INTERNAL DEFENSES OF CRUSTACEA: A REVIEWl

CARL J. SINDERMANN"

ABSTRACT

Studies of the internal defenses of Crustacea have a discontinuous history, which !)('gan in the late 1800's. Elaborate early studies of phagocytes and humoral factors in the hemolymph have b('en extended with renewed vigor during the past decade. As is true for other invertebrates studied, phagocytosis of for­ eign particles by fixed and mobile cells in the crustaceans is augment('d by naturally occurring bacteri­ cidins, lysins, and agglutinins. A few instances of experimental enhancement of titers of such hu­ moral factors by previous exposure to foreign protein have been reportf'(l. Specificity of natural and experimentally enhanced humoral factors is much lower than that of vPl'tehrate immunoglobulins, but probably such factors act synergistically with cellular protective mechanisms, as they do in the \'C'rtebrates. Phagocytosis by fixed and mobile cells in gills, pericardia! sinus, and sinuses at the bases of appendages seems to be a principal defense perimeter in many crustaceans. Efficacy of phagocytes in destroying in­ vading microorganisms varies, depending on the species of the microorganism, as well as host physiology and environmental factors. Phagocytic activity is enhanced by hemolymph factors, which, in addition to immobilizing and agglutinating the invading organisms, also sensitize them to phagoc>,tosis. Hemolymph factors, most of which seem to be of cellular origin, may also have lJactericidal or lytic activity, leading to extracellular destruction of microbial invaders. A f,,\\' recent studies indicate that effects of hemolymph factors may be enhanced experimentally by injection of killed or living micro­ organisms. The number of known microbial diseases in crustaceans is greater than that known in most other in­ vertebrate groups, with the possible exception of the Mollusca and Insecta. The number and depth of studies concerned with internal defense mechanisms of Crustacea are similarly grl'ater than those of most other invertebrate groups-again with the possible exception of the mollusks and insects. One mi­ crobial pathogen of Crustacea that has received adequate attention is (;ollkyo hOJl/o1'i-a gram-positive coccus which causes a fatal septicemia in lobsters and is capable of infecting otlwr d,'capods. An elab­ orate series of studies in several laboratories has elucidated many details of the host-parasite relation­ ship and has also provided extensive information about the internal defenses of a number of the larger Crustacea. Ga//kya constitutes a test microorganism of choice for future studies of disease processes in crustaceans. Thus the available information about cellular and humoral defenses of Crustacea against invasion by foreign protein constitutes a significant part of what we know about such proceSRes in the inverte­ brates. Phagocytosis, augmented by humoral factors with low specificity, seems to bl' the fundamental means of internal protection in the crustaceans and in other groups of invertebrates as well.

Investigations of the internal defense mecha­ The large number of published reports on in­ nisms of invertebrates against disease have pro­ ternal defenses of Crustacea call provide a good gressed with renewed vigor in the past decade. indication of the extent of our knowledge of im­ Literature has accumulated to the point where munity in marine invertebrates. Beginning with condensation and summarization of information the pioneering work on phagocytosis and the en­ tire process of inflammation by Metchnikoff about certain invertebrate groups, such as the (1884, 1893, and 1905), and on humoral factors Crustacea, seem justified, described in the very extensive (but sometimes poorly documented, in terms of procedures and t Contribution No. 197, National Marine Fisheries Service, Tropical Atlantic Biological Laboratory, Miami, ctata) work of Cantacllzl'l1e (191~-3,1), the Crus­ Fla. 33149. tacea have often been animals of choice in stucties • National Marine Fisheries Service, Tropical Atlantic Biological Laboratory, Miami, Fla. 33149. of internal ctefense mechanisms. A significant

Manuscript accepted february 197/. FISHERY BULLETIN: VOL. 69, NO. J, 1971. 455 FISllERY BCLLETI:-.1: VOL. 69, NO, ]

body of literature has accumulated, with conspic­ been that much of the earlier work failed to uous bulges in the early years (1884-1930) and indicate any immunologic responsiveness in the in the past decade (1960-70), but with a very invertebrates tested. In spite of occasional suc­ narrow waist during the period 1930-60. cesses, the "inability of invertebrates to respond Studies of humoral defenses of the larger to the introduction of antigen by formation of Crustacea during the past I) years (1961)-70) ,ire antibodies" became a sort of dogma among many curiously reminiscent of work reported by Can­ of the early biologists, as was pointed out by tacllzene and his associates during the period Cantacuzene (192:3b). Failures to find responses 1912 to 1934, with the very important difference may have been due pmtly to choice of inocu­ that most of the modern work includes elements lum with negligible antigenicity in the experi­ that were largely missing from earlier reports, mental invertebrate. Two factors may have such as details of procedures, supporting data, caused the recent resul'g'ence of interest amona adequate controls, and attempts at quantitation biologists in comparative immunology (which of results. is gradually beginning to include the lower Because so much of the literature produced be­ vertebrates and the invertebrates): (1) an fore 1940 lacks adequate quantitation and fails evident need to reexamine the conceptual and to provide details of techniques used, it is often evolutionary basis of immune reS]Jonses and (2) difficult to relate results to those of more recent an interest in understanding the internal de­ studies. Bang (1967b) has deliberately ad­ fenses of invertebrates, which allow them to dressed himself to a repetition of earlier studies survive in a microbe-rich environment even but has used modern methods in an attempt to though they lack the speciflc antibody response improve the relationship. Other recent reports, characteristic of most vertebrates. even though based on species other than those The subject matter of the present review is used in earlier studies, constitute reexaminations one that has been treated previously (Cantacu­ of the concepts and general findings of the early zene, 1923b; Huff', 19,10; Baer, 1944; Bang investigators. 1967h; Levin, 1967; TripP,1969; Rabin, 1970b; It is difficult to determine why the early work Bang, 1970). Many of those papers, however, on immune responses in invertebrates so effec­ were broad considerations of the invertebrates tively begun by l\:Ietchnikoff, Cantacuzene, Cue­ as a whole, and discussions of internal defenses not, Bruntz, and others during the late 19th of Crustacea were often more or less incidental. century and the early 20th century seemed to It is interesting-as Good and Papel'master lose impetus and then virtually cease until re­ (196,1) pointed out-that no review of inverte­ cently. It is apparent from the literature, brate immunity has emphasized induced re­ though, that research on invertebrate defenses, sponses. A recent, and excel1ent, 2-volume text initiated so auspiciously, receded for a number on the ]Jhysiolog'Y of Crustacea (Waterman of decades to the backNaters and eddies of the 1%0) does not include a detailed consideratiol~ mainstream of advances in immunology, which of the vel'Y important subject of internal de­ was concentrated on the homoiothermic verte­ fenses, except for reference to hemocytes and hrates. This may be explained in part by a phagocytosis (Maynard, 1960; Parry, 1960) natural and necessary concentration of research and a consideration of hemolymph coagulatio~ interest on human and mammalian immune re­ (Florkin, 1960). sponses (most immunologists were-and still The general plan for this review is to discuss are-generally associated with medical schools some of the early literature, after a brief pre­ and hospitals). Part of the explanation also may liminary statement about concepts and termi­ be that earlier work failed to disclose any defense nology, and a summary of known diseases of mechanisms in invertebrates that seemed funda­ Crustacea. More recent studies will then be mentally or conceptually different from those considered by categories of cellular and humoral that were being elucidated for the vertebrates. systems: phagocytic, bactericidal, lytic, agglu­ More importantly, the explanation may have tinating', precipitating, phage clearing, and anti-

456 SINDERMANN: INTERNAL DEFENSES Of' CRUSTACEA toxic, A final section will attempt a detailed or extracellular clots to close gaps in the circu­ review of the demonstrated systems of intel'l1al latory system and to immobilize microorganisms; defenses of lobsters and other crustaceans and ellcu/isltlalio/l-the surrounding of large against the microbial pathogen G((/rkya homan' masses of invading material by phagocytes and -which is one of the best examples of a test fibrocytes. Humoral defense mechanisms, which system for invertebrates for which existing in­ probably depend on cellular secretions or cell formation is adequate. disruption, and act synergistically with cellular defenses, include agglutinating, lytic, precipi­ CONCEPTS AND TERMINOLOGY tating, and bactericidal systems, and other ac­ tivities (Figure 1), Before proceeding with an examination of in­ ternal defenses of crustaceans, a brief review of some of the terminolog'y may be relevant. POTENTIAL PATHOGEN IN "Resistance"and "susceptibility" have often been EXTERNAL ENVIRONMENT used interchangeably and reciprocally, but as J I Schneider (1951) and Stauber (l9G1) pointed I I out, insusceptibility and resistance should prob­ INSUSCEPTIBILITY ably be considered as separate biological phe­ I nomena. Insusceptibility refers to those exist­ I f ing external or internal features of an animal RESISTANCE FACTORS -morphological and physiological-which deny INNATE ANDIOR access to a potential pathog'en, 01' prevent its ~ successful establishment and survival. Resist­ HUMORAL ance, on the other hand, has been defined by Phagocytosis ~ Agglutinins Read (1958) as "those changes in the physio­ Hemocytosis Lysins Coagulation Preclpltins logical state of the host which represent a re­ EncapSUlation Bactericidlns S/iOllse to present or previous contact 'with the ... other parasite or with a similar chemical entity." Resistance (and its equivalent-"immunity"), defined as host response, can then be considered as "innate" (natural) 01' "acquired" (induced). Innate resistance includes responses to primary contact with a pathogen, and acquired resistance includes responses that develop after primary contact. The distinction between the two types Fll:l'IU: 1.--1\lechanisms of intel'lla! defense. of response is not, however, always as definitive as might be preferred. Acquired resistance is often characterized by enhanced responsiveness Antibodies (in the vertebrate sense of specific to subsequent contact with a pathogen. immunoglobulins) have not been demonstrated Resistance, whether innate 01' acquired, may in invertebrates, although less specific antibody­ be cellular or humoral. The cellular defense like activity is common. Since antibodies have methanisms are based larg'ely on activities of not been demonstrated, it is probably technically leucocytes (hemocytes of invertebrates) and in­ incol'l'ect to use the term "antigen" with in­ clude: phallocytosis-engulfment and often di­ vertebrates. The semantics involved will be con­ gestion of foreign particles, lel/cocytosis (helno­ sidered in the discussion section, but for con­ cylosis) , and lellco('ylic (hell/ocylic) intill/'((­ venience the tel'm will be used in this paper. lioll-the mobilization of hemocytes in the blood Furthermore, it must be made clear that when stream and their migration to invaded or injured lysins, pl'eeipitins, agglutinins, etc. of inverte­ tissues; ('OU [/1I1u tio II-the formation of cellular brates are discussed, no attempt is made to

457 FISHERY BULLETIN: VOL. 69, NO. J

homologize them with vertebrate factors. The of Ga./11I11I1I'IIS nut rlIIIIS lVas reported from Eng­ terms merely indicate the type of activity pro­ land by Tait (U1l7), in wh ich sig-ns of disease duced. included chang'c in coloI' of the infected amphi­ pods fro111 brown to opaque yetlowish-white, re­ DISEASES OF CRUSTACEA duction in numbers of blood cells, and absence of coagulation of hemol.\·mph. Among the larger An impressive array of diseases afflicting the decapod Crustacea, a Sf'vere bacterial disease of Crustacea has been described (summarized in lobsters caused by gram-positive cocci, Gafjkya Sindermann, 1970, and Bang, 1970). A number hOIlUll"i,'was recognized in 1917 (Snieszko and of these diseases are of microbial etiology, and Taylor, 19·17) and has been the subject of in­ Koch's postulates have been satisfied for several tensive studies since then (to be considered in of them. Some of the published reports con­ detail later in this paper). Experimental in­ cerning crustacean diseases include information fections and resultant mortalities of blue crabs, about host defenses against infection, others do C((/lill('ctcs s((/JidIlS, from Chesapeake Bay were not. The literature also contains information reported by Krantz, Colwell, and Lovelace about a variety of experimentally induced infec­ (1969) with Vibrio ]ta/"UhelllOlyticus. The micro­ tions in Crustacea, many of them produced by organism has been isolated from mollusks, fishes, microorganisms not known as pathogens in and sediments in various parts of the world and natural populations. Such experimental studies is known as a cause of human gastroenteritis in have been particularly useful in elucidating pos­ the Orient. Additionally, several examr)les of sible internal defense mechanisms-augmenting "shell disease"-erosion of the exoskeleton by studies with known pathogens. The following chitin-destroying baderia-are known in lob­ summary is just that and is not intended as a iters, crabs, auo shrimps (Hess, 1937; Rosen, detailed treatment of crustacean diseases. Some 1967, 1970; Anderson and Conroy, 1968). general background information on known di­ Fungus dise:lses of Crustacea are sUrj}l'isingly seases seems important, hO\vever, to any con­ numerous in reports dating back to Metchnikoff sideration of internal defense mechanisms. (1884), who described fatal infections of Da]lh­ The only virus disease of invertebrates re­ Ilia causec1 by MOllos]JOra bicltsj!ida.ta and who ported in the scientific literature is one that oc­ first emphasized the crucial role of phagocytosis curs in crabs, Portnnus dC]Jurator, on the French in determining the outcome of infection. A Mediterranean coast (Vago, 1966). Disease yeast infection in sand hOTll)ers, Tulitrus, from signs mentioned in his very brief paper included the coast of France, was reported by Herrmann progressive darkening of the exoskeleton, paral­ and Canu (1891). Experimental infections from ysis, and death. exposure to cultured microorganisms were fatal Bacterial infections of various Crustacea have to T((litl"lls in 20 to 2G days. Phagocytosis was been described, beginning with a disease of beach marked in such infections, and the hemolymph hoppers on the French coast caused by lumines­ became milky in advanced cases. Crabs (Car­ cent bacteria «;iard and Billett, 1889). Exper­ cinusm((cnas), prawns (l'ala(,!IIollctc81'al'ialls) , imental infections were obtained by injecting and crayfish (J1.~t(lnls flilrialillis) were not sus­ cultured microorganisms, and some of the crus­ ceptible to the expel'imental infections. PixelI­ tacean species tested exhibited varying degrees Goodrich (1928) rlescl'ilJed another yeast infec­ of resistance to experimental infection. Another tion which was epizootic in (;llIlIInaI'l/8 from a luminescent disease was reported in sand fleas stream in England. The pathogen C/"m)toC()CCll.,~ (Talol"chestia lonuic{)l'lIis and Ol'chestia Ul({ti­ f/(Linnwl'i l'eproduced in the hemolymph and /IllS) from Wooos Hole, Mass., by Inman (1927). rendered it milky in color, coagulation Was re­ Luminiscent bacilli were cultured. and experi­ tarded, and heavily infected individuals died. mental infections ohtained. Luminiscent hac­ Phagocytosis was activc and som€times success­ teria were also isolated from the digestive tracts ful in overcoming infections. Hypertrophy of of nonluminous sand fleas. A bacterial disease fixed phagocytic cells was common.

458 SINDERMANN: INTERNAL DEFENSES OF CRUSTACEA

One of the most severe, widespread, and long­ Goodrich, 1928). Other pathological effects of continuing epizootics known in invertebrates has microsporidans on gammarids have been recent­ affected and still affects European crayfish. It ly reported by Bulnheim (1967) and Bulnheim is caused by the fungus Aphanomyces astaci (al­ and Vavra (1968). Crayfish muscles are at­ though other microorganisms have been var­ tacked by Microsporida of the genera Thelohania iously associated with mortalities). Known as and Nosema (Sprague, 1950b; Pixell-Goodrich, "Krebspest," the disease swept through cray­ 1956; Sogandares-Bernal, 1962). Microsporida fish populations of Europe beginning about the are also significant pathogens of shrimps. turn of the century (Schikora, 1906, 1926; Sprague (1950a), Woodburn et al. (1957), Iver­ Schaperclaus, 1935; Nybelin, 1935; Mannsfield, sen and Manning (1959), Iversen and Van Meter 1942; Unestam, 1965; Gordon, 1966). Appar­ (1964), and others have described infections of ently resistance differs among species-the body muscles and gonads of shrimps from the American crayfishes, for example, seem less seri­ Gulf of Mexico and European waters, caused ously affected by the pathogen in experimental by a number of representatives of the genera studies. Thelohania and Nosema. Other fungus diseases of Crustacea include Other protozoan diseases of crustaceans in­ a systemic infection of pea crabs, Pinnotheres, clude those caused by ciliates, an ameba, and from English sea mussels by Leptolegnia marina gregarines. A ciliate, Anophrys sarcophaga, (Atkins, 1929, 1954a); a systemic disease of causes a fatal disease in shore crabs, Carcinus cultured prawns, Palaemon serratus, in England, maenas, of Europe. The disease, and host re­ caused by Pythium sp. (Anderson and Conroy, sponses to infection, will be considered in detail 1968); a gill infection of pandalid shrimp, in a later section. Other parasitic ciliates occur Dichelopandalus leptocerus, from the western in the hemolymph of Crustacea. Paradinium sp. North Atlantic, caused by a chytrid-like micro­ and Syndinium sp. occur in calanoid copepods. organism (Uzmann and Haynes, 1969); and gill Syndinium causes gonad destruction, while Par­ infections of lobsters, Homarus vulgaris and adinium colors the host a deep red (Gordon, Palinurus vulgaris, in Italy, caused by Ramularia 1966). Hematodinium sp. has also been reported branchialis and Didymaria palinuri-both Fungi by Gordon in Carcinus. A suctorian, Ephelota Imperfecti (Sordi, 1958). gemmipara, can seriously reduce production of Fungi also infect and destroy egg masses of lobster larvae (Dannevig, 1928, 1939). An Crustacea. Eggs of blue crabs, Callinectes sa-· ameboid parasite, Paramoeba perniciosa, causes pidus, from Chesapeake Bay may be infected by a fatal disease (called "gray crab disease") in Lagenidium callinectes (Couch, 1942; Newcombe blue crabs from the Atlantic coast of the United and Rogers, 1947; Rogers-Talbert, 1948); and States (Sprague and Beckett, 1966, 1968; eggs of pea crabs are often infected by Plec­ Sprague, Beckett, and Sawyer, 1969; Sawyer, to spira dubia and Pythium thalassium (Atkins, 1969). Hemolymph of infected crabs becomes 1954b, 1955). cloudy and often incoagulable; in some individu­ Among the many protozoan diseases of Crus­ als most of the cells in the hemolymph are amebae tacea, those caused by microsporidans are prob­ (Sawyer. Cox, and Higginbottom, 1970). A ably the most destructive. Nosema sp. and great number of gregarines occur in Crustacea Plistophom cargoi destroy body muscles of blue of all kinds, but their pathogenicity seems slight, crabs (Sprague, 1965, 1966); Nosema 7J11lvis except for some destruction of the digestive and Thelohania maenadis infect muscles of green epithelium resulting from heavy infections (Ball. crabs, Carcinus maenas (Perez, 1905a, 1905b, 1938; Theodorides, 1961, 1962; Tuzet and 1907). Other microsporidan infections of body Ormieres, 1961; Kruse, 1959a, 1959b). muscles in Crustacea include those produced in Helminth diseases of crustaceans seem less Gammarus by Theileria sp. and Nosema sp. abundant and less severe in their effects than Necrotic muscle fibers containing microsporidan those of microbial etiology. Trematode metacer­ sporp.s were destroyed by phagocytes (Pixell- cariae encyst in muscles and hepatopancreas of

459 nSIlERY BCLLETIN: VOL. 69. NO. -' crabs, and larval cestodes, acanthocephalans, host died in a few days. If all the fungus spores nematodes, and leeches occasionally have been were phag-ocytized and destroyed, the infection reported from crabs, shrimps, and lobsters was al'l'ested. Thus the speen and effectiveness (Sindermann, 1970). of phag'ocytic action in some individuals, possibly Crustaceans are frequently parasitized by mediated by humoral factors, determined sur­ other crustaceans-sometimes with serious ef­ vival. Absence of phagocytosis inevitably led fects 011 the host. Rhizocephalan barnacles are to death. endoparasites of crabs, causing gonad degener­ Hemocytes of Crustacea were investigated by ation and other morphological changes. Epi­ Cattaneo (188Rb), Cuenot (18%, 1897, 19(5), caridean isopods may produce similnr changes in and Bruntz (1907). Cattaneo described the crabs and shrimps. Copepods sometimes para­ amebocvtes of Ca/,p/Illts nWPIlr.lS; Cuenot re­ sitize crab eggs, as well as the gills of lobsters ported blood forming tissues-nodules of lymph­ (Sindermann, 1970). oid cells in the blood sinuses-in decapods and described "phagocytic organs" in the hepato­ pancreas of decapods and amphipods; and INTERNAL DEFENSE SYSTEMS Bruntz published an extensive paper on the hemocytes of many of the crustacean groups, Studies of crustacean internal defenses pub­ distinguishing granular and hyaline hemocytes. lished during the last decade have augmented Bruntz also described a "phag-ocytic organ" in earlier studies and have provided additional data gammarids; his 1907 paper reviewed an exten­ to support generalizations and principles already sive series of his own studies (15 reports) pub­ enunciated, but none has yet provided the factual lished during the period 1903-1907 by the Societe basis for new or different concepts. Because Biologique de Paris. Other early studies of crus­ additional precision in terminology is now avail­ bcean hemocytes include those of Hardy (1892), able, it is possible to consider the internal defense Tait (l918a, 1918b), and Tait anrl Gunn (1918). systems of Crustacea under the following head­ Following the classical early studies of Metch­ ings: cellular (phagocytic), bactericidal, lytic, nikoff, Cuenot, Bruntz. and others, which elu­ agglutinating, precipitating, phage clearance, cidated the critical role of phag-ocytes in the in­ antitoxic, and others. It should be obvious that ternal defenses against microorganisms, phago­ these systems are not mutually exclusive and cytic cells have received greatest attention from may often interact or even share components vertebrate immunologists. General principles to protect the individual animal from invasion that have emerged from the more recent studies by potential pathogens. Largely for ease of de­ of phagocytosis in vertebrates undoubtedly apply scription, the systems will be considered con­ as well to invertebrates. Among the papers that secutively, even though many may act either have contributed to understanding of phag-ocy­ in concert or simultaneously. tosis are those of Wright and Douglas (1903), Wood, Smith, and Watson (1946), Wood (1953), PHAGOCYTOSIS AND OTHER Robineaux ana Frederic (1%5), Suter (1956), CELLULAR DEFENSES Rowley (1960), Rogel'S (1960), Evans and Karnovsky (1961), and Spector and Willoughby The earliest study of phagocytosis in Crustacea (1963). Reviews of phagocytosis have been pub­ concerned infections of Daphn;(I by the fungus lishen by Hirsch (19GG) and Aarum (1967). Mmws]Jom bicns]Jidata (Metchnikoff, 1884). The mechanism of intracellular degradation The fungus spores in the haemocoel were phago­ of phagocytized microorganisms has been de­ cytized and digested; the rapidity and vigor scribed in general terms for the vertebrates with which phagocytosis occurred determined (Fig-ure 2). Lysosomes-granules in the cyto­ the outcome of the infection. If some spores plasm of phagocytes-contain antibacterial sub­ escaped phagocytosis, germinated, and formed stance; and hydrolytic enzymes (Cohn, Hirsch, conidia, the infection became generalized and the and Wiener, 19(3). The lysosome membrane

460 SINDERMANN, INTERNAL DEFENSES OF CRt.;STACEA fuses with the vacuolar membrane within the ley, 19(iO). Sensitization of bacteria with serum phagocyte, releasing antimicrobial components factors is not always a necessary prelude to into the vacuole (Robineaux and Frederic, 19GG; phagocytosis, however, as was pointed out by Hirsch, 1965; Aarum, 19(7). Evidence for Wood, Smith, and Watson (1946) and Wood comparable intracellular events in invertebrates (19G3). In the absence of other host responses, is sparse, but Janoff and Hawrylko (1964) re­ early phagocyte activity may he important in ported lysosomal enzymes in clams and starfish, preventing infection. and Eble (1966) found hydrolytic enzymes in Phagocytosis, then, constitutes the keystone oyster phagocytes. to resistance. As Aarum (1967) mentioned, Invading microorganisms are subjected to "... the organism's ability to oppose infection antimocrobial factors both inside and outside the precisely follows the phag'ocytes' ability to func­ phagocytes. Substances of presumed cellular tion optimally. llesistance is lowered by a low­ origin, such as lysozyme, occur in the phagocytes ering of phag-ocytic activity." Phagocytosis can and the plasma. A great array of such antimi­ occur at the site of a lesion, in the filtering tissues crobial factors was identified in vertebrates and organs of the circulatory system, and (to a (Skarnes and Watson, 1957; Elberg, 1960; lesser extent) in the body fluid itself. Groups Hirsch and Cohn, 1960; Landy, 1960; Coombs, of fixed phagocytic cells are present in many Coombs, and Ingram, 1961; Mackaness, 1962; crustaceans, most commonly in the sinuses and Miles, 1962), and some counterparts were rec­ lacunae of the gills and at the bases of the legs. ognized in invertebrates. McDade and Tripp Phagocytes agglutinate, aggregate, and co­ (1967) , for example, reported Iysozymes in oys­ operate in defense-forming nodules (the "nod­ ter hemolymph. ules leucocytaires" of Cuenot, 1898), which are In the vertebrates, specifi.c and nonspecific also known in annelirls, mollusks, echinoderms, serum proteins increase the speed and effective­ and other invertebrates. In a number of ani­ ness of phagocytosis-the opsonizing effect mals the nodules are brown, due to presence of (Wright and DouglaR, 1903; Suter, 195G; Row- large numbers of brown granules in the phago­ cytes, which may be excretion products or de­ composition products. In GamnWI'IIS, the phago­ 1 2 3 4 cytes composing the nodules secrete a clear yellowish chitinoid substance around the para­ sites. This secretion gradually becomes dark brown. The nodules appear as conspicuous a-.4J-.~-.~ black spots in infected individuals (Pixell-Good­ rich, 1928) and are found frequently in gills and appendages. It should be clearly understood, however, that there are few detailed modern , studies of phagocytosis in crustaceans or other invertebrates. Data from in vitro studies are particularly scarce, so there should be no impli­ cation that the kinetics, energetics, or other as­ pects of phagocytosis are fully understood. Hemocytes of crustaceans and other inverte­ brates also act in other ways to protect the in­ dividual from overwhelming microbial invasion. Hemocytosis and hemocytic infiltration have 5 6 7 heen described in a number of invertebrate g-roups. Manifestations in inveI-tebrates and 2.-Rol~ c~ll FWIIRE of nlf'mhraJH' and Iysosonw hrpak­ vertebrates involve proliferation of hemocytes, dowll in phap;ocytosis. 1-1; phap;ocylm;is; 5-7: lysosoll1P activities. (Redrawn from Hirsch, 1965.) changes ill permeability of blood vessels, leakage

461 F1SHERY BULLET1N: YOLo 69, NO. 3

of blood fluids into tissues, adherence of hemo­ conclusion that plasmatic coagulation was a one­ cytes to blood vessel walls, and migration of step process in which fibrinogen of the plasma hemocytes into tissues around areas of injury was acted upon by a coagulin released by the he­ or parasitic invasion. mocytes. It seems equally possible, however, that The involvement of hemocytes in coagulation more than one type of coagulable protein exists or clot formation is a complex one, inasmuch as and that the categories of clots may be complex either cellular or extracellular clots may be rather than simple. Florkin also reviewed the formed. Intravascular cellular clots adhere to role of cellular clots in wound repair, emphasiz­ walls of blood vessels and spaces, producing ing the importance of secretion of a chitin film stasis, and once they are formed, persist for over the wound area by underlying coagulated some time. Extracellular clots, resulting from phagocytes. release of constituents of hemocytes, can inhibit Encapsulation is also a common form of cellu­ microbial motion and thus render microorgan­ lar internal protection in invertebrates. Invading isms more vulnerable to phagocytosis. Since organisms, often relatively large, are surrounded Fredricq (1879) first pointed out that in Crus­ by phagocytes and flbrocytes. The onset of tacea coagulation of hemolymph involves cell encapsulation may be rapid, and the cellular agglutination as well as plasma coagulation, aggregates may be resolved only very slowly. others have demonstrated similar characteristics In summary, the hemocytes function in a num­ in a number of invertebrate groups. The release ber of ways beyond phagocytosis, although the of a component from hemocytes and the role of latter must be considered the dominant cellular this component in initiating coagulation of plas­ defense mechanism: ma were reported by a number of authors be­ ginningwith Halliburton (1881'5). L6wit (1889) 1. Hemocytes are important in cellular infil­ observed the rapid disruption of hemocytes and tration of injured or diseased tissue. the rapid clotting characteristic of most Crus­ 2. They are important in clotting-either as tacea and concluded that a causal relation ex­ participants in cellular clots, or by release of se­ isted. Hardy (1892), Tait and Gunn (1918), cretions or injury lwoducts which combine with Tyler and Scheer (1941'5), and George and Nich­ plasma components to form extracellular clots. ols (1948) all provided data which supported 3. The hemocytes are of primary importance the conclusion that a component from certain to encapsulation. hemocytes acts with fibrinogen of plasma to form fibrin clots. A great variety of crustacean hemocytes have Bang (1967c, 1968) demonstrated that in the been described during the past several decades. hermit crab, EUlm,qurus lon,qicarpus, clots Animals studied included crayfishes (George and formed in at least two stages following injury­ Nichols,1948; ToneY,1958; Wood and Visentin, first a clumping and stickiness of hemocytes 1967), blue crabs (George and Nichols, 1948; without change in shape or loss of granulation, Toney, 191'58), lobsters (Toney, 1958; Hearing then retraction of the clot and the development and Vernick, 1967), and brine shrimp (Lochhead of a network of fibrous cell projections contain­ and Lochhead, 1941). Except for size differ­ ing microtubules. ences, the principal distinction seemed to be pres­ The abundant and elaborate literature on he­ ence or absence of granules in the cytoplasm. molymph coagulation in Crustacea was admir­ The hyaline hemocytes are usually smaller than ably summarized and evaluated by Florkin the granular, and some of the hyaline cells (1960). As he pointed out, coagulation has been probably develop into the granular types, since considered by some authors to occur in two dis­ the intergrades have been noted (Cuenot, 1895). tinct phases-cellular coagulation and then plas­ As was aptly pointed out by Rabin (personal ma gelation-while other workers view coagula­ communication), "The developmental relation­ tion as a continuous process in which plasma gel­ ships of one form of hemocyte to another which ation begins around hemocytes. It was Florkin's have been made amount to little more than edu-

462 SINDERMANN: INTERNAL DEFENSES OF CRUSTACEA cated guesses, since they have been based largely vide an added perimeter of defense. Antimi­ on static images which may not even represent crobial capacities of tissues as a whole, and of the true cell pictures as they occur in vivo." Both nonphagocytic cells in particular, may be a main­ types of cells (hyaline and granular) probably stay of nonspecific resistance-in both primary have physiological subtypes, as suggested by the invasion and the determination of subsequent work of Fisher-Piette (1931) in which explants courses of infection. Tissue defenses of this of lobster hemopoetic tissues resulted in multi­ nature in the invertebrates may be of great sig­ plication of two types of hyaline cells-adhesive nificance. ameboid and non-adhesive non-ameboid. Inclu­ sions of granular cells, in addition to their de­ HUMORAL DEFENSE SYSTEMS fensive function mentioned earlier, may also pro­ vide nutrient material-as suggested by release Early studies of humoral factors in Crustacea of this material into the hemolymph during ovar­ produced significant, but at times ambiguous, ian development (Lochhead and Lochhead, results. Noguchi (1903) found that sera of 1941). Hyaline hemocytes may also be trans­ lobsters and horseshoe crabs possessed natural formed into connective tissue or endothelial cells agglutinins against various vertebrate erythro­ of blood vessels (Danini, 1925, 1927; Debaisieux, cytes. After repeated injections, he was able to 1952a, 1952b; Demal, 1953). Hemocyte physi­ demonstrate an induced hemolysin in the horse­ ology and biochemistry are areas where addi­ shoe crab but not in the lobster. Fredericq tional studies are needed, but the extreme fra­ (1910), using a variety of antigens, was unable gility of certain cells once they are removed from to demonstrate precipitins in a number of dec­ the normal animal has undoubtedly been a major apods (Homa/'ns vnlgaris, Palinnrus vulgaris, deterrent. 'Carcinus maenas, Po/·tunus ]Juber, Cancel' ]Ja- Phagocytic activity has been m;cribed in vary­ gUI'llS, and Astacus flu'L'iatilis). ing degrees to most recognized categories of The early literature on humoral mechanisms hemocytes (Haeckel, 1862; Hardy, 1892; Cuenot, of internal defense in invertebrates, and espe­ 1895; Bruntz, 1905, 1907; Kollman, 1908; Tait cially the crustaceans, was clearly dominated by and Gunn, 1918; George and Nichols, 1948; CantacuzEme and his students. Cantacuz(me, in Toney, 1958; Rabin, 1970b). a period covering almost 3 decades beginning in In recapitulation, the phagocytes of vertebrate 1912, examined the broad picture of humoral de­ and invertebrate animals have been investigated fences of a large number of marine invertebrates. widely since the late 19th century, and the blood His work with Crustacea will be summarized cells of Crustacea have received at least propor­ in the next few pages as background for a con­ tionate study. Cell ular defenses of Crustacea sideration of subsequent studies. and other invertebrates are varied but center on Cantacuzene (1912a) reported (in a paper the phagocyte and its activities. Important also consisting essentially of a series of statements are the humoral defenses, which will be consid­ but little supporting data) the presence of natur­ ered in the following sections. Before proceed­ al agglutinins, lysins, and precipitins in serum ing to considerations of other than cellular de­ of the hermit crab, Eupagurus prideauxii. He­ fenses, however, it seems relevant to include an molysins for sheep and rabbit erythrocytes oc­ often overlooked perimeter of defense suggested curred in crab serum to a maximum titer of 250 by Miles (1962). Early suppression of microbial and were destroyed by heat at 55° C. Aggluti­ numbers may be due to microbicidal activity of nins for rabbit erythrocytes persisted in dilutions the tissue cells themselves, either innate or in­ beyond those at which hemolysis disappeared. duced, or to soluble antimicrobial substances in Agglutinins for bacteria (Escherichia coli and the intercellular fluid of the integument. As Vibrio choleme) were also present, as were weak Miles pointed out, such pre-inflammatory cellu­ precipitins against horse and rabbit sera. Can­ lar defenses in no way diminish the importance tacuzene also stated that comparable agglutinins, of phagocytes and humoral factors, but only pro- lysins, and precipitins were not present in Pauu-

463 FISHERY BULLETIN: VOL. 69, NO. 3

FilS stl'iahl.s, which is closely related to EUj)(({j­ ulated with killed Vilil'io ('ho!el'ae or small doses UI'1IS jJrideauxii. of live vibrio, was able-12 days after the fifth Cantacuzene (l923b), summarizing a decade injection-to slll'vive the challenge with 20 times of study of humoral defenses in invertebrates, the dose of vibrio which had proved fatal to un­ found that the serum of the spider crab, Ma ia inoculated control crabs. s(juillado, possessed natural agglutinins for Cantacuzene also reported on studies of the mammalian red blood cells, with great individual responses of MII!a .'II/II i nado to injections of gram­ variation from crab to crab. He observed, rather positive bacteria isolated from the crab's diges­ significantly, that agglutinins weakened 01' dis­ tive tract. Inocul:ltion was followed by reduc­ appeared completely in crabs held in captivity tion in numbers of amebocytes and, within 24 hI', for long pel·iods. He noted, on injection of eryth­ by disappeanmce of most of the bacteria from rocytes, that agglutinins disappeared com­ the hemolymph. The bacteria were immobilized pletely during the first days after inoculation in the various phagocytic tissues, particularly and did not return to their original titer for in the branchial lacllnae-at first they adhered several weeks after the last dose. Cantacuzene to the cell surfaces, then amassed into small also reported that rare individuals of MaiOr-in­ granules, and were finally engulfed by fixed and variably moulting females-possessed a lysin for mobile phagocytes. The process of digestion of mammalian red blood cells. M aia serum also bacteria was slow, and still incomplete after 7 possessed a strong lytic factor, but no aggluti­ weeks. Clotting ability of the hemolymph de­ nins, against cholera vibrios. creased immediately after inoculation but re­ Cantacuzene inoculated Milia with coelomic turned to normal in 8 days. No agglutinins for fluid of SipIlIICU!US lIudus (4 to 5 injections at the bacteria could be demonstrated in vitro, but intervals of 3 to 5 days) and found that the crabs the natural agglutinins for mammalian red blood produced first agglutinins and then lysins against cells (discussed earlier) disappeared. In vitro the various injected sipuncu1id coelomic cells, studies disclosed that the hemagglutinin coated including" ova. The nature, intensity, and dura­ the bacteria but did not cause their agglutination. tion of response varied greatly among indivi­ The adhel'ence in vivo of the bacteria to fixed duals. The lytic ability seemed more pronounced phagocytic cells undoubtedly was enhanced by in females than in males, and more so in fe­ the sensitization. Some evidence for this was males approaching sexual maturity. Hemolysins gained by adding macerated hypodermal or peri­ against mammalian erythrocytes were also pro­ cardial cells to a mixture of bacteria and crab duced. serum in vitro. The cell fragments acted as When he compared the lytic ability of Maia centers for bacterial agglutination and immobili­ serum after injection with sipunculid fluid and zation, but the agglutinating ability was not mammalian erythrocytes, Cantacuzene found conferred on the serum by the addition of cell that response was more rapid and stronger with fragments. the former, and he concluded-on the hasis of A different sequence of events was described these and other studies-that mammalian red for those crabs (Milia) in \vhich the experimental cells were only mediocre antigens for marine in­ infections progressed to death. Early immobili­ vertebrates. He attributed this weak antige­ zation of bacteria in lacunar cells was followed, nicity to coating of the injected mammalian in 8 to 15 days, by the appearance of encapsu­ erythrocytes in Maia and other invertebrates lated forms. invulnerable to destruction by with a serum factor that interfered with sub­ phagocytes. The encapsulated bacteria multi­ sequent reactions. Cantacuzene aptly referred plied, phagocyte numbers were reduced, and the to this as "mummification" of the red cells by clotting ability of the hemolymph diminished. By invertebrate body fluids. 10 to 20 days after inoculation, the hemolymph Concerning acquired immunity to bacterial became incoagulable, the natural agglutinin for infections in Crustacea, Cantacuzene (l923b) red cells disappeared, the connective tissue be­ found that the crab Maia squinado, when inoc- came gelatinous, and the animal died.

464 SINDER~L\:\N, INTERNAL llEFENSI':S OF CRlSTACL\

Cantacuzene (1923b) also examinee! the in­ Agglutinins against vertebrate erythrocytes and ternal defenses of the hermit crab, EIIJlnUUrlls certain bacteria were found in sera of Homarus !lrideallxii, which he hae! reportee! earlier to vulgaris, Ellpagul'Ils !Irideauxii, and E. lierllhar­ possess strong hemolysins ane! strong antibac­ dus, but the same antigens were not agglutinated terial agglutinins. Injectee! gram-negative bacilli by sera of Cancer !Jagurus, Carcinus maenas, were entrapped and immobilized on the cell sur­ Portunus IlUber, or Galathea Jlunctata. The faces of the branchial lacunae, and then phago­ serum of EUlwgunls pridcauxii agglutinated cytized, as was the case with Mo ifL described mammalian red blood cells and amebocytes of previously. Maia and BIICcillllm, but did not agglutinate Cantacuzene (1928b) also reportee! that the the coelomic cells of sipunculids or ascidians. sera of crabs, Carcillils maenas, infectee! by the And so Cantacuzene set the scene, by the rhizocephalan Sa('culina, contained a factor ab­ early 1930's, for the continuation of broad and sent from normal crabs. Using a standard com­ elaborate studies of humoral internal defenses plement fixation test, with extract of the rhizo­ of marine invertebrates-particularly the Crus­ cephalan as antigen and with crab serum, tacea-but the stage, with only a few notable ex­ Cantacuzene (192f)b) was able to demonstrate ceptions, remained curiously empty and dark an antibody-like response in parasitized crabs. until the mid-1950's. Although research during Sheep cells were lysed in tubes with normal crab the last decae!e emphasized species other than serum but not in those containing parasitized those studiee! by Cantacuzene (except for the crab serum. Using a fine suspension of Saccll­ work of F. Bang), it reinforced many of Can­ lino. CantaCl1zime found precipitating and agglu­ tacuzene's flne!ings: that natural agglutinins tinating activity in the serum of parasitized and Iysins, with some specificity, occur in Crus­ crabs. The activity ,,"as not consistent, however, tacea and other invertebrates, and that respon­ in that some sacculinized crabs lacked it. In a ses to foreign antigens can be induced in selected concurrent study, Lt'vy (1923) found that macer­ invertebrates-responses which are only par­ ated sacculinids were toxic when injected into tially specific. The increased precision and crabs, but that no antitoxic activity could be dem­ quantitation of tests, and the careful attention onstrated in parasitized crabs. Both groups, to controls, have improved the quality of the normal and parasitized, died at about the same newer data, but have neither provided new con­ rate. cepts nor modified the general conclusions of In an earlier study Cantacuzene (1913) re­ Cantacuzene. The more recent literature on ported that inoculation of the sacculinid parasite humoral defenses of Crustacea will be summar­ with gram-negative bacteria resulted in septi­ ized by general categories in the following cemia and death of the parasite within 1 week, sections. and infection of the crab host by 10 days after inoculation. At about 5 e!ays after inoculation, Bactericidal Systems the crab hemolymph became incoagulable, and agglutinins appeared against the bacteria inoc­ Natural bactericidins have been reported from ulated into the parasite. The antibacterial a number of marine invertebrates (Bang, agglutinins were not present in sacculinized un­ 1967b). Recently, increase in titers of bacteri­ inoculated control crabs. cidal activity after inoculation with Formalin­ Appreciable evidence for some degree of spe­ ki lied bacteria was noted in West Indian spiny cificity of the natural agglutinins of Crustacea lobsters, Pan uli I'IlS arfJUS, and American lob­ was accumulated bv Cantacuzene. An agglutinin sters, HOII/aI'1l8 arnCl'iCanU8 (Evans et a!., 1968; in Maia against ~ammafjan red cells could be Acton, Weinheimer, and Evans, 1969). A bac­ absorbed from crab serum by certain gram-pos­ tericidal assay s,,'stem described by Schwab and itive bacteria but did not agglutinate them, nor Reeves (1966) was used to quantitate the degree did it agglutinate cholera vibrios. It did, how­ of response. ,In experiments with American ever, strongly agglutinate typhoid bacilli. lobsters held in seawater at 5° C, the peak of

465 FISHERY BULLETIN: VOL. 69, NO.3

bactericidal response against gram-negative ba­ The spiny lobster bactericidin was apparently cilli was reached within 48 hr after inoculation, a large molecule, as suggested by resistance to and high titers persisted throughout the obser­ dialysis and by Sephadex separations. Inacti­ vation period (11 days). In spiny lobsters, vation occurred at 65° C and activity was not presumably maintained at significantly higher restored by addition of unheated normal hemo­ environmental temperatures at Bimini, the lymph. Activity was not reduced by treatment Bahamas (reported as 26°_28° C in a later pa­ with EDTA or carageenin. These results indi­ per), the primary bactericidal response to intra­ cate dissimilarity with vertebrate complement­ cardiaIinjection of living orkilled suspensions of based bactericidal systems, but the authors sug­ the same gram-negative bacilli (originally iso­ gested that the bactericidin may represent a lated from the digestive tract of spiny lobsters) primordial immunoglobulin. reached a peak at about 36 to 48 hr after in­ Noteworthy is that all the American lobsters jection, then declined slowly for the following 2 and a number of the West Indian spiny lobsters weeks. Partial lack of specificity of the bac­ used in the studies by Evans, Weinheimer, Paint­ tericidin was indicated by its appearance follow­ er, Acton, and Evans ('1969) had demonstrable ing injection of gram-positive bacilli and by its pre-existing titers of bactericidins against the activity against Salmonella typhosa and Esch­ gram-negative bacillus used in the experiments. erichia coli, as well as against the unidentified Possibly the remainder of the spiny lobsters used gram-negative bacillus used as the homologous in the studies could have had titers of bacteri­ test organism. The bactericidin was not active, cidins lower than were demonstrable by the however, against Pseudomonas aeruginosa or methods used. Thus inoculation may not have against three species of gram-positive bacteria. "induced" the bactericidin but instead may have A subsequent study (Weinheimer, Acton, merely enhanced or increm:ed the titers. A num­ Sawyer, and Evans, 1969) of the specificity of ber of explanations were offered for the pre-ex­ the spiny lobster bactericidin further indicated isting titers of bactericidal activity, including that the response was partially nonspecific. Low trauma because of handling, and response to pre­ titers of the bactericidin against gram-negative vious bacterial infection. Two relevant obser­ bacilli could be demonstrated after injections of vations are that in many American lobsters, a Formalin-killed type 2 pneumococci and bovine rapid increase in bactericidal titer preceded serum albumin. Formalin produced a pro­ death, and in studies of SIJiny lobsters, the bac­ nounced adjuvant effect. tericidal activity was found to be partially non­ Secondary responses-those following rein­ specific, in that activity against other gram­ jection of the same antigen after a lapse of time negative bacteria was enhanced. -of spiny lobsters to killed suspensions of gram­ The conclusions reached by the research group negative bacilli were also examined by Evans, that examined the spiny lobster bactericidin Cushing, Sawyer, Weinheimer, Acton, and Mc­ (Weinheimer, Acton, Sawyer, and Evans, 1969) Neely (1969) . Titers of bactericidin were are in accord with findings for other phyla: slightly but significantly higher after reinocula­ "These data suggest that, although invertebrates tion than after primary inoculation, and the rate appear capable of antigenic recognition, the mol­ of secondary response (the number of hours to ecules synthesized may have broad specificity reach peak titer) seemed somewhat accelerated covering a wide range of antigenic determinants. when compared with the primary response. Un­ Further studi~s will be necessary to ascertain fortunately, the number of animals tested was whether these\ inducible substances represent small. It seems important that similar obser­ primitive immunoglobulins. In any event, it vations be greatly extended-since, as the au­ would be surprising if they did not have a major thors pointed out, the results were reminiscent role in defense of the animal against pathogenic of the specific anamnesis or immunological mem­ microbes." ory df'lmonstrable in the immunoglobulin An important qualification was pO!,nted out by responses of vertebrates. Aarum (1967) regarding results obta~ned by ex-

466 SI:\DFR:I,IA:\"i, I"iTER:\AL DEFEl'SES OF CRl:STACEA perimental inoculation of large numbers of bac­ rws, multiplies and causes death, was immo­ teria. Infectious agents are found in large bilized, agglutinated, and lysed in vitro by serum numbers in the circulatory system only in septi­ of several other crabs-particularly Maia squi­ cemia, which occurs only after a growth phase nado, E1lpagurus prideauxii, andPortuf1us Imber. of a virulent pathogen within the host, often Experimental inoculations of ciliates were within fixed or immobilized phagocytic cells. cleared usually within several hours. It is in­ Rapid increa~e in pathogen numbers in the cir­ teresting that the parasite, when experimentally culating body fluid is usually a precursor to death introduced in another member of the Portunidae, and indicates a failure of host defenses. Thus Pol'tlll1U,'; dejJllmtor, multiplied and killed the the artificial introduction of large numbers of experimental hosts just as it did in Carcinus microorganisms should not be expected to elicit maenas. Poisson attributed the reactions of normal responses in an animal. It should also crabs to the ciliate to expressions of a natural be noted that insusceptibility barriers to infec­ immunity, effected by agglutinating and lytic tion, operative in the normal animal, may be by­ activity of the hemolymph. passed by experimental inoculation, resulting in Other lytic systems of Crustacea have received massive infection and death from microorgan­ passing attention. Cantacuzene (1913) found isms not otherwise known as pathogens. A that hemolymph of the hermit crab, Ellpagurus further qualification mentioned by Aarum is that lJrideauxii, possessed a heat-lahile hemolysin, as even the simplest experimental manipulation well as precipitating and agglutinating activities. may change phagocytic abilities, so that the nor­ Cantacllzene (1921, 1923b) stated that injection mal phagocytic response of an animal to path­ of the spider crab, Maia s(juinado, with sheep ogens may be far different from responses elic­ erythrocytes produced hemolysins as well as ited experimentally. agglutinins.

Lytic Systems Agglutinating Systems A natural hemolysin for sheep erythrocytes Natural hemagglutinins of the Australian in the hemolymph of West Indian spiny lobsters freshwater crayfish, Pa /'Gchaemps b/carinatus, was reported by Weinheimer, Evans, Stroud, were examined by McKay, Jenkins, and Rowley Acton, and Painter (1969). Low temperatures (1969). Absorptions of hemolymph by erythro­ (0° and 4° C) inhibited lysis, which was best cytes of four mammalian and one avian species demonstrated at 25° and 37° C. Heating the disclosed specificity, in that absorption by cells lobster hemolymph to 52° C destroyed lytic ac­ of one species still left agglutinins for cells of tivity. The hemolysin could be adsorbed on red other species. The crayfish hemagglutinins were cells or cell stroma at low temperatures. Red nondialysable and were inactivated at 57° C. cells with the adsorbed lysin were lysed when In vitro studies with crayfish phagocytes and the temperature was raised to 37° C, which in­ mouse erythrocytes disclosed that the crayfish dicated a possible enzymatic type of activity of hemagglutinins greatly enhanced the adhesion this hemolytic system. The authors suggested of the erythrocytes to the phagocytes-a specific a multiple step system, analogous to mammalian opsonic effect. A similar effect was observed in hemolytic systems, consisting of a single protein vivo, apparently as a prelude to phagocytosis. species which is first adsorbed on the surface The recent studies support earlier reports of of the sheep erythrocyte, and then-in one or specific hemagglutinins in a number of inverte­ more steps-lyses the celt brate phyla (Tyler and Metz, 1945; Tyler, 1946; A detailed study of the presence or absence Sindermann and Mairs, 1959; Cushing, Cala­ of natural immunity to invasion of the hemo­ price, and Trump, 1963; Tripp, 1966) with spe­ lymph of crabs by a parasitic ciliate, Anophrys cificities somewhat comparable to those of verte­ saJ'cophaga, was reported by Poisson (1930). brate natural isohemagglutinins and with some The ciliate, which in shore crabs, Carcinus mae- biological properties (such as enhancement of

467 FISlIERY Bl:LU:TIN, VOL. 69, 1';0. J

adhesion and phag-ocytosis of erythrocytes by and to a lesser extent for types A and E, was phag-ocytes) similar to those of vertebrate anti­ reported by Cushing- (1967). A number of in­ bodies. dividuals lacker the hemag-g-Iutinin, and some of A study of natural ag-g-Iutinins in the serum of these possessed a serum factor which inhibited California spiny lobsters, Panulirus interruptus, the action in vitro of the positive sera. The for blood and sperm cells of 54 species (repre­ analogy to specific soluble substances found in senting 7 phyla) was published in 1945 by Tyler sera of certain vertebrates was pointed out by and Metz, and is still cited as one of the most Cushing, with the sug-g-estion that further studies comprehensive examinations of its kind for a of this kind with other inveitebrates might prove marine animal. Ag-g-Iutination was not found in instructive. Cohen (1968) found agg-Iutinins 37 other species tested. Titers for positive re­ for human and other vertebrate erythrocytes in actions ranged from 8 to 256. The most inter­ sera from coconut crabs, BII'{/lls latro. Young esting- phase of the study was an extensive series crabs lacked the agg-Iutinins. of absorptions of spiny lobster serum by cells The serum of the American lobster contains of many of the species tested. In each case, ab­ strong and specific natural agglutinins for an sorption of serum with cells of any single species antigen present on the red blood cells of sea removed agg-Iutinins for all the species tested herring, Clllpea harell.qlls (Sindermann and that belonged to the same g-roup (Class) but left Mairs, 1959). Detection of blood groups in this ag-glutinins for the cells of all other g-rou~s fish was aided by the use of the hemagg-Iutinin_ tested. Tyler and Metz concluded, on the baSIS individual herring either had the antigen or of absorptions, that at least 10 class-specific lacked it. Titers reached as high as 256, and agglutinins were present in the serum of the reactions paralleled those obtained with absorbed spiny lobster. A few cross-reactions occurred, rabbit antisera and plant lectins (Sindermann and some reduction in titers resulted from ab­ 1963). Erythrocytes of other clupeoid fishe~ sorptions with cells of other species, which sug­ tested were also strongly agglutinated by lobster g-ested the presence of a number of reacting sites sera (Sindermann, 1962). on the cells. Bang (1967b) examined the responses of the Precipitating Systems spider crab, Maia sl/uil1ado, to injections of A noph,'Ys, a large ciliate pathogenic for shore Production of precipitins by invertebrates has crabs Carcinus maenas, as part of a laudable been reported only rarely. Osawa and Yabuubhi attem'pt to repeat with modern methods some of (1963), in a very brief paper, found that the the early French studies of invertebrate de­ "Homard americain, Camba rus clarkil" [prob­ fenses. Sera of some spidel' crabs strongly ably HomarU8 amerl('ol/Us] did not produce agglutinated the ciliates, but that of others did agglutinins or lysins when injected with red not. Ag-glutination resulted from formation of blood cells but did produce weak lwecipitins (de­ a mucoid substance around the tail cilia. Crabs tectible by immunoelectrophoresis) after in­ that lacked the ag-g-Iutinin died from overwhelm­ ~ection with serum from rabbits and goats. No ing- infections of the hemolymph, whereas those mformation was given about dosage injection with the ag-g-Iutinin survived (Bang-, 1962). schedules, time for response, or titer~. When present, the ag-glutinin was apparently Stewart and Foley (1969) sugg-ested that fairly constant, except that in some crabs it "precipitin-like principle already present in th: was lost spontaneously but temporarily at time ~emolymp~" of the American lobster might be of molting-. Poisson (1930) had noted earlier InlJlO~·tant In removal of foreign protein. Fluo­ that the hemolymph of Mala lysed the ciliates rescein-labelled bovine. serum albumin (BSA) and that the hemolymph of the hermit crab, and lobster serum IJrotems were injected into lob­ FJ/lpa{/1I1'I1S llrideallxii, ag-glutinated them. sters held at 5° C, and the flUorescence II An agglutinin in the hemolymph of the hermit . d' eve checked per.1O lcally for 6 days. With lobster crab, PII{/lll'lstes 1l1/'eYI, for human type 0 cells, serum protems, an initial decline in fluorescence

468 SINDERMANN: INTERNAL DEFENSES OF CRUSTACEA within 2 hI' after injection was followed by a ance rate. Teague and Friou did not observe plateau that remained constant for the duration precipitin activity against injected bovine and of the experiment; with labelled BSA, however, human serum albumins but concluded that clear­ the rate of clearance was concentration depen­ ance resulted from nonspecific degradation of the dent, but the foreign protein declined to very low foreign protein. levels by 3 days after injection. Fluorescent Other evidence of clearance of injected pro­ material was apparently excreted in proportion teins but failure to induce heightened responsive­ to its disappearance from lobster hemolYmph. ness in Crustacea was reported by Campbell and Pinocytosis by phagocytes was not demonstrated, Garvey (1961). They mentioned that "It is also but tiny granular fluorescent accretions w. of interest that we have made many attempts observed in the hemolymph of lobsters injected to induce antibody formation in invertebrates, with labelled BSA, beginning about 8 hI' after in­ e.g., lobsters. We have been unsuccessful so far, jection. In vitro studies, in which a standard but in every instance the antigens remained un­ ring test with labelled or unlabelledBSA and digested and unchanged in the circulation and lobster serum was used, disclosed a clearly. dis­ tissues for many months." Although not men­ cernible ring after 16 hI' at room temperature, tioned specifically, the lobsters were probably and a precipitate at the bottom of the tube after California spiny lobsters and the test antigens 36 hr. Precipitin titers of individual lobster probably included BSA, since this was the prin­ sera ranged from 2 to 16 and were independent cipal antigen used in other studies reported in of the total protein concentration of the lobster the same paper. . serum. Dialysis of lobster serum did not change the titers, but precipitin activity was destroyed Phage Clearance by heating above 50° C. A number of interesting implications in the Taylor, Taylor, and Collard (1964) and Nel­ study were pointed out by Stewart and Foley. strup, Taylor, and Collard (1968) presented The clearance mechanism seemed able to dis­ some evidence (from two crabs) of an increase tinguish between foreign and native proteins, in the rate of secondary clearance of injected and the capacity for clearance seemed high, as T 1 bacteriophage in the shore crab, Curcinus indicated by accelerated clearance of larger doses maenus. Clearance was not complete until after of BSA. Attempts to increase the levels of pre­ 2 weeks at 16° to 18° C, and no neutralizing cipitin by previous injection of lobsters with antibody to T 1 phage was detected in the hemo­ BSA did not succeed, and in fact resulted in de­ lymph. Primary inoculation with To phage did creased precipitin levels in some individuals. not increase clearance rates for T 1 secondary The authors suggested that the hemolymph fac­ injections. The small number of animals used tor responsible for clearance of foreign protein in these experiments makes the conclusions may be maintained normally at low levels and highly tentative. The authors suggested the ex­ may be supplemented by further secretions when istence of a "phylogenetically more primitive required. They also suggested thatthe precipitin type of immune response than the production of principle in the hemolymph may be the first of humoral antibody," but did not state clearly what several steps or possibly the primary removal the response was-except possibly that it was factor and that digestion and excretion may take "an apparently purely cellular secondary re­ place elsewhere-probably in the hepatopan­ sponse." creas. Studies of phage clearance by Cushing and The results of the studies of Stewart and Foley McNeely (reported in Cushing, 1967) led to neg­ are in agreement with those of Teague and Friou ative conclusions. Phage T4 persisted for up (1964), who observed that injected foreign pro­ to 168 days in the California spiny lobster and tein was rapidly removed from the hemolymph disappeared at a steady rate, uninfluenced by of the crayfish Cambu1'us Vi1·ilis. Previous in­ the size of the original inoculum. Two species jection of the protein did not increase the clear- of crabs tested also failed to clear bacteriophage.

469 FISIlERY BCLLETIN: VOL. 69, 1';0. i

Other negative findings for increased rate of brates. Reaction on the part of invertebrates phage clearance following inoculation in cray­ could be identical to reaction against any other fish were reported by Teague and Friou (1964). introduced foreign material. Invertebrate responses to gram-negative bac­ terial endotoxins were the subject of a review Antitoxic Activity by Levin (1967). The most striking activity of such endotoxins is the production, after ex­ Little definitive information is available about perimental inoculation, of intravascular clots antitoxic activity in invertebrates. As Huff and the ensuing death of various crustaceans (1940) pointed out, "Experimental demonstra­ and other invertebrates. Antitoxic immunity tion of antitoxic action in invertebrates has has not been demonstrated, but, as Levin stated: failed for the most part because of lack of "Endotoxin appears capable of activating com­ susceptibility of invertebrate cells for known plementary defense mechanisms in inverte­ toxins." Probably the best example of antitoxic brates, including aggregation of amoebocytes, phenomena in Crustacea was described by coagulation, bacterial immobilization, and phago­ Cantacuzene (1925a) and Cantacuzene and cytosis. All these may be operative through Damboviceanu (1934a, 1934b). The hermit one type of cell-the amoebocyte." crab, EupagU1'1lS ]Jl'ideauxii, exhibited resist­ ance to nematocyst toxin of Adamsia palliata, a commensal coelenterate commonly found on Other Protective Systems the shell of the crab. When injected, the toxin had no effect on E. prideauxii, but it was lethal McKay and Jenkin (1969) examined resist­ to many other Crustacea and to a number of ance of the Australian freshwater crayfish, Par­ other invertebrates tested, including the closely achaent]Js bica/'illatlls, to a pathogenic Pseudo­ related hermit crab, E. bel'nhardus. Cantacuzime monas sp. and concluded that the animal was also found that serum of E. prideallxii could capable of an adaptive immune response. Their neutralize the coelenterate toxin when the two findings indicated lower mortality rates (after -serum and toxin-were mixed and injected in­ bacterial challenge) in animals inoculated with to crab species susceptible to the toxin. The heat- and alcohol-killecl vaccines as well as with development of this antitoxic principle can be enclotoxin. Inoculation with vaccines prepared seen as a logical and necessary concomitant of from other gram-negative bacteria also increased the very close relationship of crab and anemone, the level of resistance to the Pseudomonas in­ but the question of whether this is an example fection, but vaccines from gram-positive bacteria of innate or acquired resistance has not been did not-indicating some degree of specificity. resolved. A positive correlation was found between sur­ Another example of coelenterate toxin lethal to vival of challenged animals and the number of crabs was reported by Lane, Coursen, and Hines exposures to bacterial antigen; after four inoc­ (1961). Biologically active peptides in Physalia ulations, the LD,;o of immunized animals was nematocyst toxins were tested, using fiddler nearly 100 times that of controls. Temperature crabs, Uca pugilator, as assay animals. also played a significant role in the onset, degree, Except for the work with coelenterate toxins, and duration of protection induced by inocula­ evidence of antitoxins in invertebrates is weak. tion of animals with killed bacteria. At 26° C, Stauber (1961) reported almost immediate re­ onset of protection was rapid (1 day), reached moval of diphtheria toxoid from oyster blood, a peak at 3 clays, and almost disappeared by 12 but Metchnikoff (1905) and Bengston (1924) days; at 19°. C, onset was slower (2 days), found that tetanus and botulinus toxins remained reached a maxImum at 4 days, and persisted for in insect body fluids for several weeks without 12 days (the duration of the experiment)· at loss of toxicity. These studies must, of course, 14° C, no protection was afforded. InocUl~tion be viewed as most indecisive, since substances of gram-negative endotoxin resulted in protec­ toxic to humans are not necessarily so to inverte- tion similar in appearance and duration to that

470 SINDERMANN: INTERNAL DEH:NSES 01' CRCSTACEA produced by killed vaccines. Although the terms when challenged by Gaffkya homari-which is "immunity" and "resistance" were used, the pre­ thus far the only bacterial pathogen known to cise nature of the protection afforded by inocu­ develop systemic infections in lobsters and to kill lation of vaccines and endotoxin was not de­ them. Probably the most extensive series of scribed by the authors. In vitro experiments reports concerned with responses of inverte­ with hemolymphs of control and resistant cray­ brates to a particular pathogen is that dealing fish disclosed no bactericidal or bacteriostatic with the lobster (and other decapods) and the ~ffects, and McKay and Jenkin suggested that highly pathogenic gram-positive micrococcus the most important effect of immunization may G. homari. "Gaffkaemia"-the disease caused have been to increase the metabolic rate of the by G. homal'i-is enzootic in both the American phagocytes [thereby stimulating phagocytosis]. lobster, Homarus americanus, and the European Barker and Bang (1966), extending the ear­ lobster, H. 'Uulr]aris, and has been reported to lier studies of Cantacuzcne (1925b) with the cause epizootics in captive populations of both shore crab, Carrinus maenas, and its rhizoceph­ species (Roskam, 1957; Goggins and Hurst, alan parasite Sacculina carcini, reported that 1960; Gibson,1961; Stewart and Rabin, 1970). inoculations of Vibrio sp. caused the hemolymph Microorganisms with characteristics of G. hom­ of the parasite to become incoagulable within 24 ari have been isolated from shrimp (Penaeus hr. Masses of gelled material containing bac­ aztecus from the Gulf of Mexico) and from crabs teria were seen within body spaces. Septicemia (Carcinus rnaerws and Libinia ernarginata from and death, first of the parasite, and then often New England and Canrer il'roratus from eastern of the crab host, followed soon after. Canada), but the disease "gaffkaemia" is known Insusceptibility factors seem operative when only in lobsters. Early descriptions of the di­ certain parasites of invertebrates fail to develop. r.ease and its etiological agent (Hitchner and Michajlow (1938) and Baer (1944), for in­ Snieszko, 1947; Snieszko and Taylor, 1947) have stance, found that larval cestodes, Triaenophorus been followed during the past decade by studies and Ligula, penetrated the intestinal wall of a in several laboratories, which used the lobster number of copepods, but developed only in cer­ and the pathogen as a test system to elucidate tain species. In others, the larvae died and were responses to infection and other aspects of the phagocytized. Hedrick (1935) observed similar host-parasite relationship. The possible course differences in survival of larval nematodes. of infection in lobsters is summarized in Table l. Leger and Duboscq (1908) reported earlier that Snieszko and Taylor (1947) first satisfied Koch's sporogony of the sporozoan Agregata eberthi postulates for the pathogen and demonstrated (which occurs in the intestinal wall of crabs high mortality following inoculation of cultured of the genus Portunus) took place readily in all G. hamari. Stewart and MacDonald (1962) and species except P. Imber, in which the parasite Stewart et al. (1966) found that 40 to 60';:, was quickly phagocytized after invading the in­ of lobsters they examined from certain locations testinal wall. on the Canadian east coast were infected. Studies by Harvey Rabin at Woods Hole and The Johns Hopkins University (Rabin, 1965; INTERNAL DEFENSE MECHANISMS Rabin and Hughes, 1968) confirmed that lobsters INVOLVED IN GAFFKAEMIA inoculated with Gaffkya became septicemic with­ OF LOBSTERS in 2 days and died a few days later. Inoculation of gram-negative endotoxin 10 hI' before ex­ The American lobster, Homarus americanus, posure to the pathogen did not alter the course has an effective internal defense system, consist­ of infection. Prior inoculation of heat-killed ing of active phagocytosis as well as agglutin­ Gaffkya cultures (24 hI' before challenge) pro­ ating and bactericidal (or bacteriostatic) activ­ duced no protection. ity, against a number of injected bacteria. The In vitro studies with lobster serum as a cul­ protective system seems to fail completely only ture medium disclosed that Gaffkya growth was

471 FISHERY BULLETIN: VOL. 69, NO. J

TABLE i.-A proposed hypothesis to explain the course capture the lobster was free of the pathogen. of Gaffkya homari infections in lobsters at 15° c. The animal was inoculated twice with increas­

Day Development of~ gaff~aemia in lobsters ingly larger dosages of G. horrwl'i and cleared o Bacteria gain access to tissues of lobsters as a result of the bacteria within 6 days-but died on the 11th injury which destroys the integrity of exo~kelefon (or p~s­ day following the second challenge. The reac­ sibly the gut epithelium). lobster hemocytes phagocytize tions indicated a partial resistance and an ability G. homari. Phagocytized bacteria may multiply within hemocytes. Hemo­ in some individuals to recover from gaffkaemia. (ytes containing engulfed bacteria lodge in capillary and It is interesting that serum from this presum­ lacunar areas (heart.. hepatopancreas, gills) of the lobster. ably resistant lobster was similar to that of other mnri 4 Hemocytes may be disrupted, releasing G. ho . in hemo­ lobsters tested in that it did not inhibit growth lymph. This may result in rapid decrease ,10 hemocyte numbers and logarithmic increase in bacterial numbers. of G. homal'i in vitro. Hemolymph stimulates multiplication of released bacteria. 6 Rabin and Hughes statecl that the presence Hemocyte numbers seem to be gradually reduced by con­ of Gaffkyrt infections did not damage the clotting tinued phagocytosis and disruption of phagocytes. Clotting mechanism (release of coagulin from hemocytes) mechanism-an observation quite different from 8·10 is affected-possibly by reduction of hemocyte numbers­ that of Goggins and Hurst (1960), who found and clotting time is greatly prolonged. that reduction in amebocytes and a much pro­ Lobsters die from depletion of nutrient stores and utilization 12·14 longed clotting time were distinctive features of this material by Galf/.:ya. Injured gaffkaemic lobsters may bleed to death. (The possibility of exotoxin h~s not of the disease. Stewart et al (1969) and Stewart been entirely eliminated, but there is no present eVidence and Rabin (1970) clarifiecl these seemingly dis­ to suggest its existence.) parate observations by reporting that "coagulin" is released to initiate clotting by rupture of stimulated, while growth of a Vibrio (nonpath­ hemocytes and that the "concentration of plasma ogenic to lobsters) was usually i!lhibited. Serum proteins, including fibrinogen, does not appear to from lobsters which had been moculated 24 hr decline significantly in gaffkaemic lobsters." An earlier with killed Gaffkya still stimulated earlier report by Rabin and Hughes (19G8) growth of the pathogen in vitro. stated that when extract of lobster muscle was Rabin and Hughes (1968) tested resistance used as a coagulin source, recalcified clotting to Gaffkya in a valiety of studies ~it~ lobst~rs times wel'e the same in diseased and normal and other marine arthropods. Fmdmgs WIth animals. When these facts are combinecl, it can spider crabs (Libinia emar,qinata), rock. crabs be concluded that the abl10rmally and persistently (Cancer borealis), and horseshoe crabs (Llmulus low hemocyte content of the hemolymph results polyphemus) were that most.of the t:st anim~ls in ]wolonged clotting time and does not indicate cleared inoculated G. hamar'/. In VItro studIes any deficiency in plasma constituents other than with hemolymph disclosed either no apparent coagulin (Figure 3). effect or only slight inhibition of growth of the Studies carried on by James Stewart and his pathogen by sera of spider and horseshoe crabs, associates at the Halifax (Nova Scotia) Lab­ and a slight stimulation of growth by sera of rock oratory of the Fisheries Research Board of Can­ crabs. ada have extended the work of Rabin and have The possible role of exotoxin was tested in provided the greatest number of contributions lobsters by Rabin and Hughes with inoculation t~ the literature about the effects of Gafflcya of filtrates of G. homari cultures. The filtrate cllsease on lobsters. In accord with earlier stucl­ had no effect when it was injected into the ab­ ies, infections usually were fatal, although rare domen, but injection into the major joint of the individuals infected with Gaffkya-like organisms chela induced autotomy or abnormal movements did survive (Stewart et aI., 1966). in over 50 'It of the lobsters treated. Cornick and Stewart (1968a) provided con­ Evidence of resistance to gaffkaemia was siclerable relevant information about the host­ noted by Rabin and Hughes in a single lobster, parasite relationships of Ca!flcya ancl lobsters. which had been infected naturally before it was Experimental infections by inoculation, in which brought to the lahoratory. Twelve days after dosages as low as approximately!) bacteria per

472 SINDER~ANN: INTERl':AL Df<:fENSES Of CRCSTAO:A

500, genera), except for all strains of G. homari. 4.0 10"1 BACTERIAL NUMIIRI I I ...... Such agglutinins were of low titer, nondialy­ I I " i sable, inactivated at 56° C, and seemed to be non­ " ~ I ~ if 4001 5 a-j I (lOITlNG liME specific (as suggested by the limited observation •0 I I 3.0 :! I ~ I ~ 5 I I that a single absorption of serum by Flavobac­ • I <; I :; ~ I terium rna /'illllm removed agglutinins for all <; 300-1 ~ I E I I ~ / other bacteria tested except Brevibacterium sp.). -! ~ I . 61 I "2 2.0 z I I Cornick and Stewart's observations on phago­ e I I I § I 200.., cytosis of G. are interesting and war­ ~ 4~ I homari ~ ;;; I c I ;:: I I rant further investigation. They found no e I I I ~ phagocytized bacteria in hemolymph prepara­ 1.0 '" I OJ.. ~ 100-1 0 ~ I <; 21 tions 1S min after inoculation, but they did find •~ I I ~'" ftuorescent-dyp-Iabelled bacteria in hemocytes in I I heart, liver, and gill tissues of experimental lob­ 0 0 4 a 12 16 sters soon after inoculation. Circulating hemo­ DATI AITIR INlICIION cyte numbers were reduced significantly with­ in 1S min after bacterial inoculation but returned FIGURE 3.-Relations of hemocyte counts, clotting time, and bacterial numbers to time from experimental ex­ to normal levels after 5 hr. These data are in posure of lobsters to Gaffkya. (Redrawn from figures agreement with the statements of Maynard in Stewart, Arie, Zwicker, and Dingle (1969) and Stew­ (1960), that phagocytes which have engulfed art and Rabin, 1970.) foreign material lodge in capillary and lacunar areas of the crustacean body, resulting in re­ lobster at 15° C were used, killed 90~; of the duction in numbers of circulating hemocytes. test animals within 17 days. The absence of Bang (1%6) observed in tissues of Limulus in­ an effective host defense against Gaffkya was jected with gram-negative bacteria a similar re­ strongly indicated by the fact that the mean duction in circulating hemocytes. In Cornick time to death was almost constant, regardless and Stewart's study long-term infections of lob­ of dosage (Figure 4). Cornick and Stewart's sters were characterized by the presence of black studies disclosed additional facts that help to ex­ nodules containing G. homari in tissue cells in plain the pathogenicity of the bacterium to lob­ the gills, swimmerettes, and ventral abdominal sters: Gajjkya resisted digestion in phagocytes and multiplied in the hemolymph; growth of

Gaffkya was stimulated in vitro by serum of 25 lobsters while growth of several other bacteria Gaffkya was inhibited; and was not agglutinated ;;;- 20 0 by lobster serum, though all other bacteria tested e.< were agglutinated. I < 15 Presence in lobster hemolymph of effective de­ ~ fenses against bacteria other than G. homari was Q o 10 ~ o indicated by clearance within 30 days of inocu­ ;:: lated suspensions of Mic/'ococcus conIllomeratus, Z ~ M. sedentarius, Ach/'omobactcr thalassius, and ~ Gaffkya tetragena. Since several of these bac­ 0 teria are closely related to G. homari (which was 0 4 6 9 10 not cleared), some specificity of the phagocytic lOG DOSE (BACTERIA / KG WEIGHT) or humoral protective mechanisms is strongly indicated. FlGlilU: -t.-Relation of dosage of Gaffkya to mean time to death (MTIl) in 10bRters (calculated line of best fit Natural agglutinins in lobster serum were for llwan tillH' to death, uRing expprimental groups of demonstrated against all bacteria tested (six 10 lobsters each). (From Cornick and Stewart, 1968a.)

473 FISHERY BULLETIN, VOL. 69, NO.3

sinuses of the lobster. As Parry (1960) had mic individuals contains about 10 9 organisms per pointed out earlier, this type of aggregation in ml (Stewart, Arie, Zwicker, and Dingle, 1969); gills is a common phenomenon in Crustacea. and Gafjkya can be isolated consistently from lob­ Poisson (1930) observed that, in the few crabs ster pounds, sea water, bottom mud, and slime of (Carcinus maenas) resistant to the parasitic holding containers (Goggins and Hurst, 1960). ciliate Anophrys sarcopha.qa, masses of dead cil­ A thorough study of the effects of temperature iates occurred in branchial lacunae, pericardial on experimentally induced Gafjkya infections in sinus, and hepatopancreatic sinuses. Degener­ American lobsters was reported by Stewart ating ciliates eventually formed brownish cysts. Cornick, and Zwicker (l9(i9). Mean time t~ Cantacuzi'me (l923b) observed a similar phe­ death was inversely related to temperature nomenon in Maia squinado inoculated with bac­ (Figure 5). At 1 0 C no deaths attributable to teria. He pointed out that the lacunar tissue of experimental infections occurred; at 3 0 C mean the branchial lamellae of decapod crustaceans, time to death was 172 days; and at intermediate with its many fixed phagocytes, acts as an ex­ higher temperatures mean time to death de­ tensive and effective bacterial filter. creased drastically to a minimum of 2 days at Cornick and Stewart suggested that the devel­ 20 0 C (which approaches the upper lethal tem- opment of polysaccharide capsules by G. homari in later stages of infection could be an important device that prevented destruction of the phagocy­ tized bacteria and allowed multiplication of the pathogen. They pointed out, as evidence, that unencapsulated G. homari grown in culture were 175 actively phagocytized. As Stewart and Rabin 0, (1970) later reported, however, the unencapsu­ , I lated cultured bacteria were also virulent. It 150 I may be that the capsule forms soon after the , I, organisms are injected into the host. This ob­ , V> I servation of survival and growth of phogocy­ ~ 125 , tized encapsulated bacteria in lobsters is a direct .e. , counterpart of the inability of vertebrate phago­ I , cytes to destroy many encapsulated microor­ ~ , w 100 , ganisms, and parallels the earlier findings by 0 , CantacuzEme (1923b) of a fatal disease in crabs 0 , >- 0, induced by encapsulated gram-positive bacteria. w , ~ 75 , Stewart, Dockrill, and Cornick (1969) exam­ ;:::: , ined certain insusceptibility factors affecting z 0, GajJkya disease in lobsters. Destruction of the

474 SINDERMANN: INTERNAL DEFENSES OF CRUSTACEA perature for American lobsters). It is important lobster for its own readily available storage ma­ to note (in view of the very low seasonal tem­ terial." peratures of waters in which lobsters live na­ On the basis of experimental inoculations and turally) that at 1 0 C the pathogens persisted subsequent mortalities, Bell and Hoskins (1966) in the host in low numbers but with virulence suggested that Gofjkya might be pathogenic for unchanged, and they produced mortalities when the Dungeness crab, Cant~er magister, and the the temperature increased. Experimentally in­ shrimp Panda/us platyceros from the Pacific fected lobsters were also sensitive to and died coast. That observation could be important in from rapid increases or decreases in enviroll­ view of recent introductions of American lob­ mental temperatures-although the temperature sters (some possibly carrying Gajfkua) on the changes used in the experiments were probably Canadian west coast. Other experimental greater than those that would normally be ex­ studies (Cornick and Stewart, 1968b) indicated perienced in nature. that the bacterium may also be pathogenic for Findings in vivo were paralleled by in vitro east coast crabs (Cancer il'l'ol'atus, C. borealis, results of growth of Gojfkua in lobster serum­ and ]Juas coarrtatus). In vitro growth of the with a more rapid increase to a peak of bac­ pathogen in crab sera was similar to that in terial numbers with increasing temperature. lobster serum, suggesting susceptibility of the The organism grew in culture at all the exper­ crabs. However, agglutinins for G. hamari, imental temperatures (within a range of 1 0 to which were demonstrated in the sera of one of 20 0 C); at 1 0 C the bacterial growth curve was the crab species (C. il'l'IJratus) , might counteract erratic-it decreased in numbers to the 30th day, the favorable bacterial growth in vivo and reduce then a log increase progressed to the 60th day, the severity of infections in the crab. Cornick followed by a substantial decline. The impor­ and Stewart extended their observations by inoc­ tant observation, of course, is that G. hamari can ulations of C. irral'atus with suspensions of G. survive within the range of environmental tem­ homal'i. After 49 days the surviving crabs peratures experienced by American lobsters and (three) were found to be heavily infected (l09 that the pathogen causes mortality more rapidly organisms/ml hemolymph). Passage through as temperature increases. the crabs did not alter pathogenicity of Gajfkya A concurrent physiological and biochemical to lobsters. A repetition of the crab inoculations study by Stewart, Arie, Zwicker, and Dingle with larger numbers of crabs provided some evi­ (1969) and Stewart, Foley, and Ackman (1969), dence of greater mortalities in experimental in which an attempt was made to define features groups than in controls. Pathogenicity for rock of the infection that lead to death of lobsters, crabs was less than for lobsters, as indicated by produced several interesting results. The path­ a mean time to death of 42 days in crabs, against ogen lacked proteolytic, lipolytic, and fibrinolytic only 18 days in lobsters. The authors mentioned exoenzymes, suggesting that harmful effects are the possible role of rock crabs as reservoirs of not caused by direct destruction of tissue. The infection for lobsters. in view of reduced path­ authors observed that although in vitro growth ogenicity and prolong-ed mean time to death in of G. homari was limited by the carbohydrate crabs. level of the lobster serum medium used, pre­ From the foregoing, it is apparent that ex­ sumably such a level would be maintained in perimental studies with G. h011/O I"i have been nu­ vivo at the expense of other tissues. Drastic merous and varied and have provided significant reductions in hepatopancreatic glycogen and insights about the internal defenses of Crusta­ hemolymph nonprotein nitrogen characterized cea. Important areas for future study include later stages of the infection. No evidence of a determination of whether strains of the pathogen toxin was detected, and the conclusion was that with different virulences exist. and determina­ gaffkaemia is largely a wasting type of disease tion of whether virulence may be increased by -that death from the r1isease was "a result of rapicl passage through impounded lobster pop­ an unsuccessful competition on the part of the ulations.

475 FISHERY BULLETIN: VOL. 69, NO 3

DISCUSSION of Good and Papermaster's definition of adaptive immunity as the production of specific immuno­ In the development of information and prin­ globulins (a capacity which has been correlated ciples of invertebrate internal defenses, con­ with the occurrence of lymphoid tissue). Per­ sistent and entirely natural attempts have been haps their definition is too restrictive and rigid, made to translate findings into the concepts and since a number' of invertebrates do show re­ compartments constructed for the immune re­ sponses that protect them from pathogens (hence sponses of vertebrates. The effort has led to they are adaptive). some confusion of terminology and even to lack Earlier definitions of antibodies and immunity of agreement about definitions of immunity. allowed more latitUde for inclusion of inverte­ The concept of immunity in vertebrates has brate responses. Cantacuzeme (1923b), for ex­ been admirably stated by Good and Papermaster ample, considered as antibodies "... toute sub­ (1964), who define immunity precisely and nar­ stance albuminoide du plasma, douee ou non de rowly as "a biologic phenomenon embodying pri­ specifidte, qui, se fixant sur l'antigEme, modifie mary and secondary responses, with antibody les relations de contact de ce dernier, soit avec synthesis and release, reactions of immediate and les cellules, soit avec les ~lUtres constituants delayed allergy, and homograft immunity." chimiques des humeurs." They state: "To the time of writing [1964J, McKay, ,Tenkins, and Rowley (1969) stated adaptive immunologic responsiveness has not "... to allow comlnlrisons to be made between in­ been demonstrated in the invertebrates." They vertebrate phyla and the vertebrates ... the defi­ then define adaptive immune responsiveness as nitions of the immune response should be as "the ability to respond to antigenic material by broad as possible and emphasis placed on the production of specific combining substances, and functional aspects ...." These authors suggest to show an anamnestic res!Jonse to these same that such a definition of the immune response antigens on subsequent exposure." Their con­ might be "the ability of the animal to respond tinued exposition of immunity from the verte­ to a foreign particle (whether it be truly foreign brate point of view includes the following signifi­ or unwanted self) by the production of specific cant points: proteins capable of reacting with the inducer t~ 1. "Adaptive immunity ... is primarily a func­ and the resultant of this reaction leading phagocytosis." tion achieving full expression late in phylogeny and ontogeny." Cushing (1967), in an excellent summariza­ 2. "The lymphoid cell family is the primary tion of invertebrate immune mechanisms, stated cellular basis for adaptive immune res!Jonse in "There is a growing consensus of observations vertebrates ...." supporting the view that while vertebrates and 3. "The possibility that another cell system invertebrates may share some basic immune may mimic adaptive immune responses in an in­ competences such as 'innate immunities' and vertebrate species cannot be excluded at this phagocytic ceJls, it is indeed only within the time." vertebrates that the fuJI capacity of adaptive im­ munity exists." This statement seems reason­ A broader, more inclusive, concept of immu­ able, and fits the confines of Good and Paper­ nity has been suggested recently. Ifthe broader mas~er'snarrow definition of adaptive immunity, definitions of terms proposed by several authors but IS too negative if a broader perspective of are accepted, the words "immunity" and "im­ immunity-such as that proposed by McKay, mune response," rather than careful circum­ ,Jenkins, and Rowley-is adopted. locutions, can be used with invertebrates. As Probably greater emphasis should be placed an example, McKay and Jenkin (1969) stated on the adaldive aspects of invertebrate internal that the Australian freshwater crayfish was ca­ defense processes. Substantial numbers of pable of an "adaptive immune response." Such studies have indicated the existence of adaptive a capability is not possible within the confines responses to experimental inoculations of for-

476 SINDERMANN: INTERNAL DEFENSES OF CRCSTACEA

eign protein. Whether the response is in the adaptive immunity [in the vertebrate sense], form of specific immunoglobulin seems less sig­ namely specificity and memory, did not evolve nificant than the degree of protection afforded exclusively with the lower vertebrates." to the individual by the adaptive response. In It is obvious that modification, redefinition, or the broadest sense, the replacement of a pro­ replacement of some conventional immunological tective constituent of the hemolymph after its terminology-particularly toward broader defi­ utilization in preventing infection could be con­ niticns-is needed if the invertebrates are in­ sidered adaptive. For example, the precipitat­ cluded in comparative immunology. If we re­ ing factor for foreign protein found by Stewart move "antibodies" from invertebrate terminol­ and Foley (1969) in lobster serum (which de­ ogy we must also remove "antigen," since anti­ creases following experimental inoculation of body response is part of the definition of antigen. BSA and which is unreactive against injected An effective substitute for "antigen" (as sug­ lobster serum) would be adaptive. gested for insects by Hinton) (Chadwick, 1967) Considering immune responses of vertebrates would be "immunogen." Chadwick also sug­ and invertebrates, Good and Papermaster stated gested replacement of "antibody" with such that the presence in vertebrates of lymphoid tis­ terms as "natural bactericidal substance," "spe­ sue and cells constitutes a basic distinction. On cific inducible substance," and others. Further­ this basis, as Chadwick (1967) has pointed out, more, as stated earlier, it must be made clear "It is highly unlikely that insects [or Crustacea that when "lysins," "precipitins," "agglutinins," or other invertebrates] do produce mammalian and other humoral factors of invertebrates are type antibody, or that the mechanism of any discussed, identification with vertebrate factors acquired response to antigenic stimulus could be is not intended-the terms are used merely to likened to responses in higher animals in terms indicate the kind of activity produced (i.e., "lytic of the production of specific antibody globulins." substance or activity," "precipitating substance Analogous tissues and cells exist in a number of or activity," etc.) regardless of the physiological­ invertebrate groups, however, as do analogous biochemical mechanism (s) involved. humoral responses without the extreme specifi­ When suitably qualified the "safe" general cities of vertebrate globulins. Chadwick (1967) terms, therefore, include "resistance," "immu­ also stated that the "... immune response in an nity," "immune response"; terms that can have insect is not the consequence of an antigen-anti­ general applicability and utility, if accepted in body-globulin reaction but more likely the result a general sense, include "agglutinin," "lysin," of the production of some, as yet undefined, "precipitin"; specific vertebrate terminology, principle in insect hemolymph which may con­ not applicable to invertebrates includes "anti­ tribute to its resistance." The same statement body," "antigen," and "serologicaL" might be made about other invertebrates in Beyond the establishment of working defini­ which an induced response has been demon­ tions of immunity in invertebrates, it seems ap­ strated. propriate to list a number of generalizations that Although somewhat beyond the confines of the seem warranted by the admittedly narrow base present consideration of internal defenses of of evielence now available. Obviously, any gen­ Crustacea, it might be well to call attention to eralization about a group as evolutionarily di­ recent tissue transplantation work of Cooper verse as the invertebrates-or for that matter (1968, 1969a, 1969b, 19fi9c, 1969d) with an­ even of the Crustacea-must be in the form of nelids, which indicates a high degree of specifi­ a tenuous anel easily retractable hypothesis city of response and which suggests some sim­ (which may at times border on speculation), ilarities to vertebrate tissue graft responses. and the following statements are offered with Cooper's (1969d) concluding statement is sig­ these qualifications: nificant: "Further clarification of anamnestic responses to tissue transplants would confirm our 1. Resistance in the vertebrates seems pri­ views that at least two of the parameters of marily related to production of immunoglobulins

477 FISHERY BULLETIN: YOLo 69, NO. ] which combine with foreign protein to enhance toxic metabolites, and the utilization of nutrient the phagocytic process. Although specific im­ derived from the host. Temperature can also munoglobulins have not yet been demonstrated affect the rate of phagocytosis and the rate of in invertebrates, an analogous protein system, production of humoral defenses against infec­ of lower specificity but with functions similar to tion. Thus the progress of infection and the out­ vertebrate immunoglobulins, is suggested. Na­ come of disease represents a composite of en­ tural (and in some cases, partially specific) ag­ hancement or inhibition partly mediated by glutinins are common in invertebrate body fluids, temperature. and their titers in some species may be increased 6. Endotoxin has been found (in the verte­ by exposure to specific antigens. brates) to increase the metabolic rate of phago­ 2. Resistance in the vertebrates, and in some cytes and stimulate phagocytosis (Jenkins and invertebrates also, includes the prqduetion of Palmer, 1960; Whitby et aI., 1961). A similar bactericidins, Iysins, and agglutinins. The ap­ effect may be produced by endotoxin in the in­ pearance of, or the increase in, titers of such vertebrates. Thus, exposure to gram-negative factors in certain invertebrates following expo­ bacteria which are so abundant in the sea (or sure to foreign proteins may account in part for to their endotoxins) may increase the level of increased resistance to certain pathogens (Mc­ nonspecific resistance to other gram-negative or­ Kay and Jenkin, 1969; Bang, 1967b). ganisms or their endotoxins. This "nonspecific 3. Many of the bactericidal, bacteriostatic, immunity," which is also known in the verte­ lytic, and agglutinating properties seem to be brates (Rowley, 1956; Landy and Pillemer conferred on the hemolymph of invertebrates by 1956), may be of great significance in the in~ release of materials from hemocytes. The sub­ vertebrates-in fact, it may be the basic mech­ stances so released often seem not only less spe­ anism of internal resistance to bacterial path­ cific in their action than vertebrate antibodies, ogens in the invertebrates. but also the stimulation of release may be much 7. Handling and experimental procedures less specific. For example, the release of a lysin rapidly induce bacteremias in a number of in­ in sipunculids for the ciliate Anophrys can be vertebrates. Rabin (1965), for example, found stimulated by inoculation of certain bacteria that almost half of all American lobsters used (Bang, 1967~~), and the release of a hemolysin in his studies had bacteremias upon arrival in in Maia squinado for sheep red blood cells can the laboratory. Cornick and Stewart (1966) be induced by injection of sipunculid coelomic found that about 20'/( of a large sample of lob­ fluid (Cantacuzene, 1920a, 1923b). sters had bacteria in their hemolymph. Isolates 4. Immune response in invertebrates, as best were principally Micrococcus, Pseudomonas, exemplified in insects and crustaceans, is often BrevilJacterium, and Arhromobacter-bacteria rapid in onset and disappearance-usually a commonly found in the marine environment, and matter of a few days. apparently nonpathogenic for lobsters. The acts 5. As has been observed by a number of of capture, transport, and impoundment of these authors (Feng and Stlluber, 1968; Stewart, and other marine animals may produce stresses Cornick, and Zwicker, 1969), environmental and physiological changes (or mechanical dam­ temperature is a critical factor in the host-par­ age) that permit entry of microorganisms com­ asite relationships of invertebrates. Temper­ mon in the surrounding sea water. ature has been found experimentally to exert a 8. There is some limited evidence that the significant effect on the appearance, degree, and process of phagocytosis, fully elaborated in the duration of resistance to infection in certain in­ Protozoa, is enhanced in the vertebrates by non­ vertebrates (McKay and Jenkin, 1969), just as specific humoral factors which sensitize, agglu­ it does in poikilothermic verte1>rates (Bisset, tinate, immobilize, or otherwise increase the t946, 1917a, 1947b, 1948a, 1948b). Tempera­ susceptibility of proteins to phagocytosis by fixed ture affects the rate cf growth and reproduction and mobile phagocytic cells. Although the op­ of microorganisms, the rate of production of sonizing substances of invertebrates have not

478 SINDER~ANN: INTERNAL DEFENSES OF CRUSTACEA been adequately characterized and the mecha­ these unusual choices are effective antigens is nisms involved have not been adequately eluci­ obviously a most important consideration. dated, it may be speculated that in the verte­ Another extremely pertinent observation brates a greater degree of specificity of humoral made by Bang (1967b) was: "The limited factors has been added to the nonspecific mech­ amount of information [concerning immunolog­ anisms found in the invertebrates. ical responses of invertebrates] is, I believe, due The proper r'ole of specific vertebrate anti­ mainly to the limited number of studies, and not body as a possible augmentation of evolutionarily to any lack of imagination on the part of evo­ older nonspecific internal defenses was alluded lutionary forces in developing protective mech­ to by Miles (1962). He stated "... it is fairly anisms." clear that antibody per se has little effect on 11. One final and very significant thought was the viability or metabolism of microbes with proposed by Stauber (1961): "That so few which it combines. It is effective in defense examples of a~quired resistance are known either because it neutralizes toxins, or because among invertebrates may even be quite logical. it makes the microbe susceptible to non-specific Because of their relatively short generation defense factors like complement or the phago­ times, their usual small size and often enormous cyte .. .. We may then properly consider anti­ reproductive capacities, subsequent epizootics body as accessory to the more fundamental non­ would be much more likely to be circumvented specific defense mechanisms ...." It should be by the appearance of resistant stocks through emphasized, however, that much remains to be natural selection. . .. Even with very high mor­ learned about the nonspecific humoral factors tality rates a residual stock of animals under of vertebrates, as well as invertebrates. favorable conditions later might repopulate an 9. Brown (possibly chitinoid) bodies or cysts area ... , Ifthis reasoning is adequate to explain in gills characterize later stages of a number of the lack of evidence for the occurrence of ac­ crustacean diseases. The sequence of events, quired resistance in most of the invertebrates, after invasion by microorganisms, may include perhaps those invertebrates with a long life span, action of toxic or inhibitory factors in hemo­ like Limulus should be investigated more fully, lymph, accretion of moribund or dead invaders as likely hosts capable of demonstrating ac­ in gill lacunae, phagocytosis of dead organisms, quired resistance." and formation of nodules or cysts containing It is interesting to note that it is precisely dead organisms, and gradual phagocytic de­ those invertebrates with a long life span which struction of necrotic material. have received increased attention during the past 10. An important point, as Bang (1967b) several years, and that a few indications of mentioned, is that the probability of discovering acquired resistance have been reported. internal defense mechanisms is greater when di­ Information about the internal defenses of sease phenomena are studied under natural con­ crustaceans and other invertebrates may be ditions. In experimental work, microorganisms summarized as follows: pathogenic to marine invertebrates should con­ stitute test organisms of choice; microorganisms The weight of evidence indicates a major de­ found in the environment (and which may be fensive role in invertebrates for phagocytosis, facultatively pathogenic) should be next in order augmented by relatively nonspecific innate or acquired humoral factors. Preformed substances of preference; and microorganisms or proteins released into the hemolymph from granular which the marine animal is unlikely to encounter hemocytes seem to playa major role in humoral seem least instructive. There are valid experi­ defenses of Crustacea, and probably other in­ mental reasons, of course-such as the ease of vertebrates as well. Thus the body fluids of recognition of bacteriophages-that often lead many invertebrates contain natural bacterici­ to selection of test microorganisms other than dins, agglutinins, lysins, and occasionally pre­ pathogens or potential pathogens. Whether cipitins. Some limited evidence for augmenta-

479 FISHERY BULLETIN: VOL. 69. NO.3

tion of such defenses by exposure to foreign and Dr. Marenes Tripp, Department of Bio­ antigen exists. This evidence is primarily in logical Sciences, University of Delaware, New­ form of increased titers following experimental ark, Del. Impetus for this review was provided inoculation. Specific acquired antibodies (im­ in part by an examination of cellular and hu­ munoglobulins) have not been demonstrated in moral responses of shrimps and crabs being con­ invertebrates, but induced antibody-like activity ducted with Dr. Ben Sloan, Carson-Newman has been demonstrated in a few species. The College, Jefferson City, Tenn. Elizabeth Leon­ fundamental difference seems to be in degree of ard, Librarian, Tropical Atlantic Biological Lab­ specificity of response, which is significantly oratory, aided immeasurably in assembling higher in the vertebrates. It is obvious that the copies of the extensive body of literature on synergistic action of phagocytes and humoral which this review is based. factors in the invertebrates, as well as in the vertebrates, constitutes the significant defense perimeter-but the degree of specificity of the REFERENCES humoral components is lower in the inverte­ brates. The master internal defense plan seems AARONSON, S. to be: foreign protein + humoral factor = 1956. A biochemical-taxonomic study of a marine recognition of foreignness and phagocytosis. micrococcus, (;a!Jklla homari, and a terrestrial counterpart. .J. Cen. Microbiol. 15: 478-484. Cooper (1969c), concluding a very though­ AARIT:VI, G. R. provoking paper, stated: "It seems reasonable 1967. Fagocytose. Nor. Tannlaegeforenings Tid. to conclude that invertebrates do possess immune 77: 243-254. systems, although the nature of the mechanisms ACTON, R. T., P. F. WEINIIEIMER, AND E. E. EVANS. is decidedly unknown. Reactions may be as 1969. A bactericidal system in the lobster Homarus amcricnnus. J. Invertebr. Pathol. 13: 463-464. numerous as the varied taxonomic groups, as is ANDERSON, .J. I. W., AND D. A. CONROY. true of most rigorously studied vertebrates. In­ 1968. The significance of disease in preliminary vertebrate cellular immunity may be closer to attempts to raise Crustacea in sea water. Proc. vertebrate reactions and may represent the 3d. Symp. Mond. Comm. Off. Int. Epizoot. Etud. more primitive responses. In the absence of Mal. Poissons, Stockholm, Sep. No.3, 8 p. ARVY, L. classic vertebrate-type immunoglobulin in in­ 1952. Contribution it I'etude du sang et de la leu­ vertebrates, a real dichotomy would be evident copoiese chez Pachygrapsus marmoratus Fabr. in the evolution of immune responses. On the Ann. Sci. Nat. Zoo!. Bio!. Anim. 14: 1-14. other hand, immunoglobulin precursors may be ATKINS, D. present." 1929. On a fungus allied to the Saprolegniaceae found in the pea-crab Pinnotheres. J. Mar. BioI. Certainly the next decade will prove to be an Assoc. U.K. 16: 203-219. exciting one in the study of invertebrate internal 1954a. Further notes on a marine member of the defense systems. The components of classical Saprolegniaceae, Leptolegnia marina n. sp., in­ vertebrate immunity-present as analogues in fecting certain invertebrates. J. Mar. Bio!. Assoc. invertebrates, and probably varying widely U.K. 33: 613-625. 1954b. A marine fungus Plectospira dubia n. sp. among phyla-provide an excellent background (Saprolegniaceae), infecting crustacean eggs and against which new findings may be evaluated. small Crustacea. J. Mar. BioI. Assoc. U.K 33: 721-732. . ACKNOWLEDGMENTS 1955. PlIthiulr/ thalrlssium n. sp. infecting the egg­ mass of the pea-crab Pinnatheres pisum. Trans. Br. Myco!. Soc. 38: 31-46. The author would like to acknowledge with AUSTREG, J. thanks the helpful comments of Dr. Harvey 1952. Contribution it I'etude morphologique et Rabin, Institute for Comparative Biology, Zoo­ histochimique du sang- de Gammarus pulex L en logical Society of San Diego, San Diego, Calif.; fonction du sexe et de la mue. Bull. Soc. Z·ool. Dr. James Stewart, Halifax Laboratory, Fish­ Fr. 77: 231. BAER, J. G. eries Research Board of Canada, Halifax, N. S. ; 1944. Immunite et reactions immunitaires chez les

480 SINDERMA!\:N: Il\:TERNAL DEFENSES OF CRljSTACEA

invertebres. Schweiz. Z. Ailg-. Pathol. Bact. 7: BRUNTZ, L. 442-462. 1905. Etude physiologique sur les Phyllopodes BALBIANI, G. Branchiopodes. Phagocytose et excretion. Arch. 1886. Etudes bacteriologiques sur les Arthropodes. Zoo!' Exp. Gen. 4: :i7-48. C. R. Seances Acad. Sci. (Paris) 103: 952-954. 1907. Etudes sur les organes Iymphoides, phago­ BALL, G. H. cytaires et excreteurs des Crustaces superieurs. 1938. The life history of Careil10eetes he81JerU8 Arch. Zool. Exp. Gen. 7: 1-67. n. gen., n. sp., a gregarine parasite of the striped BULNlIEIM, H.-P. shore crab, Pachy.qrapsus crassipes, with obser­ 1967. Mikrosporidieninfektion und Geschlechtsbet­ vations on related forms. Arch. Protistenkd. 90: immung bei Gommorus duebeni. Zool. Anz. 30: 299-319. 432-442. BANG, F. B. BULNlI~;IM, H.-P., AND J. VAVRA. 1956. A bacterial disease of Limulus polyphemus. 1968. Infection by the microsporidian Octosporea Johns Hopkins Med. J. 98: 325-351. effeminans sp. n .• and its sex determining influence 1962. Serological aspects of immunity in inverte­ in the amphipod Gammm'us duebeni. J. Parasitol. brates. Nature (London) 196: 88-89. 54: 241-248. 1967a. Pathology Society Symposium: Def''llse CAMPBELL, D. H., AND J. S. GARVEY. Reactions in Invertebrates. Introduction. Ped. 1961. The fate of foreign antigen and speculations Proc. 26: 1664-1665. as to its role in immune mechanisms. Lab. Invest. 1967b. Serological responses among- invertebrates 10: 1126-1150. other than insects. Ped. Proc. 26: 1680-1684. CANTACUZENE, J. 1967c. Blood clot formation in the antenna of the 1912a. Sur certains anticorps naturels observes hermit crab Pa,qurus IOl1.qicarpus. Bio!. Bull. chez ElIpo,qnrus prideauxii. C. R. Seances Soc. (Woods Hole) 133: 456-457. Bio!. (Paris) 73: 663-664. 1968. Cellular clots in Pa(/1wus lon,qiearplis and the 1912h. Ohsf'rvations relatives it certaines proprietes possible role of microtubules. Bio!. Bul!. (Woods du sang- df' Cal'f'inllg mlleno.~ parasite par la Hole) 1:i5: 414. sacculine. C. R Seances Soc. BioI, (Paris) 74: 1970. Disease mechanisms in crustacean and ma­ 109-111. rine arthropods. In S. F. Snieszko (editor), A 1912c. Rechf'rches sur la production experimentale symposium on diseases of fishes and shellfishes, p. d'anticorps chez quelques Invertebres marins. 383-404. Am. Fish. Soc., Spec. Pub!. 5. C. R. Seances Soc. BioI. (Paris) 74: 111-112. BARKER, W. H., JR., AND P. B. BANG. 191:3. Sur Ia production d'anticorps artificiels chez 1966. The effect of infection by gram-negative bac­ Eu]!o.qllrlls prideauxii. C. R Seances Soc. BioI. teria, and their endotoxins, on the blood-clotting (Paris) 74: 293-295. mechanism of the crustacean Sacenlina carcini, 1919a. Etude d'une infection experimentale chez a parasite of the crab Carcinus maenas. J. Inver­ Ascidia mentula. C. R. Seances Soc. Bio!. (Paris) tebr. Patho!. 8: 88-97. 82: 1019. BELL, G. R, AND G. E. HOSKINS. 1919b. Anticorps normaux et experimentaux chez 1966. Experimental transmission of the lobster quelques Invertebres marins. C. R Seances Soc. pathog-en, Gaffk1/a homori, to Pacific crabs and Bio!. (Paris) 82: 1087-1089. prawns. Can. Soc. Microbiol. Abstr. 16th Annu. 1920a. Sur quelques reactions d'immunite chez les Meet., p. 21. InvertebnSs. Rev. Gen. Sci. 81" annee: 353-357. BENGSTON, I. A. 1920b. Formation d'hemolysins dans Ie serum de 1924. Studies on organisms concerned as causative Maia sqllinado inoculees avec des hematies de factors in botulism. Hyg. Lab. Bul!. 132: 1-96. Mammiferes. Existence dans ce serum d'une sub­ BISSET, K. A. stance antagoniste qui empeche ou retarde hem­ 1946. The effect of temperature on non-specific in­ olyse. C. R. Seances Soc. Bio!. (Paris) 83: 1515­ fections of fish. J. Patho!. Bacteriol. 58: 251-258. 1514. 1947a. Bacterial infection and immunity in lower 1921. Quelques remarques au sujet d'unp infection vertebrates and invertebrates. J. Hyg. 45: pxperimentale chez Maia squinado. C. R. Seances 128-135. Soc. BioI, (Paris) 84: 1007-1009. 1947b. Natural and acquired immunity in frogs 1923a. Cytolysines et cytoagglutinins provoquees and fish. J. Patho!. Bacterto!. 59: 679-682. par I'inoculation de Sipuncnlus nudus chez Maia 1948a. Natural antibodies in the blood serum of squinado. C. R Seances Soc. Bio!. (Paris) 89: freshwater fish. J. Hyg. 46: 267-268. 266-268. 1948b. The effect of temperature upon antibody 1923h. Le problpme de I'immunite chez les Inverte­ production in cold-blooded vertebrates. J. Patho!. bres. C. R Seances Soc. BioI. (Paris) 75th An­ Bacteriol. 60: 87-92. niversary 88 (Suppl,) : 48-119.

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