Free radical scavengers in susceptible/resistant Biomphalaria alexandrina snails before and after infection

The activities of catalase (Cat), superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione transferase (GST), glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde3-phosphate dehydrogenase (G3PD) were studied in tissue and hemolymph of susceptible (S) (EgBS(2)) and resistant (R) (EgBR(2)) Biomphalaria alexandrina snails. The results showed that CAT and GST were higher in the hemolymph of snails susceptible to Schistosoma mansoni than in that of snails resistant to infestation, while SOD and G3PD were lower in the susceptible snails. The role of these enzymes as free radical scavengers was traced 1 and 24 h after infection of the two snail lines with S. mansoni. Moreover, the activities of SOD and G3PD were also measured 2 and 4 weeks post infection. The results revealed that the overall enzymatic activities were higher in susceptible than in resistant snail tissues. After 1 h of infection, all enzymes were increased in R and S snails except GST and G6PD which decreased in S snails. After 24 h of infection, GST increased in S snails and G3PD decreased in both S and R snails while other enzymes reached normal levels.     

Physiological and biochemical responses to cold and drought in the rock-dwelling pulmonate snail, Chondrina avenacea

The pulmonate snail Chondrina avenacea lives on exposed rock walls where it experiences drastic daily and seasonal fluctuations of abiotic conditions and food availability. We found that tolerance to dry conditions was maintained at a very high level throughout the year and was mainly based on the snails’ ability to promptly enter into estivation (quiescence) whenever they experienced drying out of their environment. Snails rapidly suppressed their metabolism and minimized their water loss using discontinuous gas exchange pattern. The metabolic suppression probably included periods of tissue hypoxia and anaerobism as indicated by accumulation of typical end products of anaerobic metabolism: lactate, alanine and succinate. Though the drought-induced metabolic suppression was sufficient to stimulate moderate increase of supercooling capacity, the seasonally highest levels of supercooling capacity and the highest tolerance to subzero temperatures were tightly linked to hibernation (diapause). Hibernating snails did not survive freezing of their body fluids and instead relied on supercooling strategy which allowed them to survive when air temperatures dropped to as low as ?21 °C. No accumulation of low-molecular weight compounds (potential cryoprotectants) was detected in hibernating snails except for small amounts of the end products of anaerobic metabolism.     

Austropeplea ollula (Pulmonata: Lymnaeidae): a new molluscan intermediate host of a human intestinal fluke, Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

Three freshwater snail species of the family Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula and Fossaria truncatula. Out of 3 lymnaeid snail species, A. ollula was naturally infected with the Echinostoma cinetorchis cercariae (infection rate = 0.7%). In the experiments with the laboratory-bred snails, F. truncatula as well as A. ollula was also susceptible to the E. cinetorchis miracidia with infection rates of 25% and 40%, respectively. All of three lymnaeid snail species exposed to the E. cinetorchis cercariae were infected with the E. cinetorchis metacercariae. It is evident that A. ollula acts as the first molluscan intermediate host of E. cinetorchis in Korea, and F. truncatula may be a possible candidate for the first intermediate host of this intestinal fluke. Also, three lymnaeid snail species targeted were experimentally infected with E. cinetorchis metacercariae.     

Host specificity of Austropeplea ollula (Gastropoda: Lymnaeidae) to miracidial infection with a human intestinal fluke Neodiplostomum seoulense (Trematoda: Diplostomatidae) in Korea

Three species of freshwater pulmonate snails of Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula, and Fossaria truncatula. Of these 3 species, only A. ollula was naturally infected with Neodiplostomum seoulense cercariae (prevalence, 0.3%). In experiments with the laboratory-reared snails, A. ollula was found to be susceptible to the N. seoulense miracidia (prevalence, 10%). This is the first report of A. ollula as the first molluscan intermediate host for N. seoulense in Korea.     

Fasciola hepatica: identification of molecular markers for resistant and susceptible Pseudosuccinea columella snail hosts

Protein electrophoresis, RAPD-PCR and nuclear rDNA ITS sequencing were performed to search for genetic differences between Pseudosuccinea columella snails susceptible and resistant to Fasciola hepatica infection. Of the 21 enzymatic loci analyzed in both populations, none of them exhibited neither within- or between-group variation. Such an absence of enzyme polymorphism support the hypothesis of selfing as the "prevalent" mating system for this hermaphroditic species. Conversely, the RAPD profiles displayed clear differences between susceptible and resistant isolates for 17 of the 26 primers tested while no within-group variation was detected. rDNA ITS sequence analysis from snails of each isolates showed only two bases that differed between groups accounting for a 0.17% of variation confirming that susceptible and resistant snails belong to the same species. This is the first time that a genetic variation using RAPD markers is demonstrated between susceptible and resistant lymnaeid snails vis-a-vis of F. hepatica infection in absence of experimental selection.     

Local adaptation of the trematode Fasciola hepatica to the snail Galba truncatula

Experimental infections of six riverbank populations of Galba truncatula with Fasciola hepatica were carried out to determine if the poor susceptibility of these populations to this digenean might be due to the scarcity or the absence of natural encounters between these snails and the parasite. The first three populations originated from banks frequented by cattle in the past (riverbank group) whereas the three others were living on islet banks without any known contact with local ruminants (islet group). After their exposure, all snails were placed in their natural habitats from the end of October up to their collection at the beginning of April. Compared to the riverbank group, snails, which died without cercarial shedding clearly predominated in the islet group, while the other infected snails were few in number. Most of these last snails released their cercariae during a single shedding wave. In islet snails dissected after their death, the redial and cercarial burdens were significantly lower than those noted in riverbank G. truncatula. Snails living on these islet banks are thus able to sustain larval development of F. hepatica. The modifications noted in the characteristics of snail infection suggest the existence of an incomplete adaptation between these G. truncatula and the parasite, probably due to the absence of natural contact between host and parasite.     

Evaluation of enzyme histochemical observations for metabolic studies. A combined histochemical and biochemical investigation of experimentally induced skeletal muscle-changes

Summary The reliability of enzyme histochemical observations for metabolic studies on skeletal muscle tissue was investigated with a combined histochemical and biochemical study. Specimens of musculus soleus with a predominantly aerobic metabolism and of musculus flexor digitorum longus with a predominantly anaerobic metabolism of rabbits in which both muscles were surgically cross-reinnervated or auto-reinnervated were used. For the histochemical investigation activities and localisations of succinate dehydrogenase, l-glycerol-3-phosphate: acceptor oxidoreductase, nicotinamide adenine dinucleotide: tetrazolium oxidoreductase and of -glucan phosphorylase were examined. For the biochemical investigation maximal activity of phosphofructokinase, the rate limiting enzyme for the regulation of the glycolysis was measured. In addition the activities of succinate dehydrogenase and l-glycerol-3-phosphate: acceptor oxidoreductase to characterize the aerobic metabolism and the key role in gearing energy requirements to glycolysis respectively were biochemically determined. For further information about metabolic aspects the isoenzyme ratio of lactate dehydrogenase was established. In the present paper the histochemical findings are reported and discussed.Part of this study was taken from the Ph. D. thesis of A. C. Jöbsis (1971).     

Susceptibility of Iraqi fresh water snails to infection with Schistosoma haematobium and Schistosoma mansoni Egyptian strains

A great number of Egyptian workers and farmers are seeking settlement in Iraq and some of them proved to have either Schistosoma Haematobium (S.h.) or Schistosoma mansoni (S.m) or even mixed infection. Besides, there is the possibility that some of the Iraqi fresh water snails may prove to be susceptible to infection by one or both of the Schistosoma Egyptian strains. The present study deals with investigations on the susceptibility of Iraqi B. truncatus, Gyranaulus ehrenbergi, Physa c.f. fontinalis, Lymnea lagetis, Melanoides tuberculata and Melanopsis nodes by these parasites. Egyptian S. haematobium but not Egyptian S. mansoni infect Iraqi B. truncatus and both proved to be unable to infect any of the other snails included in the study. Yet, the number of cercariae shedded by B. truncatus snails infected with the Egyptian S. haematobium strain, was much less that the number of cercariae shedded by these snails when infected with the Iraqi S. Haematobium strain.     

Larval productivity of Fasciola gigantica in two lymnaeid snails

Two groups of Galba truncatula and two groups of Lymnaea natalensis were experimentally infected with Fasciola gigantica to determine if snail species had an influence on the redial burden and cercarial shedding of this trematode when snails of both species were infected with the same isolate of miracidia. In the two groups used for the study of redial burden, the total number of free rediae was significantly higher at day 49 post-exposure in L. natalensis than in G. truncatula. In the groups used for cercarial shedding, the life-span of cercaria-shedding snails and those of infected snails which died without cercarial emission, and the duration of the prepatent period were significantly longer in L. natalensis than those noted in G. truncatula. However, the mean numbers of shed cercariae did not significantly differ and showed no differences in their daily distribution throughout the shedding period. These results demonstrate that G. truncatula might be the principal intermediate host of F. gigantica in Egypt, at least in the areas where this lymnaeid species lives.     

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.     

Anaerobic incorporation of radioactivity from 2,3-14C-succinic acid into citric acid cycle intermediates and related compounds in the sea musselMytilus edulis L.

Summary Sea mussels were exposed to nitrogen for various periods (0, 1, 3 and 6 days) and subsequently injected with 2,3-¹⁴C-succinic acid. After 2.5 h anaerobic incubation concentrations of succinate, some amino acids and volatile fatty acids were determined as well as the distribution of radioactivity.Conversion of the precursor decreased from 80 to 40%, due to increased dilution with endogenous succinate, accumulated during the anaerobic preincubation period.More than 80% of the activity of the converted 2,3-¹⁴C-succinic acid was incorporated into malate, aspartate, glutamate, alanine and propionate. This indicates that succinate is not only an end product of anaerobic glycogen breakdown, but remains an active intermediate of the tricarboxylic acid cycle, which can still operate under anaerobic conditions.Concentration and radioactivity of propionate were markedly increased after prolonged anoxia, which gives evidence that succinate is actively converted to propionate during anaerobiosis.Observed accumulation of glutamate during anoxia is explained by incomplete oxidation of pyruvate, which leaves the tricarboxylic acid cycle at the stage of 2-ketoglutarate.     

Infectivity of Echinostoma friedi miracidia to different snail species under experimental conditions

The infectivity of Echinostoma friedi (Trematoda: Echinostomatidae) miracidia was studied experimentally in a range of laboratory-reared snails that coexist in the same natural locality, namely Radix peregra, Lymnaea fuscus, L. truncatula (Lymnaeidae), Gyraulus chinensis, Helisoma duryi (Planorbidae) and Physella acuta (Physidae), and snails from different geographical origins acting naturally or experimentally as intermediate hosts of Schistosoma spp., namely Planorbarius metidjensis (from Málaga, Spain), Biomphalaria glabrata (Guadeloupe), B. alexandrina (Egypt) (Planorbidae), Bulinus cernicus (Mauritius), B. globosus (Zambia), B. natalensis (South Africa) and B. truncatus (Niger) (Bulinidae). Six species of snails were found to be susceptible, with the rate of infection ranging from 0 to 36.7%. The highest infection was detected in R. peregra. The low host specificity of E. friedi might have an epidemiological significance as a requisite for a recent establishment in a new geographical area.     

Biomphalaria tenagophila: genetic variability within intermediate snail hosts susceptible and resistant to Schistosoma mansoni infection

DNA analysis by molecular techniques has significantly expanded the perspectives of the study and understanding of genetic variability in molluscs that are vectors of schistosomiasis. In the present study, the genetic variability of susceptible and resistant B. lenagophila strains to S. mansoni infection was investigated using amplification of their genomic DNA by RAPD-PCR. The products were analyzed by PAGE and stained with silver. The results showed polymorphism between tested strains with four different primers. We found two bands of 1,900 and 3,420 bp that were characteristic of the susceptible strains with primer 2. The primers 9 and 10 identified a single polymorphic band that was also characteristic of (3,136 and 5,041 bp, respectively) susceptible snails. Two polymorphic bands were detected by primer 15: one with 1,800 bp was characteristic of the resistant strain and the other with approximately equal to 1,700 bp in the susceptible one. These results provide additional evidence showing that the RAPD-PCR technique is adequate for the study of polymorphisms in intermediate hosts snails of S. mansoni. The obtained results are expected to expand the knowledge about the genetic variability of the snails and to permit the future identification of genomic sequences specifically related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni.     

Parasite development and visceral pathology in Galba truncatula co-infected with Fasciola hepatica and Paramphistomum daubneyi

Histological investigations in Galba truncatula naturally or experimentally co-infected with Fasciola hepatica and Paramphistomum daubneyi were carried out to study parasite development and the responses of the digestive gland and kidney of snails, as larval forms of these digeneans often use these two sites for their growth within the snail's body. The number of live rediae per snail ranged from 2.4 to 4.2 for the dominating parasite (it developed in the digestive gland) and was less than 2.0 for the other species. When the dominating species was F. hepatica, most snails harboured cercariae-containing rediae; if this parasite was P. daubneyi, procercariae-containing rediae with or without free procercariae were observed in most snails. In contrast, most rediae of the other species were immature. The pathology caused by the dominating species in the digestive gland was greater than that recorded in the kidney, where the other parasite was generally located. The most frequent tissue lesions in the digestive gland were generalized epithelial necrosis and epithelial reconstitution. In the kidney, multifocal epithelial necrosis was frequently observed, particularly when P. daubneyi was the dominating species. The frequencies of lesions in the digestive gland agreed with percentages reported by our team in other snails mono-infected with F. hepatica or P. daubneyi. In contrast, multifocal necrosis in the kidney was clearly greater in the present study and this finding might be explained by assuming that a sufficient number of free larvae within the snail would be necessary for the development of epithelial necrosis in the whole kidney.     

Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solanacearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 x 10(5) in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.     

Allopatric combination of Fasciola hepatica and Lymnaea truncatula is more efficient than sympatric ones

Parasites are capable of rapid evolutionary changes relative to their hosts, due to short life cycle, short generation time, and high fecundity. The direction of the evolution of parasite virulence can be studied in cross-transfer experiments, combining hosts and parasites from different localities, and comparing the outcome of established (sympatric and potentially locally adapted) and novel (allopatric) combinations of hosts and parasites. We aimed to compare the compatibility with snails hosts, the infectivity of metacercariae in rabbits and rats, and the fitness among different combinations (French-FF and Spanish-SS sympatries and allopatry-FS). The first isolate of Fasciola hepatica and its corresponding intermediate host, Lymnaea truncatula originated from Lugo's northwestern Spain. The second isolate of parasite and snail was collected in the Limoges area in central France. The Spanish snails were more susceptible to their sympatric trematode than the French snails. The Spanish flukes were more infective to intermediate hosts (snails) than the French flukes, but subsequent definitive hosts (rats or rabbits) infections remained similar. The estimated fitness was low in sympatric infections and highly similar (from 4.7 to 5.3). The fitness similarity corresponds, however, to different variations in life-history traits that could represent different strategies among the host-parasite local combinations. The infection rate in snails, metacercarial productivity, metacercarial infectivity, and the estimated fitness were better for allopatric combination (FS). The susceptibility data showed a higher efficiency of flukes in the allopatric snail population than in their local snail population. However, our results were obtained after one generation and from a single isolate and it remains to be determined if all allopatric fluke-snail isolates may present a better fitness. Nevertheless our results indicate that introduction of liver fluke-infected cattle should be monitored carefully, as it could result in the introduction of more efficient parasites.     

Redial growth and cercarial productivity of Fasciola hepatica in three species of young lymnaeid snails

Experimental infections of 1-mm high snails using three populations of Lymnaea (L. glabra, L. ovata and L. truncatula) and a cattle strain of Fasciola hepatica miracidia were carried out under laboratory conditions to determine if the snail species had an effect on the number of free rediae, their growth, and cercarial productivity in relation to each redial category (R1a, R1b, R2a, or R2b/R3a). The total number of rediae ranged from 6.4 to 7.5 per snail. The mean body length of rediae varied from 1-1.2 mm (R1a) to 0.3-0.4 mm (R2b/R3a). The width of the intrapharyngeal lumen also varied from 26.0-38.8 microm to 3.0-4.2 microm, respectively. The redial category had a significant effect on both measurements, whereas snail species only had a significant influence on body length. The mean number of cercariae produced by all living rediae at day 49 post-exposure ranged from 63.0 in L. glabra to 87.2 in L. truncatula. In L. ovata and L. truncatula, 55.8% and 58.6% of cercariae, respectively, were produced by R2a rediae, whereas 53.9% of cercariae in L. glabra were formed by the R1b rediae. When young snails were infected with F. hepatica, the species of snail had an effect on the number of living rediae, their length and their cercarial productivity.     

Development of a Membrane-Bound Respiratory System Prior to and During Sporulation in Bacillus cereus and Its Relationship to Membrane Structure

Bulk membrane fragments were prepared from cells of Bacillus cereus ATCC 4342 harvested at different stages of growth and sporulation and examined for enzymes involved in electron transport functions. The presence of succinate: DCPIP oxidoreductase (EC 1.3.99.1), succinate: cytochrome c oxidoreductase (EC 1.3.2.1), NADH:DCPIP oxidoreductase (EC 1.6.99.1), NADH:cytochrome c oxidoreductase (EC 1.6.2.1), succinate oxidase [succinate: (O(2)) oxidoreductase, EC 1.3.3.1], and NADH oxidase [NADH:(O(2)) oxidoreductase, EC 1.6.3.1] were demonstrated in membrane fragments from vegetative cells, early and late stationary-phase cells, and in cells undergoing sporulation. During the transition from a vegetative cell to a spore, there was a significant increase in the levels of enzymes associated with energy production via the electron transport system. Cytochromes of the a, b, and c type were detected in all membrane preparations; however, there was a marked increase in the level of cytochromes by the end of vegetative growth which remained throughout sporulation; there were no qualitative changes in the cytochromes throughout growth and sporulation. Sporulation was inhibited by cyanide, stressing the significance of the electron transport system. Enzyme activities were partially masked in washed membrane fragments; however, unmasking (stimulation) was achieved by sodium deoxycholate, sodium dodecyl sulfate, or Triton X-100. The degree of enzyme masking was less in vegetative cell membrane fragments than in membranes prepared from stationary-phase or sporulating cells. Results indicate the development of a membrane-bound electron transport system in B. cereus by the end of growth and prior to sporulation, which results in an increased masking of a number of enzymes associated with the terminal respiratory system of the cell.     

Studies on resistance in snails: A specific tissue reaction to Echinostoma lindoense in Biomphalaria glabrata snails

In juvenile albino Biomphalaria glabrata snails exposed for the first time to Echinostoma lindoense miracidia, and observed to be resistant, the sporocysts migrated to the heart at the same speed as they did in susceptible snails. However, in resistant snails the sporocysts were soon destroyed in the heart by amebocyte clumps. When these snails were then re-exposed to miracidia of the same species of trematode, the sporocysts were quickly destroyed soon after miracidial penetration, chiefly in the head-foot region. This strongly accelerated tissue reaction appears to have been induced by the previous contact with the same parasite. The sensitization of the snail tissues was highly specific: the hosts remained susceptible to Schistosoma mansoni and Paryphostomum segregation (Echinostomatidae), although partial resistance was observed against Echinostoma paraensei and E. liei, which are closely related to E. lindoense.