Locomotion and feeding of Acanthamoeba at the water-air interface of ponds.

Acanthamoeba trophozoites attach to and effect amoeboid locomotion at the water-air interface of ponds. Their locomotory rate (approximately 0.8 microm s(-1)) and manner of independent movement at this interface is similar to that over solid substrata. Adhesion forces developed between amoebae and the water-air interface are greater than gravity and thus amoebae are also transported passively without detachment. Amoebae docked with the water-air interface remain and flourish here as they are shown, by using green fluorescent protein-labelled Aeromonas hydrophila, to feed on bacteria that occur at the interface, digesting them intracellularly.

The species specificity of Bulinus-Schistosoma interactions.

Despite certain inherent drawbacks, advances have been made recently in furthering our understanding of Bulinus-Schistosoma interactions. By highlighting these, Terry Preston and Vaughon Southgate hope to stimulate further research into this neglected but important area.

Purification of an agglutinin from the haemolymph of the snail Bulinus nasutus and demonstration of related proteins in other Bulinus spp.

The snail Bulinus nasutus 1214 possesses a potent haemagglutinin (end-point titre with human erythrocytes, 2(-18)) in its cell-free haemolymph which also binds to the miracidia (but not other larvae) of the incompatible parasite Schistosoma margrebowiei. We have purified a protein possessing this haemagglutinating property from the plasma of this snail. The native Mr of this protein was estimated by SDS polyacrylamide gel electrophoresis to be 210 kDa; under denaturing conditions in a 7.5% PAGE gel it ran as a major band of 135 kDa. Proteins of similar Mr were also found in the haemolymph of 16 other Bulinus spp. (the major intermediate hosts of human and veterinary schistosomiasis in Africa) although the plasma of none of these agglutinated human erythrocytes. Nonetheless, Cleveland mapping of the Mr 135 kDa bands from these different Bulinus spp. revealed 4 identical major peptide fragments (30, 28, 19 and 16 kDa) in each, thus demonstrating a similarity in the primary structure of these plasma proteins. Antisera from Balb/C mice immunized with the 135 kDa polypeptide from Bulinus truncatus 1521 cross-reacted in Western blots with the 135 kDa band of other members of the same truncatus/tropicus species complex but not with species from the africanus or forskalii species groups.

Evidence for the expression of actomyosin in the infective stage of the sporozoan protist Eimeria.

A high-speed supernatant extract was obtained from infective oocysts of Eimeria tenella homogenised in a sucrose-low ionic strength buffer. Immunoblotting showed this soluble, micropore-filtered preparation (designated E1) to be rich in actin. E1 underwent superprecipitation on addition of ATP but not its non-hydrolysable analogue AMP.PMP--behaviour typical of an actomyosin solution. The superprecipitate fluoresced strongly in the presence of rhodamine-phalloidin (indicative of the presence of F-actin) and electron microscopy of negatively-stained preparations of this flocculent matter confirmed the abundance of filamentous material within it. This is the first demonstration of a functional actomyosin isolated from a member of the economically important phylum Apicomplexa.

Biological characteristics of the Calliphora vomitoria agglutinin.

The galactose specific agglutinin from Calliphora vomitoria was found to be expressed in the haemolymph of all the larval instars, but could not be detected at any other time during the life cycle. The haemagglutinating activity was insensitive to wounding of the tegument or injection of saline; however, a significant increase in haemagglutinating titre could be induced upon inoculation of the haemocoel with biotic or abiotic particulate material. The agglutinin also actively agglutinated several bacterial species and appeared capable of playing a role in particle--haemocyte interaction. The presence of the purified agglutinin significantly increased the attachment of fetuin-derivatized beads to haemocytes in vitro, and this activity could be specifically reduced by the addition of galactose, suggesting that the agglutinin may act as an opsonin.

The in vitro transformation of the miracidium to the mother sporocyst of Schistosoma margrebowiei; changes in the parasite surface and implications for interactions with snail plasma factors.

The in vitro transformation of the miracidium to the mother sporocyst of Schistosoma margrebowiei was initiated by placing the miracidium in mammalian physiological saline. The transformation occurs in stages: the cilia cease beating; the ciliated plates become detached from the intercellular ridges and underlying muscle layers; the intercellular ridges spread over the body surface eventually forming a new tegument; the sporocyst changes from an ovoid to a tubular shape in about 48 h at room temperature. The surfaces of the miracidium, sporocyst and cercaria of S. margrebowiei display stage-specific carbohydrates on their surfaces as indicated by lectin staining. Ricin120 stains the cilia alone of the miracidium whereas peanut agglutinin stains the larval surface except for the cilia. The intercellular ridges of the miracidium stain with concanavalin A and wheat germ agglutinin, and these lectins stain the entire surface of the mature mother sporocyst. The cercaria is the only larval stage which stains positively with asparagus pea lectin. Bulinus nasutus is incompatible with Schistosoma margrebowiei; the haemolymph of this snail contains an agglutinin which agglutinates a wide variety of mammalian erythrocytes including those of human ABO blood groups. The haemagglutinin titre of B. nasutus plasma is reduced after incubation with miracidia of S. margrebowiei indicating that the agglutinin is absorbed onto the surface of this larval stage but not that of the mother sporocyst or cercaria. The possible roles of agglutinins in host-parasite interactions together with the significance of the differences in the surface carbohydrates of the larval stages are discussed.

Functional studies on Calliphora vomitoria haemocyte subpopulations defined by lectin staining and density centrifugation.

Haemocyte subpopulations of Calliphora vomitoria have been categorized by their surface staining properties using fluorescently labelled lectins, and their mobilities in Percoll density gradients. These methods of identification were exploited to determine the roles of these cell types in cellular defence reactions. Soybean agglutinin clearly defined the cell subpopulation involved in phagocytosis, while purified thrombocytoid fragments proved to be the main haemocyte population involved in encapsulation and nodule formation.

Purification and characterization of a galactose-specific agglutinin from the haemolymph of the larval stages of the insect Calliphora vomitoria.

A lectin was isolated from the haemolymph of the blowfly larva Calliphora vomitoria. It agglutinated a variety of mammalian erythrocytes with varying specificities and was strongly inhibited by D-galactose and fetuin. The activity was also sensitive to chelators of metal ions, heating above 50 degrees C and proteolytic digestion. SDS-PAGE identified a glycoprotein with an Mr of 32,000 under reducing and nonreducing conditions which resolved to a band at pH 5.4 using isoelectric focusing. Using FPLC gel filtration the activity was isolated in a fraction with an Mr of 130,000. It is suggested that the native form of the molecule is a noncovalently associated tetramer.

Amoeboid locomotion of Naegleria gruberi: the effects of cytochalasin B on cell-substratum interactions and motile behavior.

The major manifestations of amoeboid locomotion in Naegleria-cytoplasmic streaming, pseudopod production, cell polarity and focal contact production-require that the actin-based cytoskeleton be extremely dynamic. Whether these features are causally linked is unclear. In an attempt to answer this question we have used the fungal product cytochalasin B (cyt B) to dissect the motility process. This drug can perturb the organisation of actin filaments both in vivo and in vitro. Essentially cyt B acts as a molecule which can cap the barbed ends of actin filaments. Not surprisingly, therefore cyt B has an effect on rates of actin polymerization and the dynamic state of actin in the cytoplasm. We have found that cyt B has a profound effect on focal contact production and breakdown. Within minutes of addition of cyt B focal contact production ceases, existing focal contacts are stabilised but cytoplasmic streaming and pseudopod production are not blocked. In conclusion it is now clear that the state of actin required for focal contact production is different from that required for pseudopod extension and cytoplasmic streaming.

Effect of disruption of plasmatocyte microfilaments on encapsulation in vitro.

Very little is known about the functional basis of plasmatocyte motile behaviour. In this study we documented the effects of cytochalasin B (CB) on the cytoskeletal organisation and behaviour of plasmatocytes during spreading on a planar surface in vitro, and during in vitro encapsulation. CB produced a reversible and dose-dependent effect on the rate of plasmatocyte spreading. Moreover, incubation of fully spread plasmatocytes in CB caused their microfilaments to coalesce and the cells to arborise and eventually revert to the less adhesive spindle-shaped morphology characteristic of free plasmatocytes in circulation. The use of cytochalasins B and D on haemocytes encapsulating cotton fibre loops in vitro resulted in the development of abnormally flocculent capsules. The data demonstrate the importance of microfilament-dependent PL motility, both during the secondary "compaction phase" of encapsulation and in the laboratory manifestation of this process, spreading on a planar substratum.

A prominent microtubule cytoskeleton in Acanthamoeba.

A method for preparing by detergent extraction the cytoskeletons of substrate-attached, motile Acanthamoeba castellanii is described. A monoclonal antibody to yeast alpha tubulin has been used to demonstrate the presence of abundant microtubules in the cytoskeleton of this amoeba by fluorescence and whole-mount electron microscopy. Individual microtubules, often more than 10 micron long, interweave to form a well-developed 3-D network pervading the cytoplasm and embracing the nucleus. In some cases immunofluorescent staining reveals distinct nodes in the perinuclear region of this microtubular network.

Amoeboid locomotion of Acanthamoeba castellanii with special reference to cell-substratum interactions.

The amoeboid locomotion of Acanthamoeba castellanii has been studied by observation of individual cells moving on a planar glass substratum. Cell-substratum interactions involved in traction have been observed by reflexion interference microscopy. A variable part of the ventral surface of A. castellanii formed a protean platform, the 'associated contact', from which filopodia were subtended; these established stable, focal adhesions (approximately 0.4 micron diameter) on the substratum beneath. Surprisingly, acanthopodia, a prominent feature of this protozoon, did not play an obvious role in traction. The dimensions of the cell-substratum gap in the associated contact could be modulated by the concentration of ambient electrolyte. Dilution of electrolyte from 50 mM-KC1 to 2mM resulted in (i) an increase in the cell-substratum gap, (ii) a marked decrease in cell motility, (iii) reduced cell adhesion to glass.

Cell-substrate interactions in amoeboid locomotion - a matched reflexion interference and transmission electron microscopy study.

Cell-substrate separation distance were measured on Naegleria gruberi amoebae moving in deionized H2O on an untreated glass substratum (weakly adhesive) and on a polylysine treated glass surface (strongly adhesive). The values obtained by transmission electron microscopy on fixed cells and reflexion interference microscopy on live cells were in broad agreement.