The Compatibility and Incompatibility Concept as Related to Trematodes and Molluscs!

THOMAS C. CJ-IENG2

IN THIS PAPERI propose to bring together dur ing the evolutionary adaptation to para­ significant facts already known to have some sitism of the progenitors of modern day di­ bearing on the mechanisms which govern or genetic trematodes, one might expect to find influence host compatibility or incompatibility the occurrence of certain adaptive mechanisms during parasitism, to add information based on more firmly entrenched in them than in verte­ my own recent research, and to present some brate definitive hosts. Furthermore, it is easier speculations. to simulate experimentally the natural environ­ From the broad viewpoint, host compatibility mental conditions of molluscs than those of may be defined as the expression of the physical vertebrates in captivity, and, with relative ease, and physiological (including chemical) states one can test basic prem ises on a numb er of of the host and parasite which enable the para­ species from a variety of habitats, ranging from site to invade and carry out its life processes, marine to fresh water, and from various atti­ includ ing perpetuation of the species. Incom­ tudes. patibi lity refers to those factors which com­ It has long been known that compat ibility pletely or part ially prevent the establishment and incompatibility need not be "all or none" and normal development of the parasite. Both phenomena, since both interspecific and intra­ of these are highl y complex phenomena influ­ specific (o r strain) differences do occur, as is enced to one degree or another by a series of indicated by the rate of parasite developm ent, separate but commonly interrelated factors. infectivity of the cercariae or metacercariae, the As working models from which we can seek number of progeny produced by delayed poly­ evidence to support or reject various working embryony, etc. In fact, an understandin g of the hypotheses, we have chosen to examine the re­ factors governing compatibility and incompati­ lationship between molluscs and digenetic trem­ bility in turn most probably will provide an­ atodes, since this category of association, unlike swers for why these manifestations occur. a num ber of others, is an example of parasitism Since the initial host-parasite contact, the whether the definition of "parasitism" is invasion process, the establishment of the para­ couched in nutritional, pathological, ecological, site within the host, and the escape process are or immunological terms. Molluscs, except in a distinct aspects of a successful parasitic relation­ very few unusual instances, serve as interm edi­ ship (see the review by Cheng, 1967) , one ate hosts for the Digenea, but this fact does would expect factors correlated with all of these not render them less appropriate as experimental phases to contribute to some degree in regu ­ tools. In fact, inasmuch as it is generally ac­ lating compatibility and/ or incompatibility, and, cepted that molluscs were the original hosts indeed , available evidence indicates that this is so. In the following paragraphs are briefly re­ 1 Th e research reported in this pap er was supp orted viewed those facts which support this concept. by grants from the American Cancer Society and the Space does not permit the citation of all the Office of Research Administration, Un iversity of relevant literature; therefore, only selected Hawaii. Th is paper was originally presented at a symposi um sponsored by the Di vision of Invertebrate studies are cited as examples. Zoology, American Society of Zoologists, entitled "The Parasites of Invertebrates," held on December 30, 1966 in W ashington, D .C. Manuscript received INITIAL HOST-PARASITECONTACT December 13, 1966. 2 Dep artment of Zoology, University of H awaii, Recently, Timon-David (1965) has again H onolulu, Hawaii 96822 . raised the question of the importance of host- 141 142 PACIFIC SCIENCE, Vol. XXII, April 1968 attraction in governing host-specificity In speed of a single miracidium was timed, re­ mollusc-trematode relationships. This topic, corded as seconds/2.54 mm, and later expressed which has been critically reviewed by myself as mru/s ec (Tables 1 and 2) . As controls, the (Cheng, 1967), among others, is still a con­ swimming velocities of miracidia of similar age troversial one. The controversy is not whether placed in distilled water were determined. host-attraction does occur since, in my opinion, The six test media consisted of 1:10, 1:50, the studies of Faust and Meleney (1924), Faust and 1: 100 dilutions of molluscan plasma and (1934), Faust and Hoffman (1934), Barlow similar dilutions of tissue extracts. Blood was (1925), Tubangui and Pasco (1933), Mathias collected from the molluscs' body sinuses by (1925), Kloetzel (1958, 1960), Kawashima gently cracking the shell of each snail, without et al. (1961a), Campbell (1961), Davenport injuring the soft tissues, and permitting the et al. (1962) , Etges and Decker (1963), and blood to drain to the lower edge of an inclined MacInnis (1965) have demonstrated rather Stender dish from whence it was rapidly col­ conclusively that attraction between miracidia lected with a hypodermic needle and syringe. and molluscs does occur. This is a subtle phe­ The cellular components of whole-blood samples nomenon, however, which is operative only were removed by centrifugation. The tissue ex­ within very restricted distances and can be ob­ tracts were prepared by homogenizing the soft served only with the application of quantitative tissues of each snail in 1 cc of distilled water techniques. The controversy is over the question after the tissues had been completely desan­ whether chemotaxis is in any way related to guinated and perfused with running distilled host-specificity and hence influences compatibil­ water for 15 minutes. After homogeniz ation in ity. Although the studies of Faust and Meleney an ice bath, the homogenates were centrifuged (1924) , Barlow (1 925), Neuhaus (1953) , and and the aqueous extracts collected were con­ Etges and Decker (1963) suggest that rniracid­ sidered the "concentrated" extracts. ial attraction is host-specific, the results of Sudds All snails used were laboratory-raised and ( 1960) , Kawashima et al. (1 961a) , and Bar­ known to be parasite-free. The concentrated bosa (1965) suggest that attraction of miracidia plasma and tissue extracts of each species were to a specific mollusc need not be correlated with pooled and the desired diluti ons were made subsequent compatibility. from the pooled samples. All observations were Experiments carried out in our laboratory made at 22 -+ 10 C. have revealed that the miracidia of Fasciola From the data presented in Tables 1 and 2, gigantica are stimulated by the plasma and tissue it is evident that all three dilutions of the extracts of laboratory-raised Galba ollula, the plasma and tissue extracts of G. ollnla (the natural host in Hawaii. These reactions, how­ natural host), H. dltryi normale, and T . grani­ ever, are not specifically elicited by G. ollula, [er« stimulated F. gigantica miracidia to increase since similar reactions were observed when their swimming velocities. In addition, the mira­ miracidia were exposed to the plasma and tissue cidial swimming pattern was conspicuously extracts of two other species of freshwater altered. Miracidia in distilled water usually gastropods, T arebia granifera mauiensis and swam linearly, rotating along their longitudinal H elisoma dllryi uormale. axes. Divergences from such a course were grad­ In the first series of experiments, F. gigantica ual rather than abrupt. When placed in plasma miracidia, between 15 and 25 minutes post­ or extracts, their swimming behavior became hatching, were placed in shallow Petri dishes erratic. They turned abruptly and frequently. (50 mm in diameter, 15 mm high) in which In the second series of experiments, 10 mira­ had been placed specific dilutions of the plasma cidia, 10-15 minutes post-hatching, were placed or tissue extracts of H. dllryi normale, T. grani­ in small Petri dishes (6 0 mm in diameter, 13 fera manlensis, or G. ollula. Each dish was mm high ) which contained 10 cc of distilled placed over a grid marked off in 2.54-mm water. In the center of each dish was placed an squares. Using a 1'lo-second-interval stop-watch agar block of three mm" which had been pre­ and observing under a dissection microscope soaked in concentrated plasma or tissue extracts illuminated by indirect lighting, the swimming of G. olinl«, H . dNryi normale, or T . granifera () ~ I"..... TABLE 1 [

COMPARISO NS OF THE SWIMMING V ELOCITIES OF Fasciola gigantica MIRACIDIA IN DISTILLED WATER (CONTROLS) AND IN THREE DILUTION S OF q' M OLL USCAN P LASMA (THE STUDENT t T EST WAS EMPLOYED TO D ETERMIN E SIGNIFICAN CES) S'

DISTILLED W ATER ~ ( CON TROLS) 1 :10 P LASMA 1 :50 PL ASMA 1:100 PL ASMA I"3 S- M OLLUSCAN NO . OF VELOCITY N O. OF VELOCITY SIG· N O. OF VELOCITY SIG- N O. OFVELOC ITY SIG· c, SPECIES TRIALS ( MM /SEC) TRIALS ( M M/ SEC) P N IF . TRIALS (MM/SEC) P N IF. TRIALS (MM/SEC) PN IF. Dl I" G. ollula! 40 0.79 25 1.65 > 0.001 30 1.88 0.01 20 1.91 0.01 ;:l + + + 0.. H. duryi normals- 101 1.96 81 2.70 > 0.001 + 81 2.80 > 0.001 + 118 2.85 > 0.001 + T . g1'anijera3 60 2.30 60 3.5 < om 60 3.5 < om 60 3.5 < 0.01 ~ + + + 2­ I Th e miracidia used were 25 minutes post-hatching , C 2 Th e miracidia used were 18-20 minutes post-hatching. en 3 The miracidia used were 15 minutes post-hatching. Cl I () :r: t11 oZ TABLE 2

COMPARISONS OF THE SW IM MING V ELOCITI ESOF Fasciola gigantica MIRACIDIA IN DISTILLED WATER (CON TROL S) AN D IN THREE DILUTIONS OF M OLL USCAN A QUEOUS T ISSU E E XTRA CTS (TH E STUD ENT t T ESTW AS U SED TO D ETERMI N E SIGNI FICANCES)

DISTILL ED WATER (CONTROLS) 1:10 EXT RACT 1 : 50 EXTRACT 1:100 EXTRACT

MOL LUSCAN NO . OF VELOCITY N O. OF VELOCITY SIG- NO . OFVELOCITY SIG· N O. OFVELOCITY SIG- SPECIES TRIALS ( MM/ SEC) TRIALS (MM/ SEC) PN IF. TRIALS (MM/SEC) P NIF. TRI ALS (MM/SEC) P NIF. G. ollula! 40 0.79 20 1.69 > 0.001 + 20 0.99 > 0.001 + 20 1.26 > 0 .0001 + H. duryi normale- 101 1.96 80 0.94 < 0.001 + 80 0.81 < 0.001 + 80 1.25 < 0.001 + T . g1'anije1'a3 60 2.30 60 3.30 > 0.01 + 60 3.33 > 0.01 + 60 3.10 0.05 + 1 T he miracidia used were 25 minutes post-hatchi ng . ~ Th e miracidia used were 18- 20 min utes post-hatching. 3 Th e miracidia used were 15 minutes post-hatching.

...... ~ '-" 144 PACIFIC SCIENCE, Vol. XXII, April 1968 maniensis for 10 hours. Although exact be­ tration and development. It is noted that the havioral patterns exhibited by miracidia in the velocity-increasing stimulant and the attractant proximity of an agar block were determined, as may be the same factor. well as the number of miracidia in each con­ As has been pointed out in an earlier review centric zone at various time intervals, these (Cheng, 1967) , the effectiveness of the "host results will not be reported at this time. On ly factor " (a term introduced by Davenport the total number of contacts made by the mira­ (1955) to designate the stimulatory material of cidia per 5 minutes during the initial 10-minute host origin) in guiding miracidia to their mol­ period are reported (Table 3). The same num ­ luscan host is doubtful under certain circum­ ber of miracidia placed in distilled water with stances. For example, Etges and Decker (1963) untreated agar blocks served as contro ls. All have poin ted out that the naturally-occurring observations were made at 22 ± 10 C. negative geotaxis and postive phototaxis of From the data presented in Tab le 3, it is Schistosoma mansoni miracidia most probably apparent that significantly more contacts were eclipse the chemotactic effect of the "host fac­ made between miracidia and plasma- and tissue tor." Even between these taxes, Chernin and extract-soaked agar blocks than with control Dunavan (1962) have demonstrated that the blocks. The exception was in the case of T. negative geotaxis is a more powerful determi­ graJlijera tissue extract-soaked blocks. nant of miracidial behavior than is positive It may be concluded then that the miracidia phototax is. Thus, it is only under those condi­ of F. gigalltica respond to a velocity-increasing tions where naturally-occurring taxes guide the stimulant and an attractan t in the plasma and miracidia to the immediate proximity of the tissue extracts of certain species of molluscs, mollusc that the influence of the "host factor, " and that these factors are not host-specific and which is operative only within short distances, is need not indicate successful subsequent pene- effective (Fig. 1). ------======------PELECYPOD GASTROPOD MOLLUSC _-- --_ MOLLUSC

'\:... ------~.~~O~:;: ; · ·

II --_--=:::::===~s:=--d-'-g e-st , /c t . ..:: - ---

- --.. Host's - ~ " Ch toxl ------incurrent PASSIVE emoo x ~s ------. I ~R AC I D I U M .. - Geol ro p l s ~ __ .. + Phototroplsm .. Optimal ombient factors - _- __-=====-=- ::-.-." (salinity. current, etc.) ~-- .. Absence of inhibitory factors ------~ Q ~ ------V ACTIVE ------MIRACIDIUM ------::::=---

FIG . 1. D iagrammatic drawing illustrating factors which can govern or influence miracidium-mollusc con­ tact during the pre -invasion phase. The terms "tropism" and "taxis" are used interc hangeably. on 3 'U g~ q' 5' ~ (b 3 S-'" c, ~

'"::J T ABLE 3 0... ~ COMPARISONS Of TH E N UMBER OF CO NTACTS B ETWEE N Fasciola gigantica MIRACIDIA AN D N O NPRESOAKED AGAR BLOCKS (CONTROLS ) AND BLOCKS 2­ P RESOAKEDIN CONCENTRATED P LASMAAND T ISSUE E XTRACTS OF THREE SPECIES OF S NA ILS ( THE STUDENT t T EST W AS EMPLOYE D Cen TO D ETERMIN E SIGN IF ICANCES) Cl I CO NTROL BLOCKS PLASMA-SOAKED BLOCKS TISSUE EXTRACT-SOAKED BLOCKS n :r: tti MEAN N O. OF M EAN N O. OF MEAN NO . OF Z MOL LUSCAN N O. OF CONTACTS N O. OF CONTACTS SIG- N O. OF CONTACTS SIG- Cl SPECIES TRIALS (5 MIN . ) TRI ALS ( 5 MI N .) P N Il' . TRIALS (5 MIN. ) p N Il'. G. ollula 40 1.55 60 6. 35 < 0.0001 + 30 4.30 < 0.0 1 + H . dur yi norma!e 117 1.65 117 6.58 < 0.000 1 + 97 5.40 < 0.0001 + T . gfani!efa 60 0.9 60 4.80 > 0.001 + 60 0.4 > 0.5

..... ,l.., v ' 146 PACIFIC SCIENCE , Vol. XXII, April 1968

It should also be mentioned that it is highly oirginic«, T apes philippinamm, M ytillts ednlis, doubtful if chemotactic forces are influential and Modiolm demissns, will stimulate the cer­ during the pre-invasion relationship between cariae of Himastbla quissetensis to encyst; as miracidia and pelecypod molluscs, since the the result of the rapidity of this process in the ability of this group of molluscs to effect instances of C. gigas and C. virginica, the cer­ incurrents, through either their siphons or shell cariae are prevented from penetrating these edges, undoubtedly results in the passive intake oysters, especially C. gigas, and hence these of miracidia via the currents (Fig. 1). pelecypods may be considered incompatible Besides innate taxes, the nature of the en­ hosts (Chen g et al., 1966b). Th ese findings vironment is known to determine whether at­ demonstrate how in some instances a factor of traction between mollusc and miracidium can host origin operative during the pre-invasion be effective (Fig. 1). An excellent example of phase of host-parasite relationship could deter­ this has been contributed by Kawashima et al. mine the incompatibility of the host. (1961b). Earlier, these investigators (1961a) It should be pointed out that the tissue ex­ had demonstrated that, although the miracidium tracts of the same seven species of pelecypods of Paragonimus ohirai is attracted to three have a negative effect on H . quissetensis cer­ species of brackish water snails of the genus cariae, reducing their life spans significantly. A ssiminea ( A. parasitologica, A. japonica, and However, the fact that the cercariae are stimu­ A . latericea miyazakii) , only one of the three, lated to encyst in M . mercenaria, M . arenaria, A . parasitologica, is a compatible host. A. T . philippinamm, M . edulis, and M . demissus latericea miyazakii is an incompatible host, before the tissue component (s ) could cause while A. japonica can be infected experimen­ their death is believed to be responsible for the tally but the level of infection is consistently compatibility of those pelecypods as second low. Thus, it would appear that in nature some intermediate hosts. Th is is because the cyst other factor or factors must be operative to wall serves as a protective layer against con­ bring about the selection of A . parasitologica tinued contact with the tissue component (s ) . by miracidia. It was subsequently shown, in a Wright (1959) has suggested that the "host study of the locomotive speed and survival of factor" may be in the form of species-specific P. ohirai miracidia in various concentrations of substances incorporated in the body-surface NaCI, that the lower the salt concentration is mucus of molluscs; Kawashima et al. (1 961a) the more active the miracidia become, with the have demonstrated that Paragonim us ohirai optimum salinity being 0.25% N aCI or less. miracidia are attracted to amino acid mixtures Concurrent studies on the ecology of the three placed in cellophane bags ; and MacInnis species of snails revealed that the optimum (19 65) has found that butyric acid, galactose, salinity for A . parasitologica is 0.25% , that for L-cysteine HCI, and even 1.0 mM HCl will A . latericea miyazakii is 0.4%, and that for A . stimulate "contact with return" of over 80% of japonica is 0.6%. These findings demonstrate S. mansoni miracidia in an artificial test system. that an environmental factor, salinity in this However, no direct evidence is yet available to case, can serve as a mechanism determining host indicate the nature of "host factors." Moreover, selection. Thus, these investigators have demon­ it should be pointed out that although organic trated that the influence of the molluscs' at­ molecules, possibly amino acids, fatty acids, and tractants can be masked by an ambient factor sugars, are the attractants, there is also some and have also releaved fur ther evidence that evidence which indicate that the pH or some attraction of miracidia to mollusc need not other physical property of the host may be re­ mean successful subsequent development. sponsible, at least in part, for the attraction Substances of host origin need not always en­ (Kawashima et al., 1961a) (Fig. 1). This most hance establishment of the parasite. Some may probably explains the attraction of S. mansoni be inhibitory. For example, Cheng et al. miracidia for dilute HCl , as demonstrated by (1966a) have demonstrated that the plasma of MacInnis. seven species of marine pelecypods, Mercenaria In the case of those species of trematodes tnercenaria, Mya arenaria, Crassostrea gigas, C. which do not include a free-swimming mira- Compatibility in Trem atodes and MolluscS-CHENG 147

cidial stage, successful host-parasite contact is The question may be asked whether the mira­ depend ent upon the ingestion of the egg by the cidial cytolytic enzymes must be chemically spe­ mollusc, followed by hatching within the latter's cific for the integument of specific species of alimentary tract (F ig. 1). Although some infor­ molluscs. If this is the case, the compatibility mation is available pertaining to the hatching of enzyme to substrate could serve as a factor process of eggs in water (Standen, 1951; governing successful penetration, hence host­ Rowan, 1956, 1957) , surprisingly little infor ­ compatibility (Fig. 2) . Ind eed, Lie ( 1963) be­ mation is available on the factor or factors which lieves that the prevention of penetration of influence hatching of trematode eggs in mol­ certain echinostome miracidia by unnatural snails luscs. It is generally believed that the mollusc's is responsible for incompatibility. digestive juices in some manner stimulate hatch­ In addition to the miracidium's cytolytic en­ ing (Krull and Mapes, 1952; Timon-David, zymes, Dawes (1960c) has expressed the view 1965 ; and others), although the exact mech­ that the shedd ing of the ciliated epidermis by anisms have not been studied. Nevertheless, it the miracidium, thus transforming it to a sporo­ is apparent that the biochemistry and physical cyst, is a prerequisite for successful infection of properties of the mollusc's digestive tract could the snail. This hypothesis has been challenged serve as determinants of compatibility, as mani­ by Lengy (1 962) , who has demonstrated that fested by hatching, or of incompatibility, as Schistosoma bovis miracidia do not shed their manifested by nonhatching (Fig. 1). ciliated epidermis prior to penetrating. Similarly, Maldonado and Metienzo (1947) demonstrated earlier that S. mansoni miracidia do not shed THE INVASION PROCESS their plates until after successful penetration; Because of obvious medical implications, con­ and Heyneman (1 966) has successfully initiated siderable information is available about factors the infection of Lymnaea mbiginosa with Echi­ governing the penetration of cercariae into nostoma attdyi miracidia and of Indoplanorbis mammals and other vertebrate hosts. On the exustus with Echinostoma malavanum miracidia other hand, little is known about miracidial by inoculating these miracidia through the penetration into molluscs, either from the ex­ mantle via a minute hole drilled in the molluscs' terior or through the gut wall. Nevertheless, shells, thus suggesting that the shedding of the this barrier could be a factor determining com­ miracidial epidermal plates is not a prerequisite patibility or incompatibility. to successful infection, at least in these species. Certain aspects of the processes involved dur­ It remains true, nevertheless, that certain species ing the successful penetration of Lymnaea of fasciolid miracidia may shed their plates (= Limnaea) trnncatula and L. auricularia by prior to penetration. Campbell and Todd Fasciola hepatica and F. gigantica, respectively, have been studied by Dawes (1959, 1960a, b, c). According to him, the miracidium first becomes attached to the molluscan host's integu­ ment by suctorial action resulting from applica­ tion of the "cup" formed by the inversion of the anterior papilla assisted by mucus. Thi s is followed by the secretion of cytolytic enzymes from ".. . the gut and the unicellular pharyn­ geal 'glands' into the 'cup.''' The subsequent enzymatic activity results in the lysis of the host's epithelial and subepithelial tissues. On ly • Stimulation to .shed epidermal plates (toctorts) in plasma) • Stimulat ion to invagi nate apical papilla after the host's integument has been perforated • Stimulat ion to secrete cytolytic enzyme does the parasite enter, but not before it has • Specif icity of cytoly tic enzyme (?) sloughed its ciliated epidermis. For this reason, FIG. 2. D iagrammatic drawing illustrating factors Dawes considers the penetrating form to be a which may govern or influence successful penetration sporocyst and not a miracidium. of mollusc by miraci dium. 148 PACIFIC SCIENCE, Vol. XXII,Apri l 1968

(1955) , for example, have repor ted that Fascio­ lytic enzyme, were also noticed in F. gigantica loides magna miracidia shed their ciliated miracidia exposed to plasma fro m C. ollula and plates on the exterior afte r a brief contact with H. dllJ')'i normale (Fig. 4 ). Thus it would ap­ the molluscan host's tissues, and Barlow (1925) pear that the forma tion of the apical "cup" as has found the transformation of F. bllski mira­ well as secretion are stimulated not by physical cidia into sporocysts when bathed in "snail tissue contact but by some factor (s) present in the juices." I observed that F. gigantica miracidia mollusc's plasma (Fig. 2). shed their plates when placed in concentrated and 1 : 10 dilutions of the plasma of Calba ESTA BLISHMENT OF THE PARASITE ollnla, the natural host (Fig. 3), and in similar concentrations of the plasma of H elisoma duryi Successful establishmen t of germinal sacs normale, an incompatible host, but not in the (sporocysts, rediae, or both) within the mollusc plasma of Tarebia grani/ era mauiensis and Lit­ implies that the form wh ich has invaded the torina pintndo, both of which are also incom­ host will reach a suitable site, overcome the patible hosts. Similar phenomena were not host's internal defense mechanisms, be the target observed when miracidia were placed in tissue of host-elaborated growth and development­ extracts of all four species of snails, nor were stimulating factors, be able to obtain its required they observed in greater dilutions of C. ollula nut rients, and at the same time not kill its and H . dllr)'i normale plasma. Thus, this ph e­ host (Fig. 5) . Th ese requirements are con­ nomenon is apparently not related to host com­ sidered separately. patibility. Rather it suggests that miracidia Reaching a Suitable Site which possess the innate ability to shed their epidermal plates prior to penetration can be Although tissue specificity still remains one stimulated to do so by some factor (s) .N ever­ of the unsolved problems in parasitology, it is a theless, it would appear that the occurrence of well-documented phenomenon. For example, for the stimulatory factor (s) in the natural hosts a large num ber of species among the Digenea could influence compatibility dur ing this phase the molluscan host's hepatopancreas or gonads of host-parasite relationship, especially if Dawes' appear to be the preferred sites of normal larval contention is true among fasciolid trematodes development. Th is does not mean that aberrant (Fig. 2) . parasites cannot develop in ectopic sites. Indeed, N o information is yet available relative to such exceptions to the rule are known. Investi­ the nature of the stimulatory factor (s) ; how­ gations into the nature and development of ever, the fact that F. gigantica miracidia did not larvae which grow at ectopic sites not only can shed their plates when placed in extracts of provide insights into the physico-chemical re­ desanguinated and aqueously perfused snails quirements of these parasites but also can reveal indicates that the factor is present in plasma some of the factors which inh ibit or preven t the rather than in tissue fluids. parasite from reaching its normal developmental It is also significant that F. gigantica miracidia site. A series of such studies is summarized to were stimulated to shed their plates only in con­ illustrate this poin t. centra ted plasma and in a 1: 10 dilution of Among the Plagiorchioidea, the mother spor­ plasma. Thi s may be interpreted to mean that ocysts of certain species are known to be at­ in nature the stimulatory effect would occur only tached to the external surface of their molluscan when miracidia become intimately associated hosts' alimentary tracts ( Cort et aI., 1954; with or are in actual contact with the snail, Rankin, 1944 ; l eigh, 1946 ; Schell, 1961, since plasma, seeping from the wound resulting 1962a,o Cheng, 1961a, b; and others) .Sur­ fro m the parasite's lytic enzymes, would be rounding each of the daughte r sporocysts arising rapidly diluted as it diffuses though the aqueous from these mother sporocysts is a so-called medium. paletot. The question is: what is the origin of In addition to the shedding of epidermal this paletot ? Accordin g to Cort and Olivier plates, invagination of the apical papilla as well (1943), Cort and Ameel (1944), and Cort et as the secretion of some substance, perhaps the al. ( 1954) , the paletot is formed from the Compatibility In Trematodes and MolluscS-CHENG 149

FIG. 3. Photomicrograph show ing Fasciola gigantica miracidium which has shed its epidermal plates after exposure to a 1:10 dilu tion of Galba ollula plasma; 1 minute after expos ure . se, Shed epidermal plates.

FIG. 4. Phase-contrast photomicrograph showing invagination of apical papi lla of Fasciola gigantica miracidium and secretion of lytic enzyme( ?) after exposure to concentrated Galba ol/ula plasma. ip , Invagin ated pap illa ; s, secreted material. mult iplication of the cells of the mother sporo­ eration of the basement membrane surrounding cyst wall which invaginates and surrou nds each the snail's gut, thus preventing furt her invasion daughter sporocyst. More recently Schell (1961, by the mother sporocyst beyond that space de­ 1962a), who studied the sporocysts of Hap. limited by the intestinal epithelium on one side lometrana intestinalis and GlypthelmillS quieta, and the basement membrane on the other. If has expressed the opinion that the paletot is not Schell's observations are correct, the reason why of parasite origin but represents an enveloping mother sporocysts of H. intestinalis and G. membrane which has resulted from the prolif. qltieta are foun d abutting upon their molluscan 150 PACIFIC SCIENCE, Vol. XXII, April 1968

~ Host elaborated stimulat ing "_'. and inhibiting substances ...... :(.=::..)...... (horrnonesj) l::~:?::>. -: "'· NJtrition ~ Reaching a suitable site ~:(':: ... ~ Non-lethal to host (absence of barriers) ;X;.",::,:,:, . '::':" ~ Overcoming host's defense mechanisms \ :.:..: ~ . :><..\:.:./\ (innate and acquired cellular reactions; .....,.. -.:.-'; ;:;.::: :?~ innate and acquired (1) hUmOrO? ~{)

',_ /f1J ... _ -- ~

FIG. 5. D iagrammatic drawing illustrating factors which can govern or influence the successfu l establish­ ment of trematode larvae in a mollus c. hosts' alimentary tracts rather than between the have demonstrated that the physical and chem­ hepatopancreatic lobules-as is the case with ical natures of the basement membrane and certain other plagiorchioid trematodes (Mattes, ground substance do determ ine whether the 1936 ; Maldonado, 1945 ; Cort et aI., 1952) ­ entrance of invasive forms of helminths will be is that the migratio n of these sporocysts be­ successful. comes restricted by their hosts' basement mem­ The evidence presented indicates that bar­ branes. Although the mother sporocysts of both riers, especially in the form of basement mem­ H . intestinalis and G. quieta become successfully brane and perhaps ground substances, situated established at this site and produce daughter in the path of invading trematode larvae could sporocysts, the migration of the mother sporo­ prevent them from reaching a satisfactory site cysts does become restricted. Schell (1962a) for further development and thus serve as has stated: "In following the development of G. determinants of host incompatibilty. quieta it became evident that the thin basement membrane beneath the intestinal epithelium H ost's Dejense M echanisms of the host snail plays an important role in pro­ What is known about the nature of internal tecting the snail from invasion by the parasite." defense mechanisms in molluscs, both cellular Is it possible, then, that certain species of trema­ and humoral, has been reviewed in recent years todes, which could not undergo normal develop­ by Stauber (1 961), Cheng and Sanders (1962), ment at the site restricted by the basement mem­ Tripp (1 963), and Cheng (1967). The role of brane, would in this way be prevented from molluscan leucocytes (amoebocytes or phago­ becoming established ? Although examples of cytes) and fibrous elements in innate immun ity this, as far as I have been able to determine, by causing the encapsulation of trematode lar­ have not yet been described among mollusc­ vae, is well established. The results of Newton larval trematode relationships, the condition ( 1952, 1954), Brooks (1953), Sudds (1 960), known as schistosome dermatitis caused by avian and others strongly indicate that encapsulation schistosome cercariae in human skin is an ex­ generally occurs around larvae in unnatural ample of the inability of these carcariae to suc­ hosts. However, slight and restricted encapsula­ cessfully penetrate the depth of the abnormal tion may also occur around larvae in their host's skin : being unable to survive in the natural hosts (Cheng and Cooperman, 1964; surfacial areas of the skin, they die. In this con­ Probert and Erasmus, 1965 ; Schell, 1961, nection, Lewert and Lee ( 1954),Lewert 1% 2a, b; Pan, 1965) , but these extremely light (1 958) , and Lewert and Mandlowitz ( 1963) capsules usually inflict little or no damage upon Compatibility in Trematodes and Moll uscS- CHENG 151

the parasites. On the other hand, the extensive Occasionally suggestions have appeared in capsules which surround parasites in unnatural the literature (Sogandares-Bernal, 1965) that hosts usually result in destruction of the para­ snails at different ages present different degrees sites. The chemical basis for this destruct ion of susceptibility to infection by trematode larvae. remains undetermined ; nevertheless, it may be Most, if not all, of these reports have resulted generalized that encapsulation by leucocytes from either field studies (where the ages of and/ or fibers resulting in death is by far the snails have been estimated by their sizes) or most effective form of innate defense mech­ qualitative assays of infectivity. Whether such anism in molluscs against incompatible trema­ age-correlated resistance is due to some innate tode larvae. humoral factor or even to a cellular factor re­ Alth ough supposedly innate humoral factors mains unknown. in molluscs have been reported by various work­ The only evidence of acquired cellular im­ ers (see the review by Cheng, 1967), their ef­ mun ity I have been able to find is that presented fectiveness as defense mechanisms against by Barbosa and Coelho ( 1956). They dem­ invading trematode larvae is unkn own or un­ onstrated that, although Biompbalaria glabl'ata certain. Recently, however, Heyneman ( 1966) previously "cured" of Schistosoma mansoni in­ has successfully demonstrated in transplantation fection can be reinfected, some tissue reaction studies that the inability of Echinostoma malay­ involving leucocytes and fibrous elements is anttm to become established in Lymnaea ru­ evoked in reinfected snails, a phenomenon not biginosa and of E. a"dyi in Indoplanorbis found in initial infections. This finding could exustus is due to "physiological rejection within mean that some type of incomplete acquired im­ snail tissues distinct from the factors responsible munity exists in B. glabl'ata after the initial in­ for failure of miracidia to attach to or penet rate fection and is manifested during reinfection as the body wall of the nonadapted host." Al­ cellular response. though it would be tempting to interpret Heyne­ The belief held by some workers that ac­ man's findings to indicate the occurrence of an quired humoral immunity can occur in molluscs innate humoral factor, the destruction of E. stems primarily from the reports of Winfield malayanunz and E. at/dyi larvae could very well (1932) and N olf and Cort ( 1933). These in­ have resulted from encapsulation . Unfortu­ vestigators reported that the presence of Coty­ nately, follow-up histological studies which lurus flabellif ol'mis sporocysts in varieties of would confirm this conclusion are not available. Lymnaea stagnalis prevents almost all of the Two other examples of possible occurrence of cercariae of this trematode from successfully innate humoral immunity which prevents the penetrating and encysting as metacercariae. establishment of larval trematodes have been Later, Cort et al. (19 45) repeated these studies reported . Benex and Lamy (1 959 ) showed that and reported that the same phen omenon occurs tissue extracts from the planorbid snail Planor­ in Stagnicola emarginat« angnlata parasitized by bis corneus will immobilize S. manso ni mira­ C. flabellif ol'mis sporocysts. They noted that the cidia, and these French workers suggest that few cercariae which did succeed in penetrating species of snails which are refr actory to S. man­ were . inhibited from developing into meta­ soni infection may possess "immune-like" im­ cercariae unless they entered sporocysts and were mobilizing substances. Sudds (1960) has shown thus presumably protected from the host's anti­ that when Tricbobilbnrzia eluae miracidia pene­ bodies. On the basis of these reports, Culbert­ trate two abnormal hosts, Bulim naea megasoma son (1941) concluded that "... it is clear that and Fossaria abrussa, the parasites die and begin snails acquire an immunity after infection by to degenerate within 1.5-6 days, without any trematodes... ." Several later authors, especially indication of a host tissue reaction. Again, it Michelson ( 1963) and Cheng (1967) , have would be tempti ng to interpret these findings as cautioned that this generalization is unwarranted indications of the presence of innate humoral since, as of this date, the ability of molluscs to immunity but, under the conditions of the ex­ produce antibodies has not been conclusively periments, other possible explanations cannot demonstrated. be completely ruled out. The results of two other studies suggest the 152 PACIFIC SCIENCE, Vol. XXII, April 1968 occurrence of acquired humoral immunity. In fluence of H ost-Elaborated Groiotb- and Chowaniec (1961) reported that 'only a small Development-Stimulating or -Inbibiting proportion of snails already harboring Fasciola Substances hepatica could be infected with the same para­ This vast area of host-parasite relationships site, while most of the control snails could be has hardly been touched. From what is known readily infected. In the second study, Lie et al. about the metabolic interaction between larval ( 1966) demonstrated that only 5% of Lymnaea trematodes and molluscs, it is inconceivable that mbiginosa infected with one species of echino­ compatible hosts do not influence in some man­ stome could be superinfected with a second, ner the grow th and differentiation processes of while 89% of uninfected control snails could their parasites and thus enhance their normal be infected. In neither of these studies, however, sequence of development , or, conversely, that in­ were antibodies demonstrated. It is of interest compatible hosts do not in some manne r inhibit that, in the case of L. rubiginos«, Lie et al. the normal developmental sequences of their stated another possible explanation: young in­ parasites. vading sporocysts of the second species are Meade and Pratt (1 966) have reported that, killed and ingested by the rediae of the first when rediae of M etagonimoides oregouensis are species. experimentally transplanted from naturally in­ The most convincing evidence of acquired fected Oxytrema silicula, in which the gonads humoral immun ity in molluscs is that contrib­ had been destroyed, to young uninfected snails uted by Michelson (1 963, 1964) , who dem­ with healthy gonads, a certain number will onstrated that Schistosoma mansoni miracidia­ survive but that differences are apparent be­ immobilizing substances occur in the tissue ex­ tween the transplanted rediae, their progeny, tracts of Biomphalaria glabrata infected with and those in naturally infected snails. They this trematode. Although Michelson found that noted that the transplanted rediae more than his controls (extracts of other species of un­ doubled their natural size, "mucus and debris" infected snails, extracts of snails infected with were included in their caeca, and the enclosed an acid-fast bacillus, an echinostome metacer­ metacercariae were no longer distingu ishable. caria, the nematode Daubaylia potomaca, snails Burns and Pratt ( 1953) had shown earlier that inoculated with bovine albumin, S. mansoni the rediae of M. oregonensis give rise to both eggs, and polystyrene spheres, uninfected B. cercariae and metacercariae within their brood glabrata, and water) all gave positive results, in chambers and that no daughter rediae occur. no instance did the percentage of immobiliza­ Furthermore, although some metacercariae, re­ tion reach the level observed in extracts of S. leased from transplanted rediae into the body mansoni-infected snails. Michelson cautiously cavities of acceptor snails, survived for up to 6 states: "Although the suggestion that the im­ weeks, none of these were infective when fed mobilizing phenomenon might be associated to a known compatible experimental definitive with an antigen -antibody interaction is an ap­ host, the golden hamster. These uninfective pealing one, data are lacking to substantiate this metacercariae also exh ibited some behavioral and hypothesis. The possibility that the immobilizing morphological peculiarities. They displayed substance(s) might be related either to parasite­ greater activity, their eyespots disappeared, and produced toxins or to products resulting from the prominent Y-shaped excretory vesicle which normally appears black was often enlarged and alternations in the snail' s metabolism cannot be possessed fewer granules. Meade and Pratt are excluded ." of the opinion that these differences in the It may be concluded, then , that innate cellu­ transplanted rediae and metacercariae had re­ lar immun ity appears to be the most efficient sulted from the influence of their new host's mechanism by which trematodes are prevented gonadal hormone (s) . The same hormone (s) from developing in incompatible molluscs, al­ presumably was present at a very much lower though acquired cellular immunity may occur. level or not present at all in the original cas­ The role of humoral factors, either innate or trated hosts. Whether thi s conclusion is justified acquired, remains uncertain. must await more direct evidence. Compatibility in Trematodes and MollUSCS-CHENG 153

From the study cited above, it would appear same "daughter germinal sac." These larval that some host factor (s) , perhaps hormones, in stages are found in the haemocoelic spaces of O. silicsla with healthy gonads does disrupt the the hepatopancreas and gonad of Littorinn saxa­ normal development of M. oregonensis rediae Ii/is tenebrosa. According to James, the "primary and metacercariae during the later phase of the germinal sac" could be interpret ed to be a relationship . The sign ificance of this finding to metacercaria ; while according to Cable, the our discussion is that it is an example of a host­ "daughter germinal sac" could be considered a elaborated substance which "inhibits" normal cercaria. Thus , the usual sequence of larval development and thus promotes incompatibility. stages is reversed in P. botnoeotecnnm, Since the The phenomenon reported by Meade and Pratt, usual life history pattern among related gym­ however, does not appear to hold true in all nophallids includes two molluscan intermediate transplanted mollusc-lar val trematode associa­ hosts, both being marine pelecypods (see tions. Chernin ( 1966), for example, has re­ Stunkard and Uzmann, 1958) , Cable has given por ted successful transplants of S. mansoni the following as one possible explanation for moth er and daughter sporocysts from B. glabrala this variation : " It may be sign ificant that to acceptor snails of the same species which metacercariae of other gymnophalline species were followed by norm al cercariaI formation. live in loose, even superficial association with Perhaps this difference can be explained by the their hosts whereas the species that James fact that M . oregonensis includes a redial stage ( 1964) described invades the snail to the extent while S. mansoni includ es sporocyst stages. It is commonly seen in molluscs serving as the first well known that rediae inflict significantly more intermediate host of trematodes in general. damage upon molluscs than do sporocysts. Thus, As a result, that species pro bably gets a double perhaps the donor B . glabrata is never com­ exposure of the most intimate sort to the tissues pletely castrated and S. mansoni, as the result of and body fluid of mollusks," This, of course, a long relationship with B. glabrata and ex­ implies that the tissues and body fluids of posure to its hormones, is not adversely affected molluscs may have influenced that unusual de­ by hormones during the latter phases of its de­ velopmental sequence. Although in this instance velopment, as is M. oregonensis. the presumably host-stimulated developmental Another example of possible host-stimulated alterations do not affect the parasite deleteri­ developmental alterations among larval trema­ ously, it is conceivable that such changes could todes has been reported by James ( 1964) and in certain instances deter or inh ibit delayed discussed by Cable ( 1965). James reported that polyembryony and thus render the host in­ the intramolluscan life cycle stages of the compatible. gymnophallid trematode Paroatrema bomoeotec­ Nntrient Requirements Ill/Ill include a "primary germinal sac" with adult features, includ ing an oral sucker, ventral Available evidence indicates that trematode sucker, pharynx, and bifid caeca, and a "daugh­ parasites utilize carbohydrates as their primary ter germinal sac" which is unique in that, in energy source, They acquire their carbohydrates addition to the adult features found on the in the form of glucose resulting from the degra­ "primary germinal sac," it also possesses a dation of the host's stored glycogen or, if there bifurcate tail. The "daughter germinal sacs" is no stored glycogen in the vicinity of their increase in size and lose their tails while still natural habitat, they utilize the mollusc's blood within the "primary germinal sac." Further sugars (Cheng, 1963b) . In addition to sugars, development does not occur until they rupture these larvae apparently utilize free amino acids out of primary sacs. Daught er sacs then continue from the mollusc's hemolymph and perhaps to develop in one of thre e possible ways: (1 ) even from the surrounding host cells which most produce cercariae and metacercariae; (2) are lysed or ruptured mechanically. a few produce a second generation of "daughter Lip ids, in the form of short-chain fatty acids, germinal sacs"; and (3) very occasionally, cer­ are also taken up by germinal sacs, but are cariae, metacercariae, · and second-generation primarily stored rather than utilized in cer­ "daughter germinal sacs" are produced in the cariae and in certain species in the walls of 154 PACIFIC SCIENCE, Vol. XXII, April 1968 germinal sacs. The current belief that these ents in the snail, among other factors (Kendall, stored fatty acids are not utilized in energy 1949). It would follow that, even if the host­ production while within the mollusc's hepato­ parasite interphase is favorable for nutritional pancreas stems from von Brand's (1951) be­ uptake but nutrients are not sufficiently available lief that this environment is essentially anaero­ as the result of a poorly nourished host or be­ bic and hence is not conducive to lipid metabo­ cause of competition between a large number lism. The non-occurrence of aerobic metabolism of germinal sacs, normal development could not in sporocysts and in rediae appears to be con­ occur-and this would constitute incompatibility. firmed by the electron microscopic studies of Bils and Martin (1966) , which revealed the Leth ality to H ost absence of mitochondria in sporocyst and redial Surprisingly little informatio n is available walls. On the other hand, mitochondria have pertaining to the lethality of trematode larvae been demonstrated in the wall of Parorchis to molluscs. Some investigators (Rees, 1931; acanthus rediae (Rees, 1966) , and in the walls Kendall, 1964 ; and James, 1965) have sug­ of Pbilopb tbalmus gralli rediae (Cheng and gested that during certain mollusc-trematode Hamamoto, unpubl ished data). associations death of the host does not occur. Cheng and Snyder (1962a, b; 1963) , by Yet Schreiber and Schubert (1949) and Pan employing histochemistry, arrived at the conclu­ (1965) , both working with B. glabrata para­ sion that glucose and fatty acids are taken up sitized with S. mansoni, have shown that a by sporocysts through their body walls. Electron high incidence of mortality does occur. In fact, microscope studies by Bils and Martin (1966) Schreiber and Schubert went as far as to quote tend to corroborate this with the finding of con­ a "half life" for parasitized snails. These known spicuous microvilli along the outer surfaces of mortalities, however, cannot be considered to sporocysts. In the case of rediae, Cheng (19 62, reflect incompatibility, since the parasites do 196 3c) found that the ingestion of the host's develop normally and death, as Faust and Hoff­ cells is the primary method of nutrient acquisi­ man ( 1934), Schreiber and Schubert (1949) , tion, although some absorption may occur and Pan (1965) have pointed out, has resulted through the body wall also. Th is, again, appears from the rapid multiplication of larvae and to be corroborated by the finding of microvilli mass emergence of cercariae. From the available on redial walls by Bils and Martin (1966), information, it would appear that the death of Rees (1966) , and Cheng and Hamamoto molluscs resulting from invasion by "patho­ (unpubl. ) . For a detailed account of nutrient genic" trematodes is extremely rare. No in­ acquisition and contents of intramolluscan larval disputable examples have yet been reported, al­ trematodes, see the reviews by Cheng ( 1963a, though Kendall (1950) has shown that Fas­ 1967) . ciola hepatica does inflict conspicuous deleteri­ Relative to the relationship between nutri ­ ous effects on Lymnaea stagnalis, L. palustris, tion and compatibility and incompatibility, it and L. glabra, but not on L. auricularia. The may be asked whether the introduction of fact that mass mortality seldom occurs could be germinal sacs to some site within its natural interpreted to mean that the defense mechanisms molluscan host, or into a foreign host, where of molluscs are highly effi cient. the physico-chemical nature of the host-parasite It appears appropriate at this point to interject interphase prevents the uptake of nutrients could the following comment. Death of molluscs due cause the parasite to fail to become established. to a pathogenic trematode infers extremely se­ For example, could the destruction of heavily vere pathogenicity, a topic which has been encapsulated germinal sacs in incompatible hosts reviewed recently (Ch eng, 1967). Yet, despite actually represent, at least in part, death due known instances of drastic histopathological to starvation? alterations in parasitized molluscs caused by It is known that the rate of cercarial develop­ trematode larvae, few proven cases of rapid and ment is dependent upon the amount of food virulent deaths are known. This should serve as assimilated by the snail host and upon the num­ a warning to shellfisheries biologists and mol­ ber of larvae competing for the available nutri- luscan pathologists, who have been known to Compatibility in Trem atodes and MolluscS-CHENG 155 draw unwarranted conclusions relative to the dicating the incompatibility of different strains lethality of parasites, particularly helminths, of B. glabl'ata with strains of S. mansoni does based on histopathological studies. suggest that this relationship is not completely free of factors favoring incompatibility.

ESCAP E PRO CESS SUMMARY Our present knowledge concerning the pas­ sive and/or active mechanisms employed by Compatibility between miracidia and molluscs cercariae while escaping from their molluscan may be determined prior to actual contact. In hosts is based primarily on studies carried out some instances this may be governed by the on the human-infecting species of schistosomes occurrence of host-specific "host factors" which (see reviews by Probert and Erasmus, 1965; stimulate and attract the parasite to a com­ Cheng, 1967) , although some information is patible host only. There is sufficient evidence to available on Fasciola hepatica (Kendall and indicate, however, that the attractant need not McCullough, 1951) , N eodiplostomum inter­ be host-specific and need not indicate subse­ medium (Pe arson, 1961), and Cercaria X quent successful establishment and growth. (Probert and Erasmus, 1965). All these studies Furthermore, ambient environmental conditions have been concerned with the processes which as well as innate taxes, when such occur, are make possible successful escape, and hence, in a generally stronger determinants of miracidial manner of speaking, govern compatibility. On migration and behavior than are chemotactic the other hand, if some factor or factors within forces. Thus, it is only when the other factors the mollusc interfere with cercarial escape and serve to bring miracidia into the immediate thus prevent the parasites from continuing their vicinity of the mollusc that chemotaxis becomes normal course of development, this factor or an effective attractant. Th ere is also evidence factors may contribute to host-incompatibility. which suggests that materials extruded from In seeking evidence for this hypothetical pos­ molluscs may inhibit rather than serve to en­ sibility, one should be cautious in distinguish­ hance host-parasite contact. ing between consistent barriers, either structural In the case of miracidia which do not hatch or physiological, which prevent escape and in until the eggs are ingested by the mollusc, the so doing endanger the perpetuation of the physical and chemical factors present in the parasite species, and an occasional accidental host's gut can serve as determinants of com­ arrest of the escape of a few cercariae. Examples patibility or incompatibility. of the latter include the report by Cheng and It is possible that, during the invasion process, Cooperman (1964) that occasionally an escap­ both the specificity of the miracidium's lytic ing cercaria of Glypthelmins pennsyluanien sis secretions and the specificity of substances which may accidentally migrate into the foot mus­ stimulate the miracidium to shed its epidermal culature of its snail host, H elisoma trivolvis, plates, secrete lytic enzymes, and invaginate its and become encapsulated, and the report by apical papill a could in some instances determin e Pan ( 1965) that some escaping S. mansoni the compatibility of the host, especially if these cercariae do become trapped in the loose vas­ are prerequisites of successful penetration. cular connective tissue of A . glabratns and die. Subsequent to successful invasion, the para­ Yet, in the latter case the frequency of this site generally has to reach a suitable site within event suggests that it is a normal occurrence. the mollusc for further development. If this If all of the cercariae were thus trapped, one migration is prevented by some tissue(s), such could cite this as an example of incompatibility as a basement membrane, incompatibility re­ due to prevention of escape. It is of interest to sults. If such a barrier is absent or is overcome, note that if a similar phenomenon is not the parasite must still overcome the host's in­ found in other mollusc-trematode associations, ternal defense mechanisms (immunity) . From one might consider B . glabrata as being partially the information available, cellular immunity, incompatible with S. mansoni as far as escape both innate and acquired, occurs in molluscs; is concerned. Indeed, available info rmation in- and it is pr imarily by the formation of capsules 156 PACIFIC SCIENCE, Vol. XXII, April 1968

comprised of leucocytes and/ or fibers that in­ Schistosoma mansoni in Tr opicorbis bauenen­ compatible trematode larvae are destroyed. sis and A mtralorbis glabratm. J. Parasitol. Since some indirect evidence suggests that 39 :159- 163. substances of host origin, perhaps hormones, BURNS, W. C , and 1. PRATT. 1953. The life can alter the normal developmental pattern of cycle of iVIetagonimoides oregonensis Price intramolluscan trematode larvae, it is proposed (Trematoda : Heterophyidae) . J. Parasitol. that the absence of grow th- and development­ 39:60-67. stimulating substances or the presence of CABLE , R.M. 1965. "Thereby hangs a tail." growth- and development-inhibiting substances J. Parasitol. 51:3-12. may be factors responsible for incompatibility. CAMPBELL, W . C 1961. N otes on the egg and Relative to the nutritional requirements of miracidium of Fascioloides magna (Trema­ trematode larvae, their availability in the mol­ toda) . Trans. Am. Microscop. Soc. 80 :308­ lusc, as well as deficiencies in nutriments re­ 319. sulting from competition between larvae, could CAMPBELL, W . C , and A. C TODD. 1955. In influence the compatibi lity of the association. vitro metamorp hosis of the miracidium of Furthermore, the physico-chemical nature of the Fascioloides magna (Bassi, 1875) Ward, host-parasite interphase could influence the suc­ 1917. Trans. Am. Microscop.Soc. 74 :225­ cessful or unsuccessful uptake of nutrients and 228. hence govern compatibility or incompatibility. CHENG, T. 1961a. Description, life history, Finally, in order that the relationship be con­ C and developmental pattern of Glypthelmins sidered a completely compatible one, the para­ pennsylcaniensis n. sp. (Trematoda : Brachy­ site cannot destroy its host prior to its successful escape or the escape of its germ cell-bearing coeliidae), new parasite of frogs . J. Para­ sitol. 47:469-477. progeny. Thus, factors of parasite origin which are lethal to the host and factors of host origin --- 1961b. Studies on the morp hogenesis, which prevent escape must also be considered development and germ cell cycle on the spo­ as determinants of incompatibi lity. rocysts and cercariae of Glypthelmins pennsyl­ vaniensis Cheng, 1961 (Trematoda: Brachy­ coeliidae) . Proc. Penna. Acad. Sci. 35 :10- 22. REFERENCES --- 1962 . The effects of parasitism by the BARBOSA, F. S. 1965 . Ecology of the larval para­ larvae of Echin oparyphimn Dietz (Trema­ sitic stages of Schistosoma mansoni. Rev. toda: Echinostomatidae) on the structure and Inst. Med .T rop. Sao Paulo 7:112-120. glycogen deposition in the hepatopancreas of BARBOSA, F. S., and M. V. COELHO . 1956 . H elisoma triuoluis (Say) . Am. Zool. 2:513 . Pesquisa de immunodade adquirida homo­ - -- 1963a. Biochemical requirements of larval trematodes. Ann. N .Y. Acad.Sci. 113: loga em Australorbis glabratltSJ nas infesta­ coes por Schistosoma mansonl , Rev. Brasil. 289-321. Malariol. 8:49-56. --- 1963b. Histological and histochemical BARLOW, C H. 1925. The life cycle of the studies on the effects of parasitism of iVIltSClI­ human intestinal fluke Fasciolopsis bltSki limn partnmeinm (Say) by the larvae of ( Lankester) . Am. J. Hyg., Monogr. Ser. Gorgodera amplicava Looss. Proc. Helminth. 4 :1-98. Soc. Wash. 30:101-107. BENEX, J., and L. LAMY. 1959. Immobilisation - -- 1963c. The effects of Echin opal'yphi//1ll des miracidiums de Schistosoma mansoni par larvae on the structure of and glycogen depo­ des extraits de planorbes. Bull. Soc. Pathol. sition in the hepatopancreas of H elisoma tri­ Exot. 52:188-193. volvis and glycogenesis in the parasite larvae. BILS, R. F., and W .E. MARTIN. 1966. Fine Malacologia 1:291-303. structure and development of the trematode - - - 1967. Marine Molluscs as Hosts for integument. Trans. Am. Microscop. Soc. 85: Symbioses: W ith a Review of Known Para­ 78-88. sites of Commercially Imp ortant Species. In: BROOKS, C P. 1953. A comparative study of F. S. Russell, ed., Advances in Marine Biol- Compatibility in Trematodes and MolluscS-CHENG 157

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