John T. Sullivan

MITOTIC RESPONSES TO EXTRACTS OF MIRACIDIA AND CERCARIAE OF SCHISTOSOMA MANSONI IN THE AMEBOCYTE-PRODUCING ORGAN OF THE SNAIL INTERMEDIATE HOST BIOMPHALARIA GLABRATA

Suspensions of miracidia and cercariae of Schistosoma mansoni were subjected to repeated freeze–thaw cycles and then injected into resistant Salvador strain Biomphalaria glabrata snails. A pronounced increase in the number of mitotic figures, relative to uninjected, sham-injected, or diluent (water)–injected controls, was observed in the amebocyte-producing organ (APO) at 3 days postinjection (PI). After centrifugation of miracidia freeze–thaw extract (FTE), the resulting supernatant (FTS) and pellet possessed equal stimulatory activity that was approximately half that seen with FTE. Ultracentrifugation of miracidia FTS resulted in a supernatant that retained full activity, indicating a soluble molecule. Heat treatment of miracidia FTE reduced but did not eliminate activity, suggesting a nonprotein active component. Concentration or dilution of FTS by a factor of 10 gave a nonlinear dose–response relationship. Susceptible NIH albino snails injected with miracidia FTE had increased mitotic activity in the APO, which was much less than that seen in Salvador snails, whereas injection of miracidia FTE into Helisoma duryi had no discernable effect. Measurement of mitotic activity as a function of time PI showed no increase in numbers of mitotic figures in the APO at 18 hr but a large increase at 24 hr PI. Mitotic activity returned to preinjection levels by 96 hr PI, although a subsequent increase occurred at 120 hr PI.

Haematopoiesis in molluscs: A review of haemocyte development and function in gastropods, cephalopods and bivalves

Haematopoiesis is a process that is responsible for generating sufficient numbers of blood cells in the circulation and in tissues. It is central to maintenance of homeostasis within an animal, and is critical for defense against infection. While haematopoiesis is common to all animals possessing a circulatory system, the specific mechanisms and ultimate products of haematopoietic events vary greatly. Our understanding of this process in non-vertebrate organisms is primarily derived from those species that serve as developmental and immunological models, with sparse investigations having been carried out in other organisms spanning the metazoa. As research into the regulation of immune and blood cell development advances, we have begun to gain insight into haematopoietic events in a wider array of animals, including the molluscs. What began in the early 1900s as observational studies on the morphological characteristics of circulating immune cells has now advanced to mechanistic investigations of the cytokines, growth factors, receptors, signalling pathways, and patterns of gene expression that regulate molluscan haemocyte development. Emerging is a picture of an incredible diversity of developmental processes and outcomes that parallels the biological diversity observed within the different classes of the phylum Mollusca. However, our understanding of haematopoiesis in molluscs stems primarily from the three most-studied classes, the Gastropoda, Cephalopoda and Bivalvia. While these represent perhaps the molluscs of greatest economic and medical importance, the fact that our information is limited to only 3 of the 9 extant classes in the phylum highlights the need for further investigation in this area. In this review, we summarize the existing literature that defines haematopoiesis and its products in gastropods, cephalopods and bivalves.

Involvement of protein kinase C signalling and mitogen-activated protein kinase in the amebocyte-producing organ of Biomphalaria glabrata (Mollusca)

Mechanisms that regulate hemocyte production in molluscs, at either the organismal or cellular levels, are not well understood. In the present study, 24-h saline cultures of the amebocyte-producing organ (APO) of the schistosome-transmitting snail Biomphalaria glabrata were used to test for the potential involvement of protein kinase C (PKC) signalling in hematopoiesis. Exposure to phorbol myristate acetate (PMA), an activator of PKC, resulted in an increase in the number of dividing hematopoietic cells in APOs from schistosome-resistant Salvador snails. PMA-induced cell division was blocked by treatment with U0126, an inhibitor of the mitogen-activated protein kinase kinase, MEK1/2. These results suggest that PKC-induced activation of the mitogen-activated protein kinase, ERK1/2, is involved in cell division in the APO.

Pathogen-associated molecular patterns activate expression of genes involved in cell proliferation, immunity and detoxification in the amebocyte-producing organ of the snail Biomphalaria glabrata

The anterior pericardial wall of the snail Biomphalaria glabrata has been identified as a site of hemocyte production, hence has been named the amebocyte-producing organ (APO). A number of studies have shown that exogenous abiotic and biotic substances, including pathogen associated molecular patterns (PAMPs), are able to stimulate APO mitotic activity and/or enlarge its size, implying a role for the APO in innate immunity. The molecular mechanisms underlying such responses have not yet been explored, in part due to the difficulty in obtaining sufficient APO tissue for gene expression studies. By using a modified RNA extraction technique and microarray technology, we investigated transcriptomic responses of APOs dissected from snails at 24xa0h post-injection with two bacterial PAMPs, lipopolysaccharide (LPS) and peptidoglycan (PGN), or with fucoidan (FCN), which may mimic fucosyl-rich glycan PAMPs on sporocysts of Schistosoma mansoni. Based upon the number of genes differentially expressed, LPS exhibited the strongest activity, relative to saline-injected controls. A concurrent activation of genes involved in cell proliferation, immune response and detoxification metabolism was observed. A gene encoding checkpoint 1 kinase, a key regulator of mitosis, was highly expressed after stimulation by LPS. Also, seven different aminoacyl-tRNA synthetases that play an essential role in protein synthesis were found to be highly expressed. In addition to stimulating genes involved in cell proliferation, the injected substances, especially LPS, also induced expression of a number of immune-related genes including arginase, peptidoglycan recognition protein short form, tumor necrosis factor receptor, ficolin, calmodulin, bacterial permeability increasing proteins and E3 ubiquitin-protein ligase. Importantly, significant up-regulation was observed in four GiMAP (GTPase of immunity-associated protein) genes, a result which provides the first evidence suggesting an immune role of GiMAP in protostome animals. Moreover, altered expression of genes encoding cytochrome P450, glutathione-S-transferase, multiple drug resistance protein as well as a large number of genes encoding enzymes associated with degradation and detoxification metabolism was elicited in response to the injected substances.

Effect of crude lipopolysaccharide from Escherichia coli O127:B8 on the amebocyte-producing organ of Biomphalaria glabrata (Mollusca)

Lipopolysaccharide (LPS) is a pathogen associated molecular pattern (PAMP) to which the internal defense system (IDS) of both vertebrates and invertebrates responds. We measured the mitotic response of the hematopoietic tissue of the schistosome-transmitting snail, Biomphalaria glabrata, to crude LPS from Escherichia coli 0127:B8. In a dose-response study, snails were injected with a range of concentrations of crude LPS, and mitotic figures were enumerated in histological sections of amebocyte-producing organ (APO) fixed at 24h post-injection (PI) following a 6h treatment with 0.1% colchicine. In APOs from Salvador strain snails, which are genetically resistant to infection with Schistosoma mansoni, LPS concentrations of 0.01 mg/ml and above triggered a large increase in mitotic activity, whereas in APOs from schistosome-susceptible NIH albino snails, concentrations of 0.1mg/ml elicited a much smaller, but statistically significant increase. A time course study, without colchicine treatment, revealed that in Salvador APOs the mitotic response to 0.1mg/ml occurred by 18 h PI, peaked at 24h, and returned to control levels by 72 h; NIH albino APOs showed no detectible response. When Salvador APOs were exposed to crude LPS in vitro, no increase in mitotic activity occurred, a result suggesting the possible requirement for a peripheral tissue or hemolymph factor. The increased cell proliferation induced by crude LPS represents a novel systemic response of an invertebrate IDS to one or more PAMPs from a Gram-negative bacterium.

Hematopoietic tissue allografts in Biomphalaria glabrata (Mollusca: Pulmonata) induce humoral immunity to Schistosoma mansoni.

We examined a potential mechanism for adoptively transferred resistance against Schistosoma mansoni in schistosome-susceptible snails receiving allografts of the hematopoietic amebocyte-producing organ (APO) from a schistosome-resistant strain of Biomphalaria glabrata. Susceptible NIH albino snails first were implanted with the APO from resistant Salvador strain donors. At 14 days post-implantation, cell-free plasma was isolated from APO recipients and injected into a second group of NIH albino snails. Controls were injected with plasma from NIH albino snails that previously had received implants of Salvador mantle, an immunologically inert tissue. Finally, plasma recipients, along with untampered (non-injected) controls, were exposed to miracidia of S. mansoni and subsequently monitored for infection. A significantly lower prevalence of infection occurred in the group injected with plasma from APO recipients (53%) in comparison with that in untampered controls (100%) or in snails receiving plasma from mantle recipients (97%). These results suggest that adoptively transferred resistance in B. glabrata results from secretion of soluble resistance factor(s) by the implant, rather than, or in addition to, formation of a hemocyte chimera having cytotoxic donor hemocytes.

Activation of an innate immune response in the schistosome-transmitting snail Biomphalaria glabrata by specific bacterial PAMPs

Injection of crude lipopolysaccharide (LPS) from Escherichia coli into the hemocoel of Biomphalaria glabrata stimulates cell proliferation in the amebocyte-producing organ (APO). However, it is not known if mitogenic activity resides in the lipid A or O-polysaccharide component of LPS. Moreover, the possible role of substances that commonly contaminate crude LPS and that are known to stimulate innate immune responses in mammals, e.g., peptidoglycan (PGN), protein, or bacterial DNA, is unclear. Therefore, we tested the effects of the following injected substances on the snail APO: crude LPS, ultrapurified LPS (lacking lipoprotein contamination), two forms of lipid A, (diphosphoryl lipid A and Kdo2-lipid A), O-polysaccharide, Gram negative PGN, both crude and ultrapurified (with and without endotoxin activity, respectively), Gram positive PGN, PGN components Tri-DAP and muramyl dipeptide, and bacterial DNA. Whereas crude LPS, ultrapurified LPS, and crude PGN were mitogenic, ultrapurified PGN was not. Moreover, LPS components, PGN components, and bacterial DNA were inactive. These results suggest that it is the intact LPS molecule which stimulates cell division in the APO.

Effects of abnormal temperature and starvation on the internal defense system of the schistosome-transmitting snail Biomphalaria glabrata.

Climate change may affect the internal defense system (IDS) of freshwater snails, and as a result their capacity to transmit disease. We examined effects of short-term exposure to supra- and sub-optimal temperatures or starvation on 3 parameters of the IDS of the schistosome-resistant Salvador strain of Biomphalaria glabrata - hemocyte concentrations, cell division in the amebocyte-producing organ (APO), and resistance to infection with Schistosoma mansoni. Adult snails were exposed to 1 of 3 temperatures, 20°C, 27°C (controls), or 33°C, for 1 or 2weeks, with food. A fourth group was maintained at 27°C, but without food. Compared to the controls, starved snails had significantly higher hemocyte counts at both 1 and 2weeks, although mitotic activity in the APO was significantly lower at both time periods. Exposure to 20°C or 33°C for 1 or 2weeks did not affect hemocyte numbers. However, APO mitotic activity in snails exposed to 20°C was significantly higher at both 1 and 2weeks, whereas mitotic activity in snails exposed to 33°C was significantly lower at 1week but normal at 2weeks. None of the treatments altered the resistance phenotype of Salvador snails. In a follow-up experiment, exposure to 33°C for 4-5h, a treatment previously reported to both induce expression of heat shock proteins (Hsps) and abrogate resistance to infection, caused immediate upregulation of Hsp 70 and Hsp 90 expression, but did not alter resistance, and Hsp expression levels returned to baseline after 2weeks at 33°C. Results of this study indicate that abnormal environmental conditions can have both stimulatory and inhibitory effects on the IDS in adult B. glabrata, and that some degree of acclimation to abnormal temperatures may occur.

Altered Gene Expression in the Schistosome-Transmitting Snail Biomphalaria glabrata following Exposure to Niclosamide, the Active Ingredient in the Widely Used Molluscicide Bayluscide.

In view of the call by the World Health Organization (WHO) for elimination of schistosomiasis as a public health problem by 2025, use of molluscicides in snail control to supplement chemotherapy–based control efforts is likely to increase in the coming years. The mechanisms of action of niclosamide, the active ingredient in the most widely used molluscicides, remain largely unknown. A better understanding of its toxicology at the molecular level will both improve our knowledge of snail biology and may offer valuable insights into the development of better chemical control methods for snails. We used a recently developed Biomphalaria glabrata oligonucleotide microarray (31K features) to investigate the effect of sublethal exposure to niclosamide on the transcriptional responses of the snail B. glabrata relative to untreated snails. Most of the genes highly upregulated following exposure of snails to niclosamide are involved in biotransformation of xenobiotics, including genes encoding cytochrome P450s (CYP), glutathione S-transferases (GST), and drug transporters, notably multi-drug resistance protein (efflux transporter) and solute linked carrier (influx transporter). Niclosamide also induced stress responses. Specifically, six heat shock protein (HSP) genes from three super-families (HSP20, HSP40 and HSP70) were upregulated. Genes encoding ADP-ribosylation factor (ARF), cAMP response element-binding protein (CREB) and coatomer, all of which are involved in vesicle trafficking in the Golgi of mammalian cells, were also upregulated. Lastly, a hemoglobin gene was downregulated, suggesting niclosamide may affect oxygen transport. Our results show that snails mount substantial responses to sublethal concentrations of niclosamide, at least some of which appear to be protective. The topic of how niclosamide’s lethality at higher concentrations is determined requires further study. Given that niclosamide has also been used as an anthelmintic drug for decades and has been found to have activity against several types of cancer, our findings may be of relevance in understanding how both parasites and neoplastic cells respond to this compound.

Fucoidan stimulates cell division in the amebocyte-producing organ of the schistosome-transmitting snail Biomphalaria glabrata.

Adult Salvador (schistosome-resistant) strain Biomphalaria glabrata snails were injected with 5 μl of 10 mg/ml solutions of the sulfated polysaccharides λ carageenan, dextran sulfate, fucoidan, and heparin, the nonsulfated polysaccharide laminarin, and the monosaccharides L-fucose and L-galactose, and mitotic activity in the amebocyte-producing organ (APO) was measured in histological sections at 24 h post injection. Among the substances tested, only fucoidan induced elevated mitotic activity. Desulfated fucoidan was not mitogenic, indicating that sulfate groups are required for activity. Schistosome-susceptible M-line snails possessed minimal or no hematopoietic tissue in their APO, which did not respond to fucoidan. Immersion of juvenile Salvador snails in 1 or 10 mg/ml solutions of fucoidan for 3 h did not elevate mitotic activity at 24 h post immersion, suggesting that the external and digestive tract epithelia of B. glabrata are impermeable to this molecule. These results provide support for the hypothesis that fucosylated glycans on the tegument and in excretory-secretory products of sporocysts of Schistosoma mansoni are in part responsible for increased mitotic activity in the APO of B. glabrata infected with this trematode or injected with its extracts.