Judith E. Humphries

Cellular Receptors and Signal Transduction in Molluscan Hemocytes: Connections with the Innate Immune System of Vertebrates

Abstract The involvement of circulating hemocytes as the principal cellular effector mediating molluscan immune responses is well established. They participate in a variety of internal defense-related activities including microbial phagocytosis, multicellular encapsulation, and cell-mediated cytotoxicity reactions that are presumed to be initiated through foreign ligand binding to hemocyte receptors and subsequent transduction of the binding signal through the cell resulting in appropriate (or in some cases, inappropriate) hemocyte responses. At present, however, although functional evidence abounds as to the existence of hemocyte “recognition” receptors, few have been characterized at the molecular level. Similarly, signal transduction systems associated with various receptor-mediated hemocyte functions in molluscs are only beginning to be investigated and understood. This review examines what is currently known about the molluscan hemocyte receptors and the putative signal transduction pathways involved in regulating their cellular behaviors/activities. The cumulative data implies the presence of various hemocyte-associated receptors capable of binding specific carbohydrates, extracellular matrix proteins, growth factors, hormones, and cytokines. Moreover, receptor-ligand interactions appear to involve signaling molecules similar to those already recognized in vertebrate immunocyte signal transduction pathways, such as protein kinases A and C, focal adhesion kinase, Src, Ca2+ and mitogen-activated protein kinase. Overall, the experimental evidence suggests that molluscan immune responses rely on molecules that share homology with those of vertebrate signaling systems. As more information regarding the molecular nature of hemocyte recognition receptors and their associated signaling molecules is accumulated, a clearer picture of how hemocyte immune responses to invading organisms are regulated will begin to emerge.

Receptor-ligand interactions and cellular signalling at the host-parasite interface

Although the effects of trematode infection on snail host physiology or host responses on parasite development have been well described in the literature, very little is known regarding the underlying mechanisms and specific molecules responsible for mediating those effects. It is presumed that many host-parasite interactions are communicated through receptor-mediated events, in particular those involving haemocytic immune responses to invading parasites, larval motility and migration through host tissues, and larval acquisition of host molecules either as nutrients or critical developmental factors. The intent of this chapter is to review current knowledge of molecules (both receptors and their ligands or counter-receptors) involved in molecular communication at the interface between larval trematodes, especially the mother or primary sporocyst stage, and host cells/tissues in intimate proximity to developing larvae. Information to date suggests that the molecular exchange at this interface is a highly complex and dynamic process, and appears to be regulated in specific cases. Topics discussed will focus on snail cell receptor interactions with the sporocyst tegument and its secretions, host cell-cell and cell-substrate adhesion receptors and their related signal transduction pathways, and sporocyst tegumental surface receptors and ligands involved in the binding of soluble host molecules.

Protein kinase C regulation of cell spreading in the molluscan Biomphalaria glabrata embryonic (Bge) cell line

Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMA-induced expression of activated MAPK, suggesting that PKC-, Ras- and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek.

Infectivity and growth of Echinostoma revolutum (Froelich, 1802) in the domestic chick

Infectivity and growth studies in domestic chicks were carried out on a strain of Echinostoma revolutum isolated from Lymnaea elodes snails in Indiana, U.S.A. Of 21 chicks, each fed 40 +/- 10 cysts of Echinostoma revolutum, 16 (64%) were infected with a total of 269 (32%) worms from approximately 840 cysts. Worms were found only in the ceca and rectum at 2-14 days p.i. In vivo excysted metacercariae were obtained in the lower ileum and ceca at 4 h p.i. Excysted metacercariae averaged 0.2 mm in length and 0.02 mm2 in body area. Worm length averaged 1.3 mm on day 6, 2.3 mm on day 8 and 3.6 mm on day 14. Mean body area averaged 0.29 mm2 on day 6, 0.62 mm2 on day 8 and 1.93 mm2 on day 14. Worms first became ovigerous on day 12. Growth of E. revolutum in the chick was delayed compared to previous findings on E. trivolvis, a closely related species of 37-collar-spined echinostome in the E. revolutum complex.

Neuromusculature — Structure and Functional Correlates

The adult and developmental stages of most trematodes, including the echinostomes, are highly motile and often undergo quite elaborate behavioural patterns, mediated it is assumed by well-developed sensory modalities and neuromuscular control systems for host attachment, invasion and migration. The application of immunocytochemistry and the use of the phalloidin-fluorescence staining method in conjunction with confocal scanning laser microscopy have revolutionised the provision of detailed information on neuronal pathways and muscle organisation, respectively, in trematodes and other flatworms (Halton and Gustafsson, 1996; Mair et al. 1998a). Moreover, motility studies in vitro have provided some preliminary data on the somatic motor systems of these parasites, with respect to the actions of known neuroactive substances (Pax et al., 1996; Halton et al.,1997). Research on the neuromusculature of trematodes holds the prospect of identifying pharmacologically important targets that may be exploited in the development of novel anthelmintics in the near future. Since echinostomes, such as Echinostoma caproni, are readily maintained in the laboratory (Fried and Huffman, 1996), they would seem to offer admirable model material for experimental study of the developing neuromuscular systems in trematodes. This fact has only just begun to be exploited, and the chapter herein presents what is largely unpublished and preliminary data on the structure and functional correlates of the nerve-muscle systems of adult and the developmental stages of E. caproni.

Effects of Larval Schistosomes on Biomphalaria Snails

The aim of this chapter is to provide a clearer understanding of the complex relationship between Biomphalaria snails and larval schistosome parasites. This chapter describes the numerous changes in host physiology, biochemistry, and behavior brought about by infection. Specifically, the effects of larval schistosomes on host mortality, growth, metabolism, reproduction, organic and inorganic elements, and behavior are focused on. The chapter concentrates on three Biomphalaria species; B. glabrata, B. alexandrina, and B. pfeifferi and one species of schistosome, Schistosoma mansoni.

Identification of nuclear factor kappaB (NF-κB) binding motifs in Biomphalaria glabrata.

Biomphalaria glabrata acts as the intermediate host to the parasite, Schistosoma mansoni, and for this reason, the immune system of B. glabrata has been researched extensively. Several studies have demonstrated that the transcriptome profile of B. glabrata changes following exposure to a variety of pathogens, yet very little is known regarding the regulation of gene expression in this species. Nuclear factor kappaB (NF-κB) homologues have recently been identified in B. glabrata but few functional studies have been carried out on this family of transcription factors. The aims of this study therefore were to identify NF-κB binding sites (κB motifs) in B. glabrata and examine them via functional assays. Two different κB motifs were predicted. Furthermore, the Rel homology domain (RHD) of a B. glabrata NF-κB was able to bind these κB motifs in EMSAs, as well as a vertebrate κB motif.

Characterization of a Toll-like receptor (TLR) signaling pathway in Biomphalaria glabrata and its potential regulation by NF-kappaB

HighlightsA highly conserved Toll‐like receptor (TLR) signaling pathway exists in B. glabrata.NF‐&kgr;B‐binding sites were predicted upstream of genes for TLR pathway components.Several putative &kgr;B‐binding sites were specifically bound by Rel homology domain.B. glabrata TLR pathway may be partially regulated by NF‐&kgr;B.B. glabrata NF‐&kgr;B may self‐regulate its activity via regulation of I&kgr;B expression.

Cholinergic, serotoninergic and peptidergic components of the nervous system of Haematoloechus medioplexus (Trematoda, Digenea), characterised by cytochemistry

Cholinergic, serotoninergic and peptidergic neuronal pathways have been demonstrated in whole-mount preparations of the frog-lung digenean trematode, Haematoloechus medioplexus, using enzyme cytochemical methodologies and indirect immunocytochemical techniques in conjunction with confocal scanning laser microscopy. All 3 classes of neuroactive substance were found throughout both central and peripheral elements of a well-developed orthogonal nervous system. Peptidergic immunoreactivity was particularly strong, using antisera directed to native flatworm neuropeptides, neuropeptide F, and FMRF amide-related peptides (FaRPs), and there was significant overlap in the staining with that for cholinergic components. The serotoninergic system appeared quite separate, with the staining localised to a different set of neurons.