Chemical, histochemical, and histopathological studies on corals, Porites spp., parasitized by tremato de metacercariae

lOURNAL

OF INVERTEBRATE

PATHOLOQY

23,

303-317

(1974)

Chemical, Histochemical, and Corals, Porites spp., Parasitized THOMAS Institute

for

Histopathological by Trematode

C. CHENG AND ALAN

Pathobiology, Center Bethlehem, Received

for

Health

Pennsylvania December

Studies on Metacercariae’

K. L. WONG Sciences, Lehigh

University,

15015 5, 1973

The occurrence of the metacercaria of Plagioporus sp. encysted in two species of corals, Porites compressa and P. lobata, from Kaneohe Bay, Oahu, Hawaii, is reported. This parasite is covered with a bilayered metacercarial cyst the chemical compositions of which have been ascertained by histochemistry. Both of these cyst walls are believed to be secreted by the parasite. In addition, the cytochemical properties of the metacercaria and the adjacent host cells are reported. Histo- and cytopathological alterations in the epidermis and gastrodermis of the coral hosts were studied and such changes have been interpreted to have resulted from mechanical pressure exerted by the parasite. Because gross observations had revealed the presence of elevated nodules at the sites where the metacercariae occurred, chemical analysis was conducted to ascertain whether this larval trematode had induced hypercalcification in the hosts. However, calcium determinations of Decal in which pieces of parasitized coral tissue had been decalcified revealed that hypercalcification had not occurred. Examination of the cellular reactions in both species of Porites revealed that if the gastrodermis had been ruptured, partial encapsulation of the parasite occurs. The reaction cells include free cells of the mesoglea that become fused, accidentally trapped cnidoblasts and nutritive-muscular cells, and gastrodermal interstitial cells. Despite the partial encapsulation, the encysted metacercariae did not appear to be injured. INTRODUCTION

The life cycles of many species of digenetic trematodes include a second intermediate host in which the metacercaria, encysted or unencysted, occurs. Although a variety of vertebrates and invertebrates are known to serve as second intermediate hosts, as far as we have been able to determine, anthozoan cnidarians have not been reported to serve in this capacity, although both hydrozoans and scyphozoans have been reported (Lebour, 1916; Dollfus, 1963 ; Stunkard, 1967, 1969). Hence it appeared to be of interest to report the occurrence of a species of metacercaria en-

cysted in two species of the anthozoan Porites from Hawaiian waters. In addition, both histo- and cytopathological alterations in parasitized corals were studied as well as the chemical nature of the metacercarial cyst as determined by histochemistry. Furthermore, since, as described later, the sites where the parasites occurred appeared as elevated nodules, studies were carried out to ascertain as to whether the presence of the parasites had induced hypercalcification in the adjacent host tissues. Finally, we are reporting our observations on the cellular reactions of the coral hosts to the metacercariae.

’ This research was supported in part by a grant from the American Cancer Society and in part by a grant (FD-66416-63) from the U.S. Public Health Service.

MATERIALS

The occurrence of abnormal growths on two species of madreporarian, reef-building 303

Copyright All rights

0 1974 by Academia Press, Inc. of reproduction in any form reserved.

AND METHODS

304

CHENG

corals, Porites compressa and P. lobata, in Kaneohe Bay, Oahu, Hawaii, was called to the attention of the senior author by Dr. Howard M. Lenhoff and Dr. Leonard Muscatine of the University of California at Irvine and Los Angeles, respectively, during the summer of 1968. Subsequent examination of additional specimens collected during 1968-1969 revealed the presence of identical abnormal growths associated with many individual polyps. These growths appeared as irregularly shaped, pink or yellowish nodules protruding from the polyps. Careful dissection of several revealed the presence of an encysted trematode metacercaria at the core of each nodule. Specimens of both species of Porites collected at a depth of 3 to 6 feet at low tide were usually heavily parasitized. Those collected at greater depths were either sparsely parasitized or nonparasitized. Histology. Coral heads including parasitized polyps were fixed in 10% seawater formalin for 8-12 hr, after which pieces approximately 2 cm long containing parasites were broken off and decalcified in Decal (Scientific Products, Evanston, Illinois) for S-10 hr. The tissues were subsequently washed for 3 hr in tapwater, dehydrated via a graded butanol series, and embedded in Tissumat. Each piece, 30 in all, was serially sectioned at 9 pm, and representative sections were stained with Harris’ hematoxylin and eosin, Mallory’s triple connective tissue stain, or Milligan’s (1946) trichrome stain. Histochemistry. Selected histochemical procedures were carried out. Tissues employed for these were passed through two changes of xylene, 2 hr each, during the embedding process so as to ensure the extraction of what lipids were present. The specific tests carried out were: (1) Kramer and Windrum’s (1955) toluidine blue test for the detection of metachromasy ; (2) the periodic acid-Schiff (PAS) reaction (McManus, 1946), with selected control sections pretreated with a 0.6% aqueous solution of malt diastase for 3 hr at 37°C

AND

WONG

prior to oxidation with periodic acid, and others exposed to Schiff’s solution without prior oxidation with periodic acid to detect the presence of free aldehydes ; (3) the mercuric bromophenol blue method for proteins (Mazia et al., 1953) ; (4) Baker’s (1944) modification of Sakaguchi’s (1925) test for arginine-containing proteins; (5) Baker’s (1956) modification of the Millon reaction for tyrosine-containing proteins; (6) Yasuma and Itchikawa’s (1953.) ninhydrin-Schiff test for amino groups ; (7) Constantine and Mowry’s (1966) modification of Lev and Spice& (1964) alcian blue stain at pH 0.5; (8) Mowry’s (1963) alcian blue stain at pH 2.6; (9) Scott’s (1960) alcian blue stain at pH 5.7; and (10) Kliiver and Barrera’s (1953) 1~x01 fast blue technique for phospholipids. Calcium determinations. Since gross exanlinations of the sites of parasitization had revealed elevation of the nodules above the body surface and the presence of conspicuous epidermis-covered projections of lobes of the calcium carbonate skeleton from the nodular surfaces, it was decided to ascertain whether the presence of the parasite had stimulated hypercalcification in parasitized polyps. This was accomplished by comparing the ionic calcium concentrations in parasitized and nonparasitized polyps. Specifically, both categories of polyps were excised from whole corals, weighted, and individually placed in 2 ml of a 1:20 dilution of Decal. Polyps of the control and parasitized groups used in the quantitative calcium determinations had been either fixed in 10% seawater formalin or were living when placed in the decalcifying solution. All the specimens were removed from the Decal solution after 16 hr and the amount of ionic calcium in each sample of the solvent was determined calorimetrically by employing the method of Ferro and Ham (1957)) using a Bausch and Lomb Spectronic 20 spectrophotometer read at 520

ON METACERCARIAE

rnp. No calcium was determined to be present in fresh Decal by employing the same technique. In order to be certain that all of the calcium present in each polyp had been dissolved in the solvent, three cytochemical tests for calcium were applied to selected, decalcified polyps: Pearse’s (1961) eriochrome black T test, McGee-Russell’s (1955) calcium red method, and Dahl’s (1952) alizarin red S method. In each case, no residual calcium was detected in the tissues.

305

IN CORAI+‘S OBSERVATIONS

AND RESULTS

Gross Morphology

When observed from the top, the individual polyps of P. compressa and P. lobata are hexagonal or pentagonal in outline and measure from 1.17 to 2.52 mm in greatest diameter (Fig. 1). The heavily calcified skeletal edges are slightly raised and spiculelike, multilobed extensions of the thecal edge project from the anterior and lateral surfaces. Each projection, covered by the overlying epidermis, averages 0.139

FIG. 1. Photograph of a portion of uninfected Porites compressa showing individual polyps with hexagonal or pentagonal outlines. FIG. 2. Photograph of effect of infection of Porites compressa by Plagioporzcs metacercaria. Notice outgrowths bearing skeletal protrusions along the elevated edge (EE). FIG. 3. Photograph of effect of infection of Porites compressa by Plngioporus metacercaria. Notice skeletal portrusions along elevated edges of polyps (EE). FIG. 4. Photograph of abnormal growth on Porites compressa: parasitized by Plagioporus metacercariae showing complete displacement of tentacles and distortion of parasitized polyp (EP).

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mm (0.037-0.220 mm) in greatest height. The tentacles, arranged more or less in a circle, are situated mediad to the border. The mouth-bearing hypostome is located in the center of the tentacular ring. Nonparasitized polyps are off-white. In newly parasitized polyps, abnormal growths occur primarily along the edges of each polyp (Figs. 2 and 3)) although in infections of long duration, each nodule will extend medially, reaching, displacing, and overgrowing the tentacles (Fig. 4). Each of these nodules is initially discrete and averages 0.573 mm (0.230-1.171 mm) in diameter. However, in time, these increase in size and fuse to form large irregular outgrowths. As a result, the hexagonal outline of each parasitized polyp is greatly distorted. These abnormal growths are characteristically pink and bear enlarged thecal extensions, each averaging 0.34 mm (0.146-0.585 mm) in height. Occasionally yellowish nodules also occur. Metacercarial

Cyst

The cyst surrounding each metacercaria is comprised of two walls. The inner, herein designated as the primary cyst wall, is thicker and more conspicuous. The outer, designated as the peripheral cyst wall, abuts the primary cyst wall but has an irregular outer surface. Furthermore, it is only about one-third to one-fourth the width of the primary cyst wall. Both walls are noncellular (Figs. 5 and 6). Histochemistry

The histochemical and histological staining affinities of the metacercaria and the

WONG

enveloping cyst walls are summarized in Table 1. More detailed descriptions are presented below. In PAS-treated sections most of the parasite’s body cells, including the cystogenous glands, are PAS-positive, and the tegument is intensely PAS-positive (Fig. 5j. Both walls of the metacercarial cyst are also PAS-positive, but not as intensely nor as homogeneously as the tegument (Fig. 5). Most of the PAS-positive substrates in the body of the parasite can be considered to be glycogen since these are diastase labile. On the other hand, the PAS-positive material(s) present in the cystogenous glands, tegument, and in the cyst walls is not glycogen since it is diastase resistant. The possible chemical nature of this material is considered later. Free aldehydes, as indicated by exposure to Schiff’s reagent without preoxidizing with periodic acid, do not occur in any of the tissues examined. Sections stained with alcian blue at pH 0.5 and 5.7 have revealed no binding of the parasite’s tissues or the primary cyst wall to the stain, but the peripheral cyst wall is bound to alcian blue at these two pH’s (Figs 6, 7, and 8). On the other hand, in sections treated with alcian bIue at PH 2.6, not only are most of the cells in the body of the metacercaria stained, especially the cystogenous glands (Fig. 9), but the material secreted from these glands is also stained. This cystogenous material appears as irregular strands leading from the body surface to the inner surface of the surrounding cyst (Figs. 9 and 10). The primary cyst wall is essentially unstained, but

FIG. 5. Photomicrograph of a section of an encysted metacercaria of Plngioporus sp. showing weakly PAS-positive primary metacercarial cyst wall (MC), weakly PAS-positive peripheral cyst wal1 (PC), and intensely positive metacercarial tegument (T). Periodic acid-Schiff reaction. x400. FIG. 6. Photomicrogaph of a section of an encysted metacercaria of Plagioporus sp. showing binding of alcian blue at pH 0.5 t.o the peripheral cyst wall (PC). Alcian blue pH 0.5. x400. FIG. 7. Phase contrast photomicrograph of a section of an encysted metncercaria of Plngioporzis sp. showing binding of alcian blue at pH 0.5 to the peripheral cyst wall (PC). Alcian blue, pH 0.5. x400. Fm. 8. Phase contrast photomicrograph of a section on an encysted metacercaria of Plagioporus sp. showing binding of alcian blue at pH 5.7 to the peripheral cyst wall (PC). Alcian blue, pH 5.7. x400.

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AND

TABLE HISTOLOGICAL

AND

HISTOCHEMICAL

STAINING

WONG

1 CHARACTERISTICS

OF PARASITE

Metacercarial

Stain

Cystogenous glands

Parasite”

H&E

Primary wall

AND

HOST

CELLS

cyst Peripheral wall

cells

Nuclei hematoxyphilic, cytoplasm eosinophilic

Nuclei hematoxyphilic, cytoplasm eosinophilic

Mallory

Cells red, tegument blue

Red

Blue

Light

Milligan

Red

Red

Blue

Green

PAS

+

Mostly

Weakly +

Weakly +

-

-

-

-

Nuclei hematoxyphilic, cytoplasm eosinophilic (n-mb cells with eosinophilic cytoplasmic granules) Nuclei red, cytoplasm blue (n-ma cells with red cytoplasmic granules) Nuclei red, cytoplasm chromophobic (n-m6 cells with red cytoplasmic granules) Some with + inclusions, zymog-en granules -

Labile

Resistant

Resistant

Resistant

Mostly

-

-

-

+

Maw t

+

Sme +, strands

+

Many +

Schiff’s Diastase pH 0.5 alcian blue pH 2.6 alcian blue 5.7 alcian blue Toluidine blue

+

philic

+

Eosinophilic

Host

blue

labile

pH

-

-

-

d

4”

OLC

Many + 8”

tegument dC Hg

bromophenol blue Arginine Tyrosine Amino groups Phospholipids a Predominant 6 Nutritive-muscular c Metachromasy.

-

Zymogen a”

+ -

-

+ + +

Some + Some

staining

+ +

+ + +

+ -

granules

+ Some Some + -

+ +

characteristic. cell of gastrodermis.

it does include extremely fine, radially oriented, stained strands (Fig. 10). The peripheral wall is alcian blue-positive at pH 2.6 (Fig. 10). In sections stained with toluidine blue, all the tissues of the metacercaria are oc-metachromatic (orthochromatic) except,

for the cystogenous glands and tegument, which reveal a mixture of 01 and /I metachromasy. The primary cyst wall is a-metachromatic, but the peripheral wall is p-metachromatic. Cells of the parasite’s body are rich in proteins as indicated by their being stained

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FIG. 9. Photomicrograph of a section of a metacercaria of Plagioporus sp. showing binding of alcian blue at pH 2.6 to subsurfacial cystogenous glands (CG) and strands of secreted material (CS). Alcian blue, pH 2.6. x400. FIG. 10. Photomicrograph of a section of a metacercaria of Plagioporus sp. showing binding of alcian blue at pH 2.6 to strands of cystogenous material (CS) passing from the parasite to the primary cyst wall. Also notice that the strands (CS) pass through the primary cyst wall and are deposited as a part of the peripheral cyst wall (PC). Alcian blue pH 2.6. x900.

with mercury bromophenol blue. However, the cystogenous glands and both walls of the cyst are not stained. Results with Sakaguchi’s stain for arginine-containing proteins have revealed the absence of this category of molecules in the parasite, its cystogenous glands, as well as both walls of the cyst. On the other hand, the parasite,

its cystogenous glands, and the primary wall react positively with Millon’s reagent but the peripheral wall does not. These same tissues, plus the peripheral wall, react positively with the ninhydrin-Schiff test. In addition, the primary wall includes phospholipids as revealed by the 1~x01 fast blue stain.

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the former have resulted from mechanical pressure exerted as a result of direct conThe histology of the body walls of P. tact with parasites. If the distortion is pricompressa and P. lobata, including the marily due to chemically induced lysis, one walls of the tentacles, are similar. As is would expect to find evidence of this in characteristic of all cnidarians, the wall is areas close to but not in the immediate comprised of a cellular epidermis, which is vicinity of parasites (Malek and Cheng, separated from the cellular gastrodermis by 1974). As discussed at a later point, some a Iayer of mesoglea. The latter is very thin of the ruptured portions of the gastrodermis in Porites spp. The cell types comprising is also due to ingestion by metacercariae both of the cellular layers are known (see prior to encystation. Hyman, 1940, for review). It is noted that At the cellular level, the most connematocyst-enclosing cnidoblasts occur in spicuous difference between the epidermal ‘both the epidermis and gastrodermis, in- and gastrodermal cells of nonparasitized cluding the walls of the tentacles. Based on and parasitized tentacles is the significant measurements obtained from the cross sec- compression of these two cellular layers. tions of the tentacles of 16 different polyps, Based on measurements taken from cross the thickness of the epidermis averages sections of 14 parasitized tentacles, the 0.064 mm (0.045-0.081 mm) and that of the width of the epidermal layer averages 0.031 gastrodermis averages 0.025 mm (0.021- mm (0.013-0.054 mm), and that of the gas0.032 mm). trodermis averages 0.009 mm (0.004-0.011 In parasitized polyps, the metacercariae mm). In addition, a number of the nematoare encysted primarily in the gastrovascucysts of gastrodermal origin have been dislar cavity, within the tentacles; rarely is charged into the gastrovascular cavity in one found within the main portion of this the vicinity of the metacercariae. cavity. When present within tentacles, not Except for the physical compression, no only is the diameter of the gastrovascular other cytopathological changes have been cavity greatly distended, but the cells com- observed associated with the epidermal prising both the epidermis and gastrodermis cells ; however, two other types of changes of the adjacent tentacular wall are also occur in gastrodermal cells. One of these is greatly compressed and distorted. Furtherassociated with the nutritive-muscular more, it is fairly common to find regions cells. Specifically, the cytoplasm of these in which the gastrodermis is detached from cells become highly vacuolated, resulting in the mesoglea, and isolated cells occur in the an ‘(empty” appearance. The second type gastrodermal cavity. These histopathologiof change is associated with the gland cells. cal alterations, especially the compressed Although zymogen granules occur within appearance of the body wall, are believed these cells in nonparasitized polyps, both to have resulted from the mechanical presthe number of granules and cells are greatly sure exerted by the metacercaria during increased in parasitized polyps. These cells and after encystment. This assumption is are also hypertrophied. based on the fact that the compression and In sections of both parasitized and nondetachment of gastrodermal cells is uneven, parasitized polyps stained with H and E, i.e., the cells are conspicuously more com- the nuclei of the epidermal and gastropressed and disarranged in areas where the dermal cells are hematoxyphilic while the parasite is in intimate contact with the cytoplasm is eosinophilic in varying intententacular wall. Furthermore, the demarcasities. The zymogen granules within intact tion zones between distorted regions in juxgastrodermal gland cells and in those detaposition to parasites and normal-appeartached in parasitized polyps, however, are ing regions are abrupt, thus suggesting that not stained but retain their yellowish color. Histopathology

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In sections stained with Mallory’s, nuclei of cells of the body wall of parasitized, and nonparasitized polyps are red while the cytoplasm is blue. There is, however, an increase in the number of bright red cytoplasmic globules in the gastrodermal nutritive-muscular cells of parasitized polyps. These globules are packed between the cytoplasmic vacuoles. The zymogen granules in both intact and detached gastrodermal cells are not stained. In sections stained with Milligan’s trichrome, the nuclei of the cells of the body wall of both parasitized and nonparasitized polyps are characteristically magenta in color while the cytoplasm is pale purple. The most strikingly stained material are the zymogen granules in the gastrodermal gland cells. These appear bright green. Because of this conspicuous contrast, the hypertrophied gland cells and the increased number of zymogen granules and cells in parasitized polyps are quite conspicuous.

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CORALS “.

MC-

FC

FIQ. 11. Drawing showing one type of host reaction when the gastrodermis is ruptured. The free cells of the mesoglea (FC) adhere to the peripheral cyst wall (PC) and trap cnidoblasts (CN) and nutritive-muscular cells in a syncytial network. The primary cyst wall (MC) is situated mediad to the peripheral wall.

: MCPC

;..

‘.

FIG. 12. Drawing showing a second type of host reaction. Notice gastrodermal interstitial cells (IC) adhering to the peripheral cyst wall (PC). The primary cyst wall (MC) is situated medind to the peripheral wall.

Host Reactions

Cellular reactions on the part of parasitized polyps are not consistent, but such do occur, especially in those areas in which the gastrodermis has been ruptured. Five types of host cells are involved: free cells from the mesoglea, gastrodermal interstitial cells, cnidoblasts, gastrodermal nutritive-muscular cells, and gastrodermal gland cek Little or no reaction occurs if the presence of the metacercaria does not result in the rupturing of the surrounding gastrodermis. On rare occasions, a few cells identified as gastrodermal interstitial cells have been observed adhering to the outer surfaces of metacercarial cysts in such regions. As to whether this represents true cellular reaction is dubious. Released nematocysts are fairly commonly found adhering to the outer surfaces of metacercarial cysts. The discharge of these had undoubtedly been stimulated by the presence of the parasite. Their effectiveness as a defense mechanism, however, appears to be negligible.

When the gastrodermis is ruptured, there is a conspicuous partial enveloping of the encysted metacercaria. The most dramatic encapsulation occurs when what have been identified as free cells of the mesoglea migrate through the disrupted gastrodermis and adhere to the outer surface of the cyst (Fig. 11). These cells, each with a typically oval nucIeus and measuring 3 pm in length, fuse to form a syncytial network in which are trapped isolated gastrodermal cells, especially and cnidoblasts nutritivemuscular cells. Such a partial encapsulating cyst averages 19 pm thick. In addition to certain areas of the encysted parasite being covered by the type of cellular complex described above, other areas are walled off by what is essentially a single stratum of gastrodermal interstitial cells. These are not fused and no other types of cells are involved (Fig. 12). Besides the two types of incomplete encapsulation reported, a large number of gastrodermal gland cells consistently occur

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nonparasitized polyps to verify or deny this assumption. Our results of the individual Ca?+ determinations in both categories of polyps are presented in the accompanying idiograms (Figs. 13 and 14). The Parasite

ABCDEFGHIJKLMN Mgof CarMgofTissue

FIG. 13. Idiogram showing distribution of ionic calcium concentrations in polyps of PO&es compressa parasitized by Plagioporus metacercariae. -4 = 0.171-0.180, B = 0.181-0.190, C = 0.191-0.200, D = 0.201-0.210, E = 0.211-0.220, F = 0.221-0.230, G = 0.231-0.240, H = 0.241-0.250, I = 0.251-0.260, J = 0.261-0.270, K = 0.271-0.280, L = 0.281-0.290. M = 0.291-0.300, and N = 0.301-0.310 mg of Ca’+/mg of coral tissue.

1 9

n:37 X:0.254 +O.O25mg/mg

6 i

2 1

Mg of CwMg of Tissue

FIG. 14. Idiogram showing distribution of ionic calcium concentrations in polyps of nonparasitized Porites compressn. A = 0.171-0.180, B = 0.1810.190, C = 0.191-0.200, D = 0.201-0.210, E = 0.2110.220, F = 0.221-0.230, G = 0.231-0.240, H = 0.2410.250, I = 0.251-0.260, J = 0.261-0.270, K = 0.2710.280, L = 0.281-0.290, M = 0.291-0.300, and N = 0.301-0.310 mg of Ca”+/mg of coral tissue.

in the proximity of the metacercaria gastrodermis is ruptured.

if the

Ionic Calcium Determinations As stated, since gross comparisons of parasitized and nonparasitized polyps have suggested hypercalcification, comparative, quantitative determinations of ionic calcium were carried out on parasitized and

In order to identify t’he parasite occurring in the two species of Porites, metacercariae dissected out of corals were fed to the fish Thalassoma ballieui and adults were recovered, stained, and identified. The trematode proved to be a new species of Plagioporus, and its description will be presented elsewhere. DISCUSSION

AND

CONCLUSIONS

Histochemistry of metacercarial cyst. Available information pertaining to the formation and chemical nature of metacercarial cyst has been reviewed by Thakur and Cheng (1968). Since then, Macy et al. (1968)) &h$rska (1968, 1971)) Hall et al. (1969)) Howell (1970)) Lee and Cheng (1970)) Southgate (1971)) Stein and Lumsden (1971a,b), Strong and Cable (1972)) and Laurie (1974) have reported additional information. Among those cysts that occur in invertebrate hosts, the composition of the wall (s) can be morphologically categorized as being one of three types. The first is comprised of two walls; the inner being of parasite origin and the outer of host reaction cells and/or fibers. The second type also consists of two walls, but both are of parasite origin. The third type consists of a single wall of varying complexity but it is of parasite origin. Known examples of the first type include Crepidostomum cornutum, metacercarial cysts in the cra,yfish Can1baru.s bartoni s&otensis (see Cheng, 1957), Himasthla quissetensis metacercarial cysts in six species of marine pelecypods, viz., Mytilus edulis, Modiolus desuissus, Ensis directus, Myw arenaria, Mercenaricr mercenarin, and Tapes philippinensis (we Chcng et al., 1966), and Cercwia trernnglandis cysts in naiads of the

ON

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stonefly Acroneuria carolinensis (see Hall et al., 1969). Known examples of the second type of metacercarial cyst include Paragonimus westermani cysts in various crabs (see Yokogawa et al., 1960), P. kellicotti cysts in the crayfish Cambarus sp. (see Ameel, 1934), P. ohirai cysts in brackish water crabs (see Miyazaki, 1939a-d, 1947), and Echinoparyphium aconiaturn cysts in the snail Lymnaea stagn&is (see ?J&rskB, 1968). It is our opinion that the two-walled metacercarial cyst of Plagioporus reported herein is of this type. This belief is based on the finding of material secreted from the cystogenous glands passing through the primary waI1 to form the peripheral waI1. Finally, the third type of cyst is currently represented by only one known example, Cyathocotyle bushiensis cysts in the snail Bithynia tentaculata. In this species, Erasmus (1967) has reported that the single, multilamehated cyst wall is of parasite origin. The question may be raised whether what has been morphologically categorized as the first and third types are indeed different. It is possible that the absence of an outer wall of host reaction cells and/or fibers may merely reflect the lack of host response to the encysted metacercaria. Relative to this point, it is noted that Salt (1963) has concluded that parasites of insects are more likely to elicit a defense reaction in unusual hosts than in compatible or normal ones, and Cheng (1967) has concluded that the compatibility-incompatibility spectrum between zooparasites and their molluscan hosts may be reflected by the degree of host cellular reaction. Thus, the absence of an encapsulating cyst surrounding encysted C. bushiensis metacercariae in B. tentaculata may merely represent a total or near total compatible hostparasite relationship and although morphologically distinct from the first type of cyst complex, is actually the same type. All metacercarial cysts of parasite origin are secreted by subsurfacial cystogenous

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glands. A variety of terms has been employed to designate these gland cells based upon their topography and staining characteristics (Dixon, 1966, Thakur and Cheng, 1968; %%rskB, 1968). However, until a more detaiIed study of the cystogenous glands of the species of Plagioporus under consideration, especially as found in the cercarial stage, can be conducted, no categorization is attempted. This is because it has not been possible to clearly identify these cells in encysted metacercariae since they are usually completely or partially spent at this stage. Another consequence is that it. has not been possible to account for the specific origins of the materials found to be present in the cyst walls. Nevertheless, the finding of pH 2.6 alcian blue-positive strands leading from certain of these glands to the inner surface of the primary wall (Figs. 9 and 10) indicates that at least one of the secreted materials includes compIex carbohydrates rich in acid groups (polycarboxylates) (Mowry, 1963). Such molecules, however, do not constitute the primary wall but do occur in the peripheral wall. This suggests that these complex carbohydrates, once secret.ed, pass through the primary wall by some mechanism and become deposited in the peripheral wall. That this occurs is strongly suggested by the presence of very fine radially oriented, pH 2.5 alcian blue-positive strands transgressing the primary wall (Fig. 9). Further discussion of the chemical nature of the cyst walls is presented below. As reported, the metacercarial cyst of Plagioporus sp. is comprised of a thin, irregular, outer or peripheral cyst wall and a comparatively thick primary cyst wall. The first is believed to serve primarily as an attachment mechanism. This peripheral wall is chemically distinct from the cyst proper as revealed by histochemistry. As indicated in Table 1, it is weakly PAS-positive and diastase resistant, thus indicating the presence of one or more of the following categories of compounds: neutral mucopolysaccharides, mucoprot#eins, glycopro-

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CHENG

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teins, unsaturated lipids, and phospholipids (Pearse, 1961; Bancroft, 1967). However, the fact that lipids had been extracted from the tissues prior to oxidation with periodic acid permits the elimination of unsaturated lipids. That phospholipids are absent is indicated by its not being stained with 1~x01 fast blue (Kliiver and Barrera, 1953), hence this category of molecules can also be eliminated. This wall also includes molecules which bind alcian blue at pH 0.5, 2.6, and 5.7 and reveal a mixture of LYand /3 metachromasy when stained with toluidine blue. The fact that it binds alcian blue at pH 0.5 indicates the presence of sulfated polyanions (Lev and Spicer, 1964; Constantine and Mowry, 1966) ; that it binds this stain at pH 2.6 indicates the presence of complex carbohydrates rich in acidic groups (Mowry, 1963) ; and the fact that it binds alcian blue at pH 5.7 indicates that the polyanions present are probably carbohydrate polyanions (Scott, 1960; Scott et al., 1964). Furthermore, the fact that this wall does not give positive reactions with mercury bromphenol blue, Sakaguchi’s stain, and Millon’s reaction suggest that there are few or no proteins present. Consequently, one may eliminate the PAS-positive substrate as being a mucoprotein or a glycoprotein. Hence, this peripheral wall is believed to consist of neutral mucopolysaccharides and complex carbohydrates that are sulfated and rich in acidic groups. That acidic groups in the form of carboxyl radicals are present is also indicated by its p metachromasy (Kramer and Windrum, 1955). The primary cyst wall is heterogeneously and weakly PAS-positive. The positive regions are diastase resistant, thus indicating that the substrate does not include glycogen but may include one or more of the following: neutral mucopolysaccharides, glycoproteins, unsaturated lipids, and phospholipids (Pearse, 1961; Bancroft, 1967). Again, the fact that lipids has been extracted from the PAS-positive tissues eliminates the possibility that the positive reac-

WONG

tion is due to unsaturated lipids. Unlike the condition in the peripheral wall, the presence of phospholipids is indicated by its positive staining with 1~x01 fast blue (Kliiver and Barrera, 1953). In addition, the fact that this wall is essentially not stained with alcian blue at pH 2.6 indicates the absence of complex carbohydrates rich in acid groups (Mowry, 1963), and is also not stained with alcian blue at pH 0.5 indicates the absence of polyanions rich in suifate groups and undissociated carboxyls, i.e., polyanions with carboxyls other than those of acidic groups (Pearse, 1961; Quintarelli et al., 1964; Lev and Spicer, 1964). Furthermore, the fact that it also is not stained with alcian blue at pH 5.7 indicates the absence of carbohydrate polyanions (Scott, 1960; Scott et al., 1964). Relative to proteins, the fact that this cyst wall, as well as certain cystogenous glands, give a slight positive reaction with Millon’s reagent indicates the presence of a tyrosine-containing protein, and its positive reaction with the ninhydrin-Schiff reaction indicates the presence of a-amino groups on the protein. On the other hand, its negative reaction with Sakaguchi’s stain indicates the absence of an arginine-containingprotein. That the protein concentration must be low is indicated by its not being stained with mercury bromphenol blue. As the result of the histochemical tests performed, it may be concluded that the primary cyst wall includes nonsulfated neutral mucopolysaccharides without dissociable phoscarboxyls and/or glycoproteins, pholipids, and a tyrosine-containing protein. In addition, a-amino groups are present. Histopathology and host reactions. The occurrence of large quantities of cellular debris in the intestinal caeca of most of the metacercariae examined indicates that these immature trematodes had recently fed on host cells prior to encysting. Thus, the histopathological changes, especially the sloughing of the gastrodermis, associated with the host’s body wall can be attributed, at least in part, to active in-

ON METACERCARIAE

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gestionby the parasite.As stated,mechani- slight and the partially envelopedmetacercal friction exerted by the motile metacercaria during the encystation process and by the cyst could also be partially responsible for these abrasions. It is noted that no significant cellular reactions occur in parasitized polyps when the gastrodermis is not disrupted. On the other hand, if the gastrodermis is ruptured, partial encapsulation of the encysted metacercaria occurs. This, as reported, is accomplished in one of two ways. In the first, the primary type of cell involved are free cells of the mesoglea, which fuse to form a syncytium. That such cells of cnidarians are involved in cellular reaction to foreign bodies, especially by phagocytosis, was known to Metchnikoff (1893), The concept that foreign bodies too large to be phagocytized may become encapsulated by a thin layer of phagocytic cells that become flattened against their surfaces has been proposed by Cheng and Rifkin (1970). Thus, it is not surprising to find a thin layer of syncytial mesogleal cells enveloping areas of the metacercarial cyst. The presence of cnidoblasts and nutritive-muscular cells entangled in a syncytium of this type is believed to be accidental since these two types of cells, freed from the gastrodermis as the result of mechanical disruption, are abundant in the vicinity. The second type of partial encapsulation is accomplished by interstitial cells freed from the ruptured gastrodermis. These cells are intimately adhered to the surface of the metacercarial cyst but do not fuse to form a syncytium. It is noted that it is generally accepted that interstitial cells are totipotent and are capable of differentiating into the various other types of cells, including free mesogleal cells (see Hyman, 1940, for review). Thus, it would appear that the ability to react to foreign bodies is present in interstitial cells and has been retained in the mesogleal cell line during differentiation. The fact that the two types of incomplete encapsulation of metacercarial cysts are

cariae are not in any manner injured attests to the ineffectiveness of the host’s cellular reactions. Ionic calcium studies. Analysis of the data presented in Fig. 13 and 14 has indicated that there is a statistically significant difference in the calcium contents of parasitized and nonparasitized polyps as determined by the t test (p’< 0.01). Specifically, the mean ionic calcium concentration in nonparasitized polyps of 0.254 2 0.025 (SD) mg/mg of coral tissue is significantly greater than the 0.235 & 0.025 (SD) mg/mg of coral tissue as found in parasitized polyps. Thus, what appears superficially as increased growth, including the conspicuous protrusion of skeletal lobes, does not involve parasite-induced hypercalcification, i.e., increased deposition of CaCO, skeletal material. Furthermore, our finding of compressed rather than thickened regions of the body wall surrounding metacercariae indicates that neither hyperplasia nor hypertrophy, except for the gastrodermal gland cells, occur. Hence, the only conclusion that can be drawn is that the superficial growths associated with parasitized polyps represent distended body walls and skeletons resulting from mechanical pressure exerted by the large, encysted metacercariae within the underlaying gastrovascular cavity. Furthermore, the significantly less calcium in the parasitized polyps may be attributed to the disruption of normal calcium deposition by epidermal cells. ACKNOWLEDGMENT

We wish to acknowledge that this study was carried out in the Department sity of Hawaii, Honoluiu.

of Zoology, Univer-

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