found brate premelanosomes (Novikoff et al., 1968). Taylor (1969) drew attention to the similarity between these premelanosomes and organelles of the blood cells of the cockroach Leucophaea maderae and the horseshoe crab. The release of these granules from the crayfish granulocytes often left the remaining cell ma its plasmalemma surprisingly intact. In Limulus the granule and plasma membranes apparently fuse (Dumont et al., 1966). The closest thing to such release found in vert,ebrates seems to be the emiocytosis in the islets of Langerhans (Lacy et al. 1968; Esterhuizen and Howell, 1970).

BLOOD

REACTIONS

Outside the cells, the granule’s content underwent a drastic change (Figs. 8, 9). Smaller particles were formed with a fragmented content. The particles became attached to the cell wall of the fungus by some specific meahanism and their content of polyphenol oxidase was a-tivated. That these particles are directly involved in the inhibition of the hyphal growth has been indicated in many ways during our studies but awaits confirmation. Also, oxidative blood reactions were shown to inhibit myCelia1 growth and extracellular fungal enzyme activity (unpublished). So far as known, no similar process has ever been described in vertebrate blood, but it would indeed be of great interest to know whether similar mechanisms are “hidden” behing already known immunosystems. However, a recent report of an electrondense sheath surrounding phagocytized hyphae of Aspergillus in fowl chicks (Campbell, 1970) makes a comparison with the present study very intereat.ing.

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structure of Pythium debaryanu?n. nllycologia, 59, 65%670. J. N., ANDERSON, E., AND WINNER, G. D~ONT, 1966. Some cytologic characteristics of the hemocytes of Limulus during clotting. J. Morphok, 119, 181-208. ESTERHUIZEN, A. C., 11~~ HOWELL, S. L. 1970. Ultrastructure of the A-cells of cat islets of Langerhans following sympathetjc stimulation of glucagon secretion. J. Cell Biol., 46, 593-599. FLORKIN, M.

1960. Blood chemistry. In “The Physiology of Crustacea” (T. H. Waterman, ed.), Vol. 1, pp. 141-159. Academic Press, New York. G~Tz, P. 1969. Die Einkapslung von Parasiten in der HSmolymphe von Chironomus -Larven (Diptera). Zool. Anz., Suppl. Vol. 33, 610-617. HEIMPEL, A. M., .QND HARSHBARGER, J. C. 1965. Immunity in insects. Bacterial. Rev., 29, 3974. LACY, P. E., HOWELL, S. L., YOUNG, D. A., AND FINK, C. J. 1968. New hypothesis of insulin secretion. Nature (London), 219, 1177-1179. LEVIN, J. 1967. Blood coagulation and endotoxin in invertebrates. Fed. PTOC. Fed. Amer. Sot. Exp. Biol., 26, 1707-1712. MACLEOD, D. LM., AND LOUGHHEED, T. C. 1965. Entomogenous fungi. Recent Progr. Microbial.,

8, 141-150 ACKNOWLEDGMENTS

This investigation was supported by grants from the Swedish Nat.ural Science Research Council and the Fishery Board of Sweden. We are indebted to the head of this Institute, Professor Nils Fries, for his encouragement and support, to Docents Angelica v. Hofsten and Jan Westman for help and advice in the electron microscopic work, to Mrs. Anita Grandin and iMiss Ingvor Andersson for skillful technical assistance, and to Dr. Linda Fryklund for linguistic revisions. REFERENCES BANG, F. B. 1967. Serological

responses among invertebrates other than insects. Fed. PTOC. Fed. Amer. Sot. Exp. Biol., 26, 16X&1684. BH~IGVAT, K., AND RICHTER, D. 1938. Animal phenolases and adrenaline. Biochem. J., 32, 1397-1406. CAMPBELL, C. K. 1970. Electron microscopy of aspergillosis in fowl chicks. Sabouraudia, 8, 133-140. CH,IDWICK, J. S. 1967. Serological responses of insects. Fed. PTOC. Fed. Amer. Sot. Exp. Biol., 26, 1675-1679. COOPER, B. A., AND ARONSON, J. M. 1967. Cell wall

NOVIKOFF, A. B., ALBALA, A., AND BIEUPICA,

L. 1968. Ultrastructural and cytochemical observat.ions on B-16 and Harding-Passey mouse melanomas. The origin of premelanosomes and compound melanosomes. J. Histochem. Cytochem., 16,29%319. OKIJX, M. R., EDELSTEIN, L. M., OR, N., RAMADA, G., DONNELLAN, B., AND LEVER, W. F. 1970. Histochemical differentiation of peroxidasemediat.ed from tyrosinsse-mediated melanin formation in mammalian tissues. Histochemde: 23,29&309. POINAR, G. O., JR., LEUTENEGGER, R., .~ND G~Tz,

P. 1968. Ultrastructure of the formation of a melanot,ic capsule in Diabrotica (Coleoptera) in response to a parasitic nematode (Mermithidae). J. Ultraslruct. Res., 25, 293-306. SALT, G. 1961. The haemocytic reaction of insects to foreign bodies. In “The Cell and the Organism” (J. A. Ramsay and V. B. Wigglesworth, eds.), pp. 175-192. Cambridge Univ. Press, London and New York. SALT, G. 1963. The defence reactions of insects to metazoan parasites. Parsitology, 53,527-642. SALT, G. 1970. “The Cellular Defence Reactions of Insects.” Cambridge Univ. Press, London and New York.

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TAYLOR, R. L. 1969. A suggested role for the poly-

phenol-phenoloxidase system in invertebrate immunity. J. Inverlebr. Pathol., 14, 427-428. TONEY, M. E., JR. 1958. Morphology of the blood cells of some crustacea. Growth, 22, 3550. UNESTAM, T. 1965. Studies on the crayfish plague fungus Aphanomyces astan’.. I. Some factors affecting growth in vitro. Phyeiol. Plant., 18, 483-505;

AND NYLUND UNESTAM, T. 1969. On the physiology

production Plant.,

in Aphanomyces

&a&

of zoospore Physiol.

22, 236-245.

UNESTAM, T., AND WEISS, D. W. 1970. The hostparasite relationship between freshwater crayfish and the crayfish disease fungus Aphanomyces astaci: Responses to infection by a susceptible and a resistant species. JP Gen. Microbial., 60, 77-90.