Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection

Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.     

Differences in esterase frequencies in five strains of Biomphalaria glabrata (Say)

Bair R. D. and Etges F. J. 1973. Differences in esterase frequencies in five strains of Biomphalaria glabrata (Say). International Journal for Parasitology, 3: 43–46. Vertical polyacrylamidegel electrophoresis of extracts from hepatopancreas tissue of individual snails of five laboratory strains of Biomphalaria glabrata has shown considerable variation in the frequency of occurrence of ten esterases. Differences in esterase frequencies seemed correlated with strain differences in certain morphological, physiological, and biochemical aspects of the snail strains; however, no causal relationships are apparent in these systems.     

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.     

Biomphalaria tenagophila: dominant character of the resistance to Schistosoma mansoni in descendants of crossbreedings between resistant (Taim, RS) and susceptible (Joinville, SC) strains

The aim of the present work was to study parasitological, molecular, and genetic aspects in descendants of crossbreedings between a totally resistant Biomphalaria tenagophila strain (Taim, RS) and another one highly susceptible (Joinville, SC) to Schistosoma mansoni. Descendants F1 and F2 were submitted to S. mansoni infection (LE strain). The susceptibility rates for individuals from Group F1 were 0 to 0.6%, and from Group F2 was 7.2%. The susceptible individuals from Group F2 discharged a lower number of cercariae, when compared with the susceptible parental group, and in 2 out of 9 positive snails the cercarial elimination was discontinued. In order to identify genetic markers associated with resistance the genotype of parental snails and their offspring F1 and F2 were analyzed by means of the randomly amplified polymorphic DNA method. Nevertheless, it was not possible to detect any marker associated to resistance, but the results showed that in the mentioned species the resistance character is determined by two dominant genes.     

Structural analysis of hemolymph proteins from Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda)

1. Five different molecular weight polypeptides from serum (cell-free hemolymph) of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata, were examined by two-dimensional 125I-peptide mapping and high performance liquid chromatography (HPLC). 2. Peptide mapping indicated that all five radiolabeled polypeptides within and between the two snail strains had similar migration patterns when cleaved with pepsin or alpha-chymotrypsin, thus revealing a shared structural homology. All peptides chosen for analysis appeared to be structurally similar to the 160 kDa hemoglobin molecule. 3. Separations of the radiolabeled enzyme digests by HPLC confirmed results seen in the mapping experiments since all chromatograms had similar elution patterns. 4. Minor differences in the peptide maps and chromatograms within and between snail strains may be due to quantitative differences in the amount of protein present and/or variations in the primary amino acid sequences of the proteins chosen for analysis.     

Mitotic arrest and toxicity in Biomphalaria glabrata (Mollusca: Pulmonata) exposed to colchicine

Continuous exposure of Biomphalaria glabrata snails to 0.1% colchicine resulted in a significant increase, relative to non-exposed snails, in the number of arrested mitotic figures in the amebocyte-producing organ (APO) as soon as 4 h, with peak numbers after 12 h of exposure. The number of circulating hemocytes was significantly elevated at 24 h. However, by 72 h both the number of mitotic figures in the APO and the concentration of circulating hemocytes in the hemolymph had returned to control levels. Hemocytes appeared to possess normal morphology throughout this exposure, including the formation of long filopodia with supporting rodlike structures that have been reported to contain microtubules. Snail survival decreased as a function of exposure time. Significantly fewer snails, relative to controls, survived a 48-h exposure, and only 1 out of 30 snails recovered from a 72-h exposure to 0.1% colchicine. Colchicine-exposed snails displayed intoxicated behavior, even upon removal from the colchicine solution, although no histopathology was evident in the CNS of snails exposed for 72 h.     

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: functional heterogeneity in cell subpopulations recognized by a monoclonal antibody

A hemocyte surface membrane marker (BGH₁) has been identified using hemocyte-specific monoclonal antibodies (mABs) generated by somatic cell fusion methods. The BGH₁ epitope was expressed on a subpopulation of circulating, glass-adherent blood cells from two strains of the snail, Biomphalaria glabrata. Approximately 40% of the circulating hemocytes from the PR albino (M-line) B. glabrata strain were BGH₁^?, compared to a prevalence of 10% BGH₁⁺ cells in the 10-R2 snail strain. When hemocytes were firmly attached and spread on a glass surface, BGH₁⁺ cells were morphologically distinguishable from BGH₁^? cells by their ovoid shape and the presence of short, thin filopodial projections along the ectoplasmic border. In contrast, BGH₁^? hemocytes were more pleomorphic and possessed long, spike-like filopodia. Moreover, the BGH₁ epitope was trypsin-resistant and retained its antigenic reactivity with probe mABs following fixation with paraformaldehyde or paraformaldehyde/MeOH. Fixation with glutaraldehyde, however, significantly reduced mAB binding to the BGH₁ surface epitope. There was no apparent age-dependent expression of the BGH₁ determinant since circulating hemocyte populations in very young (1–2 mm) to adult (10–12 mm) snails were composed of both BGH₁⁺ and BGH₁^? subpopulations. Quantitative shifts in the prevalence of epitope-bearing hemocytes between the smallest snail size class (1–2 mm) and the larger snails (3–4 and 10–12 mm) are believed to be due to a differential production and/or release of BGH₁^? hemocytes within the blood circulation rather than a gradual age-related change in the expression of surface antigens on individual cells. Experiments designed to assess the in vitro phagocytic capability and lysosomal acid phosphatase (APase) activity of mAB-reactive hemocytes revealed that BGH₁⁺ cells, when compared to those lacking the surface marker, were significantly reduced in both their phagocytic and APase-producing activities. Since the PR albino strain of B. glabrata possesses a higher proportion of BGH₁^? hemocytes and a lower total concentration of circulating cells than do snails of the 10-R2 strain, PR albino snails are thus potentially reduced in their natural capacity to mount cellular reactions against foreign materials.     

Biomphalaria tenagophila: dynamics of populations of resistant and susceptible strains to Schistosoma mansoni, with or without pressure of the parasite

Resistant (Taim, RS) and susceptible albino (Joinville, SC) Biomphalaria tenagophila populations were kept together, at different proportions, throughout a 18-month-period. Some of the snail groups were submitted to Schistosoma mansoni infection. The targets of this study were (a) to analyze the populational dynamics among resistant and susceptible individuals to S. mansoni; (b) to study the resistance phenotype in descendants of cross-breeding; (c) to observe whether the parasite could exert any kind of selection in those snail populations. Throughout the experiment it could be observed that the susceptible B. tenagophila strain (Joinville) underwent a selective pressure of the parasite that was negative, since the individuals showed a high mortality rate. Although B. tenagophila (Taim) population presented a higher mortality rate without pressure of the parasite, this event was compensated by a reproductive capacity. B. tenagophila Taim was more fecund than B. tenagophila Joinville and was able to transmit the resistance character to their descendants. F1 generation obtained by cross-breeding between resistant and susceptible lineages was completely resistant to S. mansoni infection, irrespective of the Taim proportion. Moreover, less than 5% of F2 progeny were susceptible to S. mansoni infection.     

Experimental exposure of Helisoma trivolvis and Biomphalaria glabrata (Gastropoda) to Ribeiroia ondatrae (Trematoda)

Experimental infections provide an important foundation for understanding host responses to parasites. While infections with Ribeiroia ondatrae cause mortality and malformations in a wide range of amphibian second intermediate host species, little is known about how the parasite affects its snail first intermediate hosts or even what species can support infection. In this study, we experimentally exposed Helisoma trivolvis, a commonly reported host of R. ondatrae, and Biomphalaria glabrata, a confamilial snail known to host Ribeiroia marini, to increasing concentrations of embryonated eggs of R. ondatrae obtained from surrogate definitive hosts. Over the course of 8 wk, we examined the effect of parasite exposure on infection status, time-to-cercariae release, host size, and mortality of both snail species. Helisoma trivolvis was a highly competent host for R. ondatrae infection, with over 93% infection in all exposed snails, regardless of egg exposure level. However, no infections were detected among exposed B. glabrata, despite previous accounts of this snail hosting a congener parasite. Among exposed H. trivolvis, high parasite exposure reduced growth, decreased time-to-cercariae release, and caused marginally significant increases in mortality. Interestingly, while B. glabrata snails did not become infected with R. ondatrae, individuals exposed to 650 R. ondatrae eggs grew less rapidly than unexposed snails, suggesting a sub-lethal energetic cost associated with parasite exposure. Our results highlight the importance of using experimental infections to understand the effects of parasite exposure on host- and non-host species, each of which can be affected by exposure.     

Mannose-binding lectin (MBL) mutants are susceptible to matrix metalloproteinase proteolysis: potential role in human MBL deficiency

Mannose-binding lectin (MBL) plays a critical role in innate immunity. Point mutations in the collagen-like domain (R32C, G34D, or G37E) of MBL cause a serum deficiency, predisposing patients to infections and diseases such as rheumatoid arthritis. We examined whether MBL mutants show enhanced susceptibility to proteolysis by matrix metalloproteinases (MMPs), which are important mediators in inflammatory tissue destruction. Human and rat MBL were resistant to proteolysis in the native state but were cleaved selectively within the collagen-like domain by multiple MMPs after heat denaturation. In contrast, rat MBL with mutations homologous to those of the human variants (R23C, G25D, or G28E) was cleaved efficiently without denaturation in the collagen-like domain by MMP-2 and MMP-9 (gelatinases A and B) and MMP-14 (membrane type-1 MMP), as well as by MMP-1 (collagenase-1), MMP-8 (neutrophil collagenase), MMP-3 (stromelysin-1), neutrophil elastase, and bacterial collagenase. Sites and order of cleavage of the rat MBL mutants for MMP-2 and MMP-9 were: Gly(45)-Lys(46) --> Gly(51)-Ser(52) --> Gly(63)-Gln(64) --> Asn(80)-Met(81) which differed from that of MMP-14, Gly(39)-Leu(40) --> Asn(80)-Met(81), revealing that the MMPs were not functionally interchangeable. These sites were homologous to those cleaved in denatured human MBL. Hence, perturbation of the collagen-like structure of MBL by natural mutations or by denaturation renders MBL susceptible to MMP cleavage. MMPs are likely to contribute to MBL deficiency in individuals with variant alleles and may also be involved in clearance of MBL and modulation of the host response in normal individuals.     

Concanavalin A-mediated phagocytosis of yeast by Biomphalaria glabrata hemocytes in vitro: Effects of temperature and lectin concentration

Exposure of either Biomphalaria glabrata granulocytes or yeast to concanavalin A (Con A) prior to being incubated together in a “microhemadsorption assay” affected the number of yeast attached to or endocytosed by the granulocytes after 60 min. The yeast-granulocyte interaction was sensitive to temperature, Con A concentration, and the cell treated. Numbers of associated (attached + endocytosed) and endocytosed yeast either increased or decreased, as compared with nontreated controls, depending on the protocol. The effect of a given protocol was not necessarily similar for attachment and endocytosis. By separately enhancing these two stages of phagocytosis, Con A may be a useful tool for studying this process of molluscan granulocytes.     

Synergism of tebufenozide in resistant and susceptible strains of obliquebanded leafroller (Lepidoptera: Tortricidae) and resistance to new insecticides

Cross-resistance of the obliquebanded leafroller, Choristoneura rosaceana (Harris), to tebufenozide was reported from laboratory studies before it had been used in commercial orchards in New York State. Bioassays with obliquebanded leafroller larvae from tebufenozide and organophosphate susceptible and resistant colonies were conducted with chlorfenapyr, emamectin benzoate, fenoxycarb, fipronil, spinosad, and tebufenozide to determine if cross-resistance was present before these new insecticides were introduced into commercial orchards. Resistance ratios ranged from 1.1 to 3.2 for all insecticides except tebufenozide (12.8). Significant differences between susceptible and resistant colonies were found with emamectin benzoate, fenoxycarb, and fipronil. The effect of the metabolic synergists piperonyl butoxide (PBO) and diethyl maleate (DEM) on tebufenozide toxicity was examined to determine mechanisms for obliquebanded leafroller resistance to tebufenozide and potential mechanisms for other new insecticides. At a concentration of 20 ppm, PBO and DEM significantly synergized the toxicity of tebufenozide in resistant and susceptible colonies (three- to fourfold). Obliquebanded leafrollers may be resistant to new insecticides with distinct modes of action even if these compounds have not been previously used in commercial orchards.     

Comparative mortality studies on Biomphalaria glabrata (mollusca: Pulmonata) exposed to copper internally and externally

Various concentrations of copper in the form of CuSO₄ were injected into the hemocoel of Biomphalaria glabrata, and mortality of this snail was subsequently monitored. The concentrations of copper in the hemolymph of injected snails were calculated, and specimens were incubated in these concentrations. Greater mortality was observed when snails were incubated in concentrations of copper than when they were injected with a sufficient amount of copper to attain these same concentrations in the hemolymph. Injection of copper into the hemocoel of B. glabrata resulted in the formation of a noncellular hemolymph precipitate, most likely denatured proteins, at the injection site, which was most noticeable with higher concentrations of copper. It has been concluded that external concentrations of copper are more cidal to B. glabrata than are internal, i.e., injected, concentrations. These data support the hypothesis that the cidal action of copper on B. glabrata is due to an attack on the mollusc's surface epithelia.     

The chemical ecology of Biomphalaria glabrata (say): Sugars as attractants and arrestants

• 1.1. The behavioural responses of the freshwater snail Biomphalaria glabrata to chemical gradients of sugars were investigated by means of diffusion olfactometers.
• 2.2. The snails proved very discriminating in their responses. Thus, only nine (39.1%) of the 23 sugars tested proved to be statistically significant attractants or arrestants. None proved to be statistically significant repellents.
• 3.3. Of all the sugars tested maltose proved to be the most potent attractant or arrestant. The lower threshold of response to this sugar lies between 5 × 10⁻⁶ and 5 × 10⁻⁷M.
• 4.4. The results are compared with those obtained for amino and carboxylic acids and their ecological relevance is discussed.