Total protein and carbohydrate content and protease and disaccharidase activities in the hemolymph of Lymnaea stagnalis naturally infected with digenean larvae

Lymnaea stagnalis is an intermediate host of many Digenea. The infestation affects host metabolism. The aim of the work was to investigate hemolymph biochemical indicators of L. stagnalis infected with four species of trematodes: Diplostomum pseudospathaceum, Paryphostomum radiatum, Plagiorchis elegans or Opisthioglyphe ranae. The protein profiles and proteinase activity in the hemolymph of sexually mature individuals of Lymnaea stagnalis maintained at 19°C were tested. As the carbohydrates are main substrates for energetic metabolism of the great pond snail their content and disaccharidase activity were also studied. Hemolymph samples were collected during weeks 3 and 4 of rearing. No significant differences in the total protein content between uninfected individuals and snails infected with the first three trematode species were detected. In the snails infected with O. ranae the quantity of total proteins was near twice higher than in those uninfected. A higher share of 70 kDa proteins in infected than in uninfected snails as well as reduction of the low molecular weight fractions of proteins for snails infected with D. pseudospathaceum and P. radiatum were detected. During week 3, carbohydrate content in the infected snails did not differ from that in the controls while during week 4 it was significantly lower in snails infected with P. elegans or O. ranae. The content of the major soluble carbohydrate in the hemolymph - saccharose — changed in a similar way. No activity of trypsin or pepsin in the hemolymph sample was detected while the activity of chymotrypsin was lower in infected snails vs. controls. On the other hand, saccharase and maltase activities were higher in infected than in uninfected snails. The biochemical hemolymph indicators in naturally infected host-snails show some differences depending on the parasite species but they are not sufficiently species-specific to offer the basis for establishing the model unique for a particular parasitosis. Our results from the field did not always coincide with those from the laboratory.     

Echinostoma paraensei: hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni

Earlier in vivo work by Lie et al. (1977) indicated that the innate resistance of the 10R2 strain of Biomphalaria glabrata to PR1 Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from 10R2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P less than 0.001) than the 90% killing rate observed with hemolymph from uninfected, control 10R2 snails. Hemolymph from the infected 10R2 snails and from schistosome susceptible M line snails did not differ significantly (P greater than 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected 10R2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected 10R2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P less than 0.001) killing of S. mansoni sporocysts than did controls containing only 10R2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts.     

Growth rate and changes in tissue carbohydrates during schistosome infection of the snail biomphalaria alexandrina

Laboratory investigations were carried out to determine the growth rate, glycogen content of different body parts and glucose uptake in normal and schistosome-infected Biomphalaria alexandrina snails over a period of 45 days post infection. Infected snails grew at a faster rate. There were no significant differences in tissue water content and tissue dry weight of control and infected ones. A marked decrease in the glycogen content of female sex organs, mantle, digestive gland and muscles was observed in infected snails. Glucose utilization was also increased in infected snails.     

Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca: Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda,Metastrongylidae) by high-performance liquid chromatography

The activity of lactate dehydrogenase and the concentrations of glucose in the hemolymph and of glycogen in the digestive gland and cephalopedal mass of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis were evaluated. Additionally, high performance liquid chromatography (HPLC) was used to determine the hemolymph concentrations of some carboxylic acids (oxalic, piruvic, lactic and succinic). After one, two and three weeks of infection, the snails were dissected to collect the hemolymph and separate the tissues. A significant reduction of the levels of glucose in the hemolymph was observed as of the first week of infection in relation to the control group. The lactate dehydrogenase activity of the infected group was significantly higher than the average of the control group. This increase was accompanied by a reduction of the levels of piruvic acid and an increase in the levels of lactic acid in the hemolymph of the parasited snails, confirming the acceleration of the anaerobic metabolism, necessary for the host to obtain energy and maintain its redox balance. In parallel, there was a decrease in the glycogen content of the storage tissues, with that reduction being significantly greater in the cephalopedal mass than the digestive gland, demonstrating that in this interaction system, the mobilization of glycogen was not sufficient to maintain and reestablish the normal glycemia of the infected snails.     

Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata

Lie K. J., Jeong K. H. and Heyneman D. 1980. Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata. Intemational Journal for Parasitology10: 183–188. More than 85% of echinostome-infected albino B. glabrata laboratory strain snails develop miracidia-immobilizing substance(s) (MIS) in the hemolymph, while less than 5% of control uninfected snails show this ability. Snails infected with Echinostoma lindoense show a strong miracidial immobilizing test (MIT) when homologous miracidia are exposed to the hemolymph and a moderate response when E. liei and Paryphostomum segregatum miracidia are used. Infection with E. paraensei results in a high level of hemolymph MIS with E. lindoense miracidia, a moderate one with P. segregatum miracidia, and a weak one when hemolymph is tested against E. liei as well as the homologous E. paraensei miracidia. Infection with E. liei induces a strong MIT with E. lindoense miracidia whereas only a moderate one was observed when using homologous or P. segregatum miracidia. Infection with P. segregatum gives a moderate MIT reaction to miracidia of the homologous species, as well as to E. lindoense and E. liei, and only a weak response to E. paraensei miracidia. Infection with S. mansoni fails to induce hemolymph that shows MIS to any of the parasites tested. Production of hemolymph MIS is temporary. It begins one day postexposure, reaches its maximum 10–14 days postexposure, and declines to the preinfection level several weeks later. Infection of snails with irradiated parasites also results in a temporary production of hemolymph MIS.Uninfected snails show a tissue-extract MIS, which is especially strong when digestive gland extracts are used. However, these snails give little or no evidence of a hemolymph MIS.     

Effect of Schistosoma mansoni infection on fecundity and perivitelline fluid composition in Biomphalaria glabrata

Egg production in the snail, Biomphalaria glabrata, infected with Schistosoma mansoni declined on day 23 postinfection, and was significantly lower than uninfected control snails by day 28 and thereafter. Protein and galactogen content of eggs produced by infected snails did not change during the period of reduced fecundity. This suggests that decreased hemolymph nutrient levels (rather than depleted albumen gland reserves) are responsible for inhibition of snail egg production. Growth rates of infected and uninfected snails were indistinguishable from days 14 through 35 postinfection. The hatching success of eggs produced by infected snails decreased slightly beginning at day 21 postinfection.     

Physiology and lipid metabolism of Littorina saxatilis infected with trematodes

Physiological and biochemical alterations in Littorina saxatilis infected with larval trematodes were investigated and compared with the metabolism of non-parasitized snails. Oxygen consumption rates of infected snails differed from those of non-infected controls in medium sized individuals (30 to 130 mg) but not in very large infected individuals (> 200 mg). Small snails (0.5 to 8.5 mg) were seldom infected by parasites, and this size-class consisted only of non-infected specimens. The specific oxygen consumption rate of infected snails was not dependent on their mass and remained constant over the size ranges investigated. Alterations in the snail metabolism appeared to be connected to injuries to digestive gland tissues caused by the parasites. The glycogen concentration and fatty acids of neutral lipids and phospholipids in the digestive gland were determined. Infected snails differed from uninfected snails in the complete absence of glycogen in digestive gland and had proportionally higher quantities of eicosenoic (20:1) acid in the total phospholipids. It remains unclear whether infection by trematodes activates enzymes in the snail's digestive gland to synthesize eicosenoic (20:1) acid, or whether the sporocysts themselves possess these enzymes. The role of phospholipid fatty acids in the regulation and maintenance of the parasite's metabolism is briefly considered. Biochemical alterations observed in the fatty acid composition may have an adaptive significance, by helping to stabilize the host-parasite system.     

Schistosoma mansoni: effect of infection on reproduction and gonadal growth in Biomphalaria glabrata

Sexually mature Biomphalaria glabrata were exposed to 12 miracidia of Schistosoma mansoni, and egg production of snails was monitored over a period of 5 weeks. During the study period, exposed snails grew at approximately the same rate as unexposed controls. Castration, as measured by a reduction in the mean number of eggs laid per snail occurred between 14 and 21 days postexposure (PE). The reduction in fecundity in infected snails coincided with the migration and establishment of daughter sporocysts in the digestive gland and gonad. Enumeration of individual oocytes in longitudinal sections of the ovotestis revealed that uninfected snails contained significantly more oocytes per section than infected snails at 27, 31, and 40 days PE. In addition, the mean area of gonadal sections of control snails increased over the 40-day experimental period, whereas there was no such increase in gonadal area of infected snails. These data suggest that there is an inhibition in gonadal growth in infected snails. When oocyte data were expressed in terms of mean gonadal area, the mean number of oocytes per mm2 of gonad of uninfected and infected snails did not differ significantly over the study period, except at Day 14 PE, when infected snails contained a significantly greater number of oocytes per mm2 of gonad than did uninfected controls. It is hypothesized that daughter sporocysts of S. mansoni are primarily responsible for the inhibition of host reproductive activity, and may be mediating their effects through mechanisms involved in the regulation of gonadal growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in body tissues and hemolymph composition of Culex pipiens in response to infection by Romanomermis culicivorax

Hemolymph composition of fourth instar larvae of an autogenous strain of Culex pipiens was examined to determine the effects of parasitism by a mermithid nematode, Romanomermis culicivorax. Mosquitoes were reared under two different pH regimens: 4.5 and 7.3. Wet and dry weight of infected mosquitoes reared at either pH were significantly lower than controls. The effects of parasitism in the development of C. pipiens were evaluated from paraffin sections of mosquito larvae 2, 4, and 6 days postinfection. At 2 days postinfection, the infected larvae showed no apparent effects of parasitism; at day 4, the fat body tissue was reduced and imaginal disc development was retarded; and at day 6, parasitized mosquitoes were smaller in cross section, fat body tissue was found only in isolated clumps, and there was a complete absence of imaginal discs. Concentrations of total carbohydrates in hemolymph from infected fourth instar mosquitoes reared at pH 7.3 were reduced. Trehalose and glucose were each reduced by more than half. Total α-amino nitrogen was significantly lower in infected mosquitoes reared at pH 7.3. However, total amino acid concentrations for hemolymph from control and infected larvae reared at pH 7.3 were the same. Methionine sulfoxide decreased 63% and proline increased 2.5 times in infected mosquitoes. Hemolymph protein concentrations were reduced 80% in infected mosquitoes reared at both pHs. The number of hemolymph proteins also declined from 35 to 22 during infection. Two host proteins, 82,000 and 158,000 daltons, remained prominent throughout the mermithid infection.     

Schistosoma mansoni: Lysozyme activity in Biomphalaria glabrata during infection with two strains

Levels of lysozyme activity were determined in the hemolymph, digestive gland, and headfoot extracts of M-line stock of snails, Biomphalaria glabrata, during infection with the PR-1 and Lc-1 strains of the trematode, Schistosoma mansoni. At 3 hr postexposure there was a 10-fold increase in the levels of enzyme activity in the hemolymph of snails infected with the Lc-1 strain to which the snail is resistant. This increase was considerably higher when compared to the threefold increase in the PR-1-infected snails. The infection also induced a gradual depletion of lysozyme activity in the headfoot muscles of the two groups of infected snails. There were no changes in the levels of enzyme activity in the digestive gland extracts of the control and the two groups of infected snails. Similar changes in the levels of enzyme activity in the hemolymph and headfoot extracts of infected snails suggest a nonspecific response to a parasite infection and do not indicate that lysozyme is primarily responsible for the destruction of schistosome parasite in a resistant snail host.     

Eimeria tenella: Cecal contraction in infected chickens

The muscle of ceca from chickens infected with Eimeria tenella had an increased amplitude of contraction when compared to the cecal muscle of uninfected control chickens. The increased amplitude was significant (P ? 0.05) at 5 days postinoculation (PI) and became nonsignificant at 7 days PI. The amplitude also increased with the severity of the infection. The sensitivity of the muscle from infected ceca to acetylcholine (ACH) was greater than the control. Infection by different strains of E. tenella also increased the sensitivity of the cecal muscle to ACH when compared to the uninfected control. The rate of spontaneous contractions was not different in any group or treatment. The wet weight of infected ceca increased with days PI and severity of infection.     

Biomphalaria glabrata amoebocytes: Effect of Schistosoma mansoni infection on in vitro phagocytosis

The rate of phagocytosis by amoebocytes obtained from hemolymph of the pulmonate Biomphalaria glabrata infected with the trematode Schistosoma mansoni for 24 hr and 2, 4, and 6 weeks has been determined using the monolayer assay system. Amoebocyte preparations from snails infected for 4 and 6 weeks showed a gradual decrease in the phagocytic rates compared to those from uninfected controls. Snails harboring the parasite for 4 and 6 weeks also showed a significant increase in the number of amoebocytes in the hemolymph. No significant changes were detected in the rate of phagocytosis or number of amoebocytes in snails infected for 2 weeks or less. Alterations in the morphology and behavior of amoebocytes from infected snails were also noted.     

Effect of xiphidiocercarial infection on oxidation of glycolytic and Krebs cycle intermediates in Lymnaea luteola (Mollusca)

With the aim of assessing the effect of xiphidiocercarial infection on its gastropod host, Lymnaea luteola, the oxidation of glycolytic and tricarboxylic acid cycle intermediates by digestive gland homogenates of uninfected and infected snails was studied manometrically. The oxidation of glycolytic and NAD-linked substrates was reduced in infected snails. On the other hand, succinate oxidation was very high in infected snails and the reasons for this are discussed.     

Effects of exogenous interleukin-1β on primary sporocysts of Schistosoma mansoni (Trematoda) incubated with plasma and hemocytes from schistosome-susceptible and resistant Biomphalaria glabrata (Gastropoda)

Abstract. The cytokine interleukin-1β (IL-1β) mediates interactions of immune and inflammatory cells in mammals. Previous reports also have linked plasma (cell-free hemolymph) levels of IL-1β in the snail Biomphalaria glabrata to resistance against Schistosoma mansoni . In the present study, fluorescent probes were used to study larval schistosome and snail hemocyte viability during in vitro encounters. Hemolymph (plasma and hemocytes) from schistosome-susceptible (M-line) and resistant (13–16-R1) B. glabrata was added to sporocysts of S. mansoni and the viability of hemocytes and parasites was assessed. Next, IL-1β was added to sporocyst-hemolymph samples, the viability of sporocysts and hemocytes determined and then compared to control assays. The number of live sporocysts present after incubation for 1 h with hemolymph from M-line snails was significantly greater than the number seen when hemolymph from 13–16-R1 snails was tested. Nearly all sporocysts survived the 1 h incubation with M-line hemolymph, and most of the hemocytes attached to sporocysts were dead. In contrast, nearly all sporocysts were dead when hemolymph from 13–16-R1 snails was tested, and most attached hemocytes were alive. Addition of IL-1β to M-line hemolymph resulted in a dramatic increase in sporocyst death. Addition of IL-1β to 13–16-R1 hemolymph produced a small but significant increase in the rate of sporocyst death. These results indicate that the concentration of IL-1β present in hemolymph from B. glabrata is directly related to the ability of this snail to kill S. mansoni sporocysts in vitro.     

Trichobilharzia ocellata: physiological characterization of giant growth, glycogen depletion, and absence of reproductive activity in the intermediate snail host, Lymnaea stagnalis

Giant growth, depletion of energy stores, and inhibition of reproductive activity are striking effects of many trematode parasites on their intermediate snail hosts. Two hypotheses have been put forward to explain these phenomena: (1) host and parasite compete for energy rich and other essential nutrients, with the parasite as the winner, and (2) the parasite intervenes in the endocrine control of reproduction of the snail. These hypotheses were tested in the present study with the Trichobilharzia ocellata/Lymnaea stagnalis association. The snails were infected at a juvenile stage, and release of cercariae started on Day 55 after exposure. It was shown that enhanced growth of infected snails is not paralleled by a greater increase in dry weight, but hemolymph volume does increase, being 35% greater than in the noninfected controls. Control snails, on the other hand, showed an increase in the percentage body dry weight during sexual maturation. The conclusion is that infected snails retain an essentially juvenile body structure. In control snails, glycogen was depleted from the mantle store at the start of egg laying but the onset of cercariae production marked a severe glycogen depletion from the headfoot and the mantle in infected snails, being nearly complete on Day 68 after exposure. The hemolymph glucose concentration was only slightly lower in infected than in control snails and it did not change (in both groups) during glycogen mobilization. This suggests that glycogen mobilization does not result from the snail and the parasite competing directly for metabolites within the hemolymph. Infection inhibited the maturation of the accessory sex organs: there was no increase in the relative wet weights nor in the amounts of DNA and secretion products in the albumin and prostate glands. Infected snails did not lay eggs. It is presumed that the parasite produces one or more agents which intervene in the action of the gonadotrophic hormones. The release of these agents commences at an early stage of infection.     

Effects of Schistosomatium douthitti infection on the growth, survival, and reproduction of Lymnaea catascopium

Lymnaea catascopium snails infected with Schistosomatium douthitti grew faster than uninfected control snails during the first 2 months postexposure, but thereafter grew more slowly, and by 8 months postexposure were significantly smaller. When reared in isolation, uninfected snails survived significantly longer (mean survival time, 515 days) than snails exposed to three miracidia each (400 days), which in turn survived longer than snails exposed to 10 miracidia per snail (223 days). When maintained in aquaria in contact with other snails, snails exposed to three miracidia each survived longer (227 days), but not significantly longer, than control snails (198 days). Production of large numbers of eggs by control snails grown under the latter conditions may account for their reduced survival. The ovotestes and accessory genitalia of snails infected with S. douthitti were much reduced in size in comparison with uninfected control snails. These effects were most pronounced in snails which had been infected for over 100 days. Egg production was normally totally inhibited if snails were infected before the onset of sexual maturity. If infected after the onset of maturity, eggs were produced only during the prepatent period.     

Alterations in the osmoregulation of the pulmonate gastropod Biomphalaria glabrata due to copper

Specimens of Biomphalaria glabrata were exposed to 0.06 ppm of copper in the form of CuSO₄, and the resulting changes in the wet and dry weights of the soft tissues and in the osmolality of the hemolymph were measured. The wet weights of snails exposed to copper increased as a function of time, while those of the controls decreased. The dry weights of both the experimental and control snails decreased equally. Finally, the ratio of wet weight to dry weight of the experimental snails was significantly higher than that of the controls after 24 and 48 hr of exposure to copper. In addition, the osmolality of the hemolymph of snails exposed to copper was significantly lower than that of the controls after 12, 24, and 36 hr of exposure. These data have led to the conclusion that exposure of B. glabrata to copper results in an osmotic influx of water into its tissues and thereby causes death.     

Carboxylic Acids as Biomarkers of Biomphalaria alexandrina Snails Infected with Schistosoma mansoni

Biomphalaria alexandrina snails play an indispensable role in transmission of schistosomiasis. Infection rates in field populations of snails are routinely determined by cercarial shedding neglecting prepatent snail infections, because of lack of a suitable method for diagnosis. The present study aimed at separation and quantification of oxalic, malic, acetic, pyruvic, and fumaric acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to test the potentiality of these acids to be used as diagnostic and therapeutic biomarkers. The assay was done in both hemolymph and digestive gland-gonad complex (DGG) samples in a total of 300 B. alexandrina snails. All of the studied acids in both the hemolymph and tissue samples except for the fumaric acid in hemolymph appeared to be good diagnostic biomarkers as they provide not only a good discrimination between the infected snails from the control but also between the studied stages of infection from each other. The most sensitive discriminating acid was malic acid in hemolymph samples as it showed the highest F-ratio. Using the Z-score, malic acid was found to be a good potential therapeutic biomarker in the prepatency stage, oxalic acid and acetic acid in the stage of patency, and malic acid and acetic acid at 2 weeks after patency. Quantification of carboxylic acids, using HPLC strategy, was fast, easy, and accurate in prediction of infected and uninfected snails and possibly to detect the stage of infection. It seems also useful for detection of the most suitable acids to be used as drug targets.     

Physiologic studies of snail-schistosome interactions and potential for improvement of in vitro culture of schistosomes

Summary Despite extensive efforts to develop suitable media for rearing the intramolluscan stages of schistosomes, successful in vitro culture of these parasites remains elusive. Recent³¹P NMR studies demonstrated that the levels of free phospholipids, particularly phosphatidylcholine, in the digestive gland of the snail,Biomphalaria glabrata, were dramatically reduced when the host was infected withSchistosoma mansoni. It was speculated that absorption of host phosphatides may be an important source of membrane phospholipid precursors and fatty acids for developing sporocysts and cercariae. During the present investigations,B. glabrata was maintained on a high fat diet of egg yolk, and the lipid composition of control uninfected and infected snails examined by³¹P and¹³C NMR. In addition, the levels of host hemolymph metabolites, including glucose and urea, considered as indicators of parasite nutrient uptake, were monitored. The lipid level of snails fed egg yolk was greatly increased, and hosts developed patent infections in approximately half the time of infected snails maintained on lettuce. The composition of the free phospholipids accumulated in the tissues ofB. glabrata fed egg yolk were the same as those previously reported in the cercarial stage ofS. mansoni. Moreover, the fatty acids ofS. mansoni and those reported here in the neutral lipids and free phosphatides in the host tissues were similar. Uninfected snails maintained on lettuce had higher hemolymph levels of glucose than those reared on egg yolk, and infected hosts on egg yolk had significantly lower levels of hemolymph urea. β-hydroxybutyrate was the principal hemolymph metabolite in snails fed egg yolk, but was not detected in snails maintained on lettuce. The level of β-hydroxybutyrate in the hemolymph of snails on egg yolk was significantly reduced by infection. The results indicated that the pattern of host hemolymph nutrient utilization by larval schistosomes may be markedly altered by host diet, and it was concluded that host lipids may directly and indirectly be important nutrients for developing schistosomes. Future studies on in vitro culture of the intramolluscan stages of schistosomes should emphasize the potential role of lipids and attempt to define the nutritive value of those medium components that presently supply lipids in culture media, most notably serum.     

Distribution and variation of hemagglutinating activity in the hemolymph of Biomphalaria glabrata

A sensitive hemagglutination assay utilizing glutaraldehyde-fixed trypsinized calf erythrocytes (GTC) is described to test for agglutinin levels in hemolymph and albumen gland extracts from nine populations of Biomphalaria glabrata, and from B. straminea and B. obstructa. High levels of GTC-reactive hemagglutinin were found in all snail populations. There was no correlation between hemagglutinin titer and innate resistance of B. glabrata strains to Schistosoma mansoni. However, an increase in hemagglutinin titer occurs in B. glabrata M-RLc snails infected with Echinostoma lindoense and in snails sensitized and reexposed to this parasite.