Phyllis T. Johnson

The coelomic elements of sea urchins (Strongylocentrotus) III. In vitro reaction to bacteria

Abstract The interactions of coelomocytes of Strongylocentrotus purpuratus and Strongylocentrotus franciscanus with various marine bacteria and one terrestrial insect pathogen were followed in hanging drops. Coelomocytes responded to the gram-negative species used by early regression of leukocytes from the bacterial area; by walling off bacteria through leukocyte clots; by congregation of red spherule cells in “palisades” on the edges of leukocyte clots; and by encapsulation of masses of bacteria by leukocytes and red spherule cells. A naphthaquinone pigment, echinochrome, which is present in red spherule cells, can be released by them in the presence of bacteria. Also, in the presence of red spherule cells, motile species of bacteria rapidly lose motility. The gram-negative species were seldom phagocytized although the two species which caused the most cell reaction would stick to cytoplasmic strands of aggregated phagocytic leukocytes. Gram-negative species were usually disposed of by lysis or by killing followed by no further obvious reaction. Two of the gram-negative species tested, including the insect pathogen, elicited practically no response from the coelomocytes; one caused a slight reaction; and three caused immediate and marked response. The gram-positive bacteria used elicited marked phagocytosis, some species more than others, but there was little other demonstrable reaction on the part of coelomocytes. Vibratile cells apparently release an acid mucopolysaccharide which immobilizes foreign material including microorganisms entering the coelom through wounds in the gut; probably they also release the mucin in bacteria-infected hanging drops.

The coelomic elements of sea urchins (Strongylocentrotus). I. The normal coelomocytes; their morphology and dynamics in hanging drops.

Abstract The cells found in the coelom (coelomocytes) of Strongylocentrotus purpuratus , the purple sea urchin, and Strongylocentrotus franciscanus , the red sea urchin, can be maintained for long periods in hanging drops. The morphological changes, clotting mechanisms, and in vitro reactions undergone by the various coelomocyte types can be followed with ease. The four cell types present are phagocytic leukocytes, vibratile cells, red spherule cells, and colorless spherule cells. On contact with a surface, the phagocytic leukocytes change from bladder amoebocytes to flattened syncytial cells. Within the liquid they form permanent aggregations composed of cells which do not lose the plasma membranes to form plasmodia. Other cell types are caught passively in the aggregations. Apparently only the phagocytic leukocyte is involved in cellular clotting, and extracellular gelling of the coelomic fluid was not observed. A special lysing cell type which initiates clotting was not found despite suggestions in the literature that cell aggregation in echinoids is dependent upon such a mechanism. Under adverse conditions small aggregations of cells will form plasmodia but in large clots only the surface cells form a syncytial membrane while plasma membranes of the interior cells appear to remain intact. It has been though that the function of vibratile cells is to mix the fluid and cells of the capacious and nonmuscular echinoid coelom. In hanging drops, vibratile cells are not efficient in mixing the fluid or in moving cells from one place to another. However, both in vivo and in vitro they will release a portion of a mucoid compound present in their cytoplasm when stressed by intrusion of fluid from the gut into the coelom or hanging drop. The temporary semigel thus formed may operate in limiting spread of gut fluid or other foreign material through the coelom.

The coelomic elements of sea urchins (Strongylocentrotus)

SummaryThe four coelomocyte classes of the red sea urchin,Strongylocentrotus franciscanus, described by light-microscope studies, are confirmed and the fine structure described. Material examined included fresh, non-aggregated cells; partially aggregated ones that had been heldin vitro up to four days; and aggregated cells heldin vitro for 40 days. Leukocytes from youngin-vitro preparations differed from most fresh leukocytes by having enlarged dense nucleoli and enlarged rough endoplasmic reticulum, which was often filled with secretion, and sometimes connected to the perinuclear cisterna. Leukocytes held 40 daysin vitro were mainly plasmodial. Unlike cells held a limited timein vitro, the 40-day leukocytes had nuclei much like those in fresh preparations.The three classes of spherule-bearing cells (vibratile cells, red spherule cells, and colorless spherule cells) differed greatly in ultrastructure, and varied in appearance according to the fixative and pH present during fixation. Vibratile-cell spherules were of biphasic construction, suggesting the condition of certain vertebrate mast cells. Red spherule cells occurred in two forms. The most common form in fresh preparations had “despherulated”,i.e., lacked material in the spherules; and the spherules of the second type were filled with either granular or homogeneous material. Colorless spherule cells had evenly and finely granular material in the spherules. Colorless spherule cells were uncommon or missing in material that had been heldin vitro. Certain unidentifiable spherule cells occurred in some preparations.Although samples are small, it is notable that in May and June, recognizable glycogen was present only in leukocytes that had been heldin vitro, not in any fresh cells. Glycogen occurred in fresh cells of all classes from samples taken in December and February (during or shortly before the normal spawning season ofS. franciscanus). Unlike the cells in fresh preparations made in May, June, and December, fresh leukocytes and vibratile cells taken in February often had extremely lobed nuclei and considerably developed rough endoplasmic reticulum.

Comparative studies on the in vitro response of bacteria to invertebrate body fluids: II. Aplysia california (sea hare) and Ciona intestinalis (tunicate)☆

Abstract Five species of gram-negative, obligately marine bacteria, one facultatively marine gram-positive micrococcus, and a strain of the gram-negative terrestrial Serratia marcescens were used in the tests. They were incubated in vitro in cell-free and whole coelomic fluid of the sea hare Aplysia californica and the tunicate Ciona intestinalis. Both fluids of Aplysia showed little or no effect on the bacteria. Both fluids of Ciona killed two of the obligately marine species and depressed numbers of two of the remaining three, but did not affect either the gram-positive micrococcus or the gram-negative terrestrial rod. There is a discussion of the possibility that the antibacterial substances in the coelomic fluids of marine invertebrates would be more likely to affect the common gram-negative, obligately marine bacteria than the rarely encountered gram-positive and terrestrial gram-negative forms.

Comparative studies on the in Vitro response of bacteria to invertebrate body fluids. I. Dendrostomum zostericolum, a sipunculid worm☆

Abstract Six species of marine bacteria, five gram-negative rods and one gram-positive micrococcus, plus a strain of the terrestrial gram-negative rod Serratia marcescens , were tested. They were incubated in vitro in cell-free coelomic fluid and whole coelomic fluid from the sipunculid worm Dendrostomum zostericolum . Incubation with seawater alone and in a mixture of seawater and bovine albumin were used as controls. The whole fluid was strongly depressive or fatal to all the bacteria except the grampositive micrococcus and S. marcescens . Cell-free fluid was less depressive or fatal in its effects. There is discussion of the possibility that some of the depressive effects of the sipunculid fluids might be similar to the effect of incubation in seawater alone. Attributes of the various bacteria that might make them susceptible or resistant to the effects of the fluids are discussed also.

In vitro studies on Patiria Mininata (Brandt) coelomocytes, with remarks on revolving cysts

Abstract Coelomic fluid from several Patiria miniata , pooled in test tubes and maintained at 4°C, will form sheet-like cell clots in about 24 hours. The clots probably begin as monolayer syncytia on the lower walls of the test tubes and later contract and thicken. The clots are reversible and in 48–96 hours, cells making up the clots begin to scatter away from each other. One-fourth of the hanging-drop preparations made from the clots and kept at 4°C stayed in good condition for three weeks. Only one type of coelomocyte occurs regularly in Patiria and probably in most other asteroids as well. It is a phagocytic amebocyte. In normal starfish the coelomocytes have bladder-like ectoplasmic extensions; under conditions which induce clotting the coelomocytes become filiform amebocytes; and finally, when present on a clot edge or when applied to a surface such as a coverslip, they develop broad, ruffled or crinkled pseudopodia. Carmine-filled coelomocytes were withdrawn from Patiria inoculated 74 models capable of yielding basic information applicable to many echinoderm groups. Origin and function of the revolving cysts remain obscure. We know some cysts contain phagocytic elements which may be coelomocytes, and the cysts must gain their propellent flagella from one of the flagellated tissues adjacent to the coelomic cavity, perhaps by phagocytes partially ingesting flagellated cells. In vitro , coelomocytes do engulf flagella-bearing cells and the flagella may remain beating for at least some time after the parent cell is incorporated into the phagocyte. Single flagella with the attendant small bits of matter must also be phagocytized as foreign bodies, for they are not normal to the coelomic fluid. If formed by coelomocytes in conjunction with many separate flagellated cells, the cyst-like nature of the completely formed bodies might be induced by the same forces which form the zooids of Volvox and related flagellated colonial protozoans. On the other hand, some uncolored cysts occured in coelomic fluid where nearly all the single coelomocytes had particles of carmine in the cytoplasm, casting doubt on the invariable presence of free coelomocytes in the cysts 1 . The occurence of cysts in coelomic fluid just removed from apparently normal Patiria deserves comment. In the process of taking starfish from their environment some tube feet are usually torn and other wounds may occur even when great care is taken. These breaks might release bits of the flagellated epithelium into the coelom where by some process they form into the revolving cysts. It is just as likely that wounds normally acquired would accomplish the same end. Judging from the long life of revolving cysts in hanging drops, one might assume they would last as long or longer in the coelomic cavity since days earlier with carmine, indicating inert foreign material may remain in this species of starfish at least 74 days. Spherical multinucleate macrophages measuring up to 275 microns in diameter occured in the coelomic fluid of carmine-inoculated starfish. They also were present in fresh clots prepared from coelomic fluid of normal starfish. Flagellated cells, minute flagellated corpuscles, and variously-shaped crystal-like bodies were found in coelomic fluid withdrawn from normal starfish. These were probably present adventitiously and were aspirated into the needle from coelomic lining or pyloric caeca. Spherical revolving cysts were found in the coelomic fluid of normal starfish. They sometimes contained coelomocytes as well as flagellated cells but we could not ascertain if they always included coelomocytes. They were not ciliates or abnormally-developing larvae. Sperm were not involved in their formation. At least part of their origin must be in cells or groups of cells from the flagellated coelomic lining. They maintained their integrity and continued revolving up to 73 days in hanging drops, even after all other cells in the drop were lysed or granular, and even in the presence of numerous apparently nonpathogenic bacteria.

Infection with diatoms and other microorganisms in sea urchin spines (Strongylocentrotus franciscanus)

Abstract Naviculoid diatoms, red and green algae, and probable blue-green algae were found on the surface of the regenerating spine tips of an abnormally pale Strongylocentrotus franciscanus . Within the tissues of the tip were unidentified multicellular or plasmodial organisms, wormlike forms, fungi, and ciliates. Echinochrome-bearing material in granular and amorphous forms invested many of the internal organisms. These granules were also found in the actively regenerating area, and isolation of heavily infected areas by the echinochrome-bearing substance, with repair by leukocyte-like cells, was taking place. Evidence presented in this paper and elsewhere indicates that the repair cells were the same as the sclerocytes of the spine and possibly related to coelomic leukocytes. It is suggested that the echinochrome-bearing material that occurs in regenerating areas of normal spines as well as in infected ones, may act as a “general disinfectant.” Two other specimens of S. franciscanus , neither particularly pale, were found to have infected spines. In addition to organisms like those in the pale spines, the diatom Licmophora flabellata colonized the surface, and blue-green algae bearing a resemblance to Oscillatoria , and large numbers of diatoms, Navicula sp., occupied the internal tissues. The Navicula is related to a species found in a brown alga. This is the second report of diatoms inhabiting animal tissues.

Abnormal epithelial growth in sea urchin spines (Strongylocentrotus franciscanus)

Abstract Abnormal dark-red tissue swellings found on the spines of Strongylocentrotus franciscanus are composed of red spherule cells and cells that appear similar to those of the spine matrix. They may be formed in response to settling and attachment of microorganisms on the spines. Detachment of parts of the swellings may serve to rid the spines of these microorganisms. The despherulation of the echinochrome-bearing, red spherule cells on the unprotected surface of the swellings could provide substances that protect the cells from invasion by algae, etc. In a different type of tissue “growth,” which was accompanied by a veillike layer of collagen-containing connective tissues, the spherule cells apparently were acting in the production of collagen.

Population crashes in the bean clam, Donax gouldi, and their significance to the study of mass mortality in other marine invertebrates☆

Abstract The bean clam, Donax gouldi, occurs in large, dense populations on exposed sandy ocean beaches. Individual populations of this species are subject to periodic sudden and complete extinction. Four populations of D. gouldi occurring on beaches ranging from Los Angeles, California to Ensenada, Baja California were kept under surveillance for a 2-year period. During this time no population crashes were observed, nor was there evidence of disease in the clams except for the presence of trematode larvae in otherwise healthy individuals. Colonies of the clams maintained in the laboratory lived varying amounts of time but each colony eventually underwent swift and total mortality in much the same manner as do natural “crashing” populations. The time of onset of general mortality in a laboratory colony did not depend on the conditions under which it was kept, the age of the clams, or their place of origin. It is suggested that since members of a population are in a similar physiological state they respond to environmental pressure more as a unit than as individuals. The possibility that some “epizootics” in other marine invertebrates, such as cultured oysters, may be a manifestation of unit reaction is discussed.

Natural bacterial flora of the bean clam, Donax gouldi

Abstract Morphological and physiological studies of 47 bacterial isolates from the digestive gland of Donax gouldi, the bean clam, indicated the predominance of gram-negative, polar monotrichous, fermentative rods of the genus Vibrio. Environmental factors apparently influence the variety of bacteria present in healthy clams from two sites, but intrinsic factors are considered more vital in the selection of the gut flora. The significance of the uniformity of bacterial types in the digestive tract of various marine animals is discussed.