Storage and incubation of Echinostoma revolutum eggs recovered from wild Branta canadensis, and their infectivity to Lymnaea tomentosa snails

Echinostoma revolutum eggs recovered from naturally infected wild Canada geese (Branta canadensis) were cold stored (4-6 degrees C) for up to 72 weeks. Successful hatching followed incubation for from 6 to 8 days at an optimum temperature of between 25 and 30 degrees C. A partial life cycle from adult worm to metacercarial encystment in Lymnaea tomentosa snails was completed in the laboratory. Snails were infected both by free miracidia and by ingestment of unhatched embryonated eggs. Infection was equally successful in environmental temperature ranges from 10 to 25 degrees C, and at challenge levels of 2, 5 or 10 embryonated eggs per snail. Exposure to 10 eggs was lethal. Ingestion by snails of embryonated eggs with successful infection at 10 degrees C suggests that embryonated eggs may be used to infect wild snails when the environmental water temperature has reached 10 degrees C.     

Effect of duration and intensity of infection with Echinostoma audyi on survival of Lymnaea rubiginosa exposed to copper sulfate.

Lymnaea rubiginosa infected with 1 to 5 miracidia of Echinostoma audyi for 1, 2, 3, and 5 weeks, and snails infected with up to 10 or 20 miracidia for 3 weeks, were exposed to copper (Cu) as CuSO4 2 hr and allowed to recover 48 hr. In general, snails ininfected with up to 5 miracidia of E. audyi were no more susceptible to Cu than noninfected snails. Snails infected with up to 10 miracidia for 3 weeks were 1.3 times more susceptible to Cu than noninfected snails. However, snails infected with up to 20 miracidia for 3 weeks were not more susceptible to Cu, possibly because infection with such a large number of parasites eliminated the most susceptible snails from the population prior to exposure to Cu.     

Influence of low temperatures on the cercarial shedding of Paramphistomum daubneyi from the snail Lymnaea truncatula

Lymnaeids in nature are subjected to temperature fluctuations that may have an influence on the shedding of cercariae. Thus, experimental infections of Lymnaea truncatula by Paramphistomum daubneyi were performed to determine whether a sudden fall in temperature--daily dipping of infected snails into spring water at 14-15 degrees C, or at 6-8 degrees C--followed by its increase at 20 degrees C in the subsequent hour, had an influence on the characteristics of snail infection and cercarial production. The immersion of infected snails in cold water during a short period delayed the first cercarial shedding, at day 66 on average, in the 6-8 degrees C group vs at day 57 in the 14-15 degrees C group, or at day 49 in the 20 degrees C group. The percentage of cercaria-shedding snails was greater in the 6-8 degrees C group than in the 14-15 degrees C and the 20 degrees C ones: 41.8% vs 17.3% and 7.1%, respectively. The total number of cercariae given by each infected snail was also higher in the 6-8 degrees C group. A fall in the temperature of water during daily change, followed by its increase at 20 degrees C within the subsequent hour, stimulated the cercarial shedding of P. daubneyi.     

Resistance to desiccation of Biomphalaria glabrata adults infested by various miracidia of Schistosoma mansoni

1200 adult Biomphalaria glabrata were submitted during 6 weeks to anhydrobiosis condition. Some snails were healthy, some were previously infected 3 days or 12 days ago with 8 +/- 2 miracidia of Schistosoma mansoni, others were shedding cercariae. The snails were put on soil or buried into hermetically closed, or ventilated, plastic boxes. There was no survival of snails kept in sealed boxes, or among positive snails, but 44% of control healthy snails and 40.6% of infected (for 3 or 12 days) snails in ventilated boxes were living at the term of the desiccation stage. Survival was better for "on soil" snails than for "buried" snails, but no difference was shown between 3-days and 12-days infection. The surviving desiccated B. glabrata had a lesser death rate and a lesser cercarial production than infected snails kept in water. An inferior production of male cercariae comparatively to female and to "mixed" cercariae was demonstrated by statistical analysis of the cercarial sheddings. In all positive snails, periodic variations of cercarial production was shown, whatever the sex of those cercariae. In addition many pauses of the sheddings were established by the authors.     

Diplostomum spathaceum (Rud. 1819): effect of miracidial age and lifespan on miracidial infectivity

Experiments were performed to study the effect of age and lifespan on the infection success of Diplostomum spathaceum miracidia. Specimens of Lymnaea peregra and L. stagnalis were individually exposed to single miracidia of D. spathaceum of different ages under conditions of constant illumination at 20 degrees C. The life-span of the free-swimming miracidium of D. spathaceum is 24 h at 20 degrees C. It was observed that as the miracidium ages, its ability to successfully infect snails declines rapidly.     

Photoperiod and temperature interaction in the determination of reproduction of the edible snail, Helix pomatia

Snails were kept in self-cleaning housing chambers in an artificially controlled environment. Mating was frequent under long days (18 h light) and rare under short days (8 h light) regardless of whether the snails were kept at 15 degrees C or 20 degrees C. An interaction between photoperiod and temperature was observed for egg laying. The number of eggs laid (45-50/snail) and the frequency of egg laying (90-130%) were greater in long than in short days (16-35/snail and 27-77%) but a temperature of 20 degrees C redressed, to some extent, the inhibitory effect of short days. At both temperatures only long photoperiods brought about cyclic reproduction over a period of 16 weeks, confirming the synchronizing role of photoperiod on the neuroendocrine control of egg laying in this species of snail.     

Rearing temperature influences flavivirus vector competence of mosquitoes

Culex annulirostris Skuse mosquitoes (Brisbane strain) were reared at 20 degrees C or 27 degrees C and the adult females were experimentally infected by feeding Murray Valley encephalitis virus (MVE). They were then maintained (a) in the insectary at 20 degrees C, after rearing at either 20 degrees C or 27 degrees C; (b) at ambient outdoor temperatures, range 12.2-28.9 degrees C, mean 19.6 degrees C; or (c) at 27 degrees C after rearing at 27 degrees C. There was no significant difference in rates of MVE infection or transmission when mosquitoes were reared and maintained constantly at 20 degrees C or 27 degrees C. However, for females kept at reduced temperature (i.e. mean = 19.6 degrees C or 20 degrees C after rearing at 27 degrees C), the infection and transmission rates of MVE were significantly reduced (2 x 8 replicates). This investigation illustrates that vector competence is depressed by decreasing temperatures for adult mosquitoes compared with those they experienced during development. Similar patterns were evident with previously published work on Japanese and St Louis encephalitis, dengue and yellow fever. The process appears to be reversible, i.e. increased temperature raises virus infection and transmission rates. It is concluded that, without incubation at warmer temperatures, flavivirus recovery from overwintering mosquitoes will be negatively biased.     

Schistosomatium douthitti: Exposure of Lymnaea catascopium to irradiated miracidia

Irradiation of Schistosomatium douthitti miracidia (4000, 5000, or 6000 rad) did not substantially alter their behavior or ability to penetrate their snail host. Treatment with 4000 rad was not sufficient to prevent all miracidia from establishing patent infections in Lymnaea catascopium, although significantly fewer snails exposed to these miracidia shed cercariae than did controls exposed to normal miracidia. Irradiation of miracidia with either 5000 or 6000 rad totally prevented cercarial production. Although destruction of irradiated mother sporocysts by encapsulating amebocytes was occasionally observed, most expanded without concomitant multiplication of germinal cells and embryo production and then collapsed. They generally persisted in this state throughout the period of observation (32 days). Snails sensitized by exposure to irradiated miracidia and challenged 2 or 10 days later with normal miracidia were as likely to develop patent infections as were snails exposed only to normal miracidia. Double sensitization of snails with irradiated miracidia also failed to confer protection upon challenge with normal miracidia. Most challenge sporocysts developed normally, often in close proximity to collapsed irradiated sporocysts.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

Radix natalensis (Gastropoda: Lymnaeidae), a potential intermediate host of Fasciola hepatica in Egypt

Experimental infections of Egyptian Radix natalensis with French miracidia of Fasciola hepatica were carried out to determine if this snail might act as an intermediate host in the life cycle of this digenean in Egypt. Single exposures of R. natalensis to miracidia (2/snail) and two successive exposures (a total of 4 miracidia/ snail) were performed using lymnaeids measuring 1 to 6 mm in height. Live larval forms of F. hepatica were noted in single- and double-exposed snails. In double exposures, a significant increase of snail survival on day 28 post-exposure (at 24 degrees C) and an decrease in prevalence were noted when the height of snails at exposure was increasing. Cercariae of F. hepatica were shed by these snails (90.7/snail) during a mean patent period of 24.3 days. All snails have released these cercariae during 2-13 waves of shedding. According to these results, R. natalensis can be considered a potential intermediate host of F. hepatica in Egypt.     

Influence of thermal and nutritional acclimatization on body temperatures and metabolic rate

1. An investigation of the influence of previous thermal and nutritional experience on body temperatures and metabolic rate has been carried out with growing piglets. Littermates were kept, from shortly after birth, at either 10 or 35 degrees C and fed either a high (H) or a low (L) energy intake. At 8 weeks of age the animals were exposed to a series of environmental temperatures of 10, 20, 27 and 35 degrees C for 1.5 hr and their rates of oxygen consumption were determined over the last 45 min. At the end of the session body temperatures were measured. 2. Rectal temperatures measured 24 hr after the start of the last meal were higher at each test temperature in piglets which had been living at 35 degrees C than in those at 10 degrees C. Also, rectal temperatures were higher in those on the H intake for animals which had been living in either the hot or the cold environment. 3. Skin temperature on the back was similar in all groups at any given test temperature although there was a tendency for those on an H intake to have the higher temperatures. Skin temperatures of the legs and ears were higher in the 10H and 10L groups than in the 35H or 35L groups at all the test environmental temperatures; energy intake had little effect. 4. Metabolic rate was greater for the animals on the H than the L intake, for those which had been living at either 10 or 35 degrees C at all the test environmental temperatures. The analysis did not reveal any significant difference related to the overall effect of living temperature, which was independent of energy intake. 5. At thermal neutrality (27 degrees C) there was a significant interaction, between energy intake and normal living temperature, on metabolic rate. Living temperature was found to modify the effect of intake: the difference between the two intakes was greater in those from the cold environment than from the hot.     

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.     

The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.     

The effect of variations in host and parasite density on the level of parasitization of Lymnaea truncatula by Fasciola hepatica

Groups of the snail Lymnaea truncatula, maintained at a range of spatial densities, were exposed to different densities of miracidia of Fasiola hepatica. The resulting degree of parasitization was measured 3--4 weeks after infection. The relationship between parasite densities and parasitization appeared to be curvilinear. The lower than expected parasitization at high parasite densities could be explained by the multiple infection of some snails by two or more miracidia. The level of parasitization was not related exponentially to the temperature at which infection was carried out. This was thought to be due to the inverse relationship between miracidial longevity and swimming speed, with respect to temperature. A depth of free water overlying a mud surface was an absolute requirement for miracidia to successfully infect snails.     

Schistosoma mansoni: influence of Biomphalaria glabrata size on susceptibility to infection and resultant cercarial production

Biomphalaria glabrata snails of the same age, but different sizes, were used to determine size-related susceptibility to Schistosoma mansoni miracidial infection and the influence of snail size on total cercarial production. Snails with shell diameters from less than 5 to greater than 17 mm were individually exposed to one or several miracidia, depending on the experiment. In snails exposed to multiple numbers of miracidia, the percentage of snails which developed patent infections was lower in snails with larger shell sizes. This was also reflected by fewer primary sporocysts per infected snail found in tissues of the larger snails. Upon determining cercarial production in these groups over a 1-month period there were no statistical differences between any groups in the numbers of cercariae produced per snail. However, upon determining the number of successful primary sporocysts found in cohort snails of each size group, cercarial production increased as a function of the number of successful primary sporocysts. This was verified by examining cercarial production in various size snails with known monomiracidial infections. Our data therefore confirm and extend earlier work using snails infected with unknown numbers of miracidia and clearly show that total S. mansoni cercarial development and decreased susceptibility of snails is a direct reflection of snail size and not necessarily age of the snail.     

Influence of temperature on survival, growth and fecundity of the freshwater snail Indoplanorbis exustus (Deshayes).

To note the effect of temperature on survival, growth and fecundity, newly hatched (zero day old) snails Indoplanorbis exustus were cultured at 10 degrees, 15 degrees, 20 degrees, 25 degrees, 30 degrees and 35 degrees C constant temperatures and room temperature (17.5 degrees-32.5 degrees C). Individuals exposed to 10 degrees C died within 3 days while those reared at 15 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees C and room temperature survived for a period of 6, 27, 18, 16, 12 and 17 weeks respectively. An individual added on an average 0.21 mm and 0.45 mg, 0.35 mm and 7.94 mg, 0.63 mm and 15.5 mg, 0.81 mm and 27.18 mg, 1.07 mm and 41.48 mg and 0.78 mm and 31.2 mg to the shell diameter and body weight respectively at those temperatures per week. The snails cultured at 15 degrees C died prior to attainment of sexual maturity. On an average, an individual produced 31.9 and 582.77, 54.86 and 902.18, 56.01 and 968.45, 49.32 and 798.68 and 62.34 and 1143.97 capsules and eggs respectively at 20 degrees, 25 degrees, 30 degrees, 35 degrees C and room temperature (17.5 degrees-32.5 degrees C).     

Supercooling ability in two populations of the land snail Helix pomatia (Gastropoda: Helicidae) and ice-nucleating activity of gut bacteria

The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gotaland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T=5 degrees C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T(c)=-6.4+/-0.8 degrees C), significantly greater, however, than the supercooling capacity of the population from France (T(c)=-4.6+/-1.4 degrees C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T=20 degrees C), both populations exhibited a similar high T(c) (-2.0+/-1.0 degrees C). The lower T(c) of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg(-1)) and active (165-215 mOsm kg(-1)) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than -9 degrees C. Only the strain Kluyvera sp. initiated nucleation at -9 degrees C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of hibernating snail populations, triggering changes in osmolyte concentration, rather than the presence of endogenous ice-nucleating-active bacteria, accounts for fluctuations in their T(c).     

Echinostoma liei miracidia and Biomphalariaglabrata snails: Effect of egg age,habitat heterogeneity,water quality and volume on infectivity

Infectivity of Echinostoma liei miracidia to NIH albino Biomphalaria glabrata declines significantly from 62% with eggs incubated for 10–24 days to 3% for eggs incubated for 30–42 days. In mass exposures of 25 snails to 125 miracidia in 1 liter of water infectivity was high (54–66 %) and not affected by the presence of lettuce, plastic sheets, chalk, detritus or snail-conditioned water. In distilled water or snail-conditioned water the proportion of infected snails exposed singly to five miracidia per snail in 5 ml was not significantly different from the results of mass exposures of 25 snails in 1 liter to the same snail: miracidia ratio. Some evidence is presented suggesting that infected snails are less likely to suffer mortality than uninfected snails during the first 7–10 days post-exposure.The results suggest that Echinostoma liei miracidial searching efficiency is robust in volumes of at least 1 liter and in a heterogeneous habitat. These aspects enhance the competitive potential of echinostomes as possible biological control agents for Schistosoma mansoni.     

On the distribution of schistosome infections among host snails

The distribution of Schistosoma haematobium infections in a population of Bulinus globosus snails collected from a field site in Zimbabwe is described. Cercariae of both sexes emerged from a proportion of patent infected snails, as was demonstrated by the detection of paired schistosomes in hamsters each exposed to cercariae from a single snail. These snails must have been infected by two or more miracidia. Analysis showed that the proportion of mixed-sex infections was higher than expected if infections were distributed at random among snails. An index of overdispersion was calculated. Overdispersion of infections results from heterogeneities in the exposure and/or susceptibility of snails to infection. The implications for the epidemiology and control of schistosomiasis haematobium are discussed.     

Effects of temperature on survival,infectivity and in vitro encystment of the cercariae of Echinostoma caproni

The effects of temperature on survival, infectivity and in vitro encystment of Echinostoma caproni cercariae in artificial spring water (ASW) were studied. Effects of aging cercariae in ASW at various temperatures showed that at 23 degrees C cercariae achieved 50% survival in 24 h, compared to 92 h at 12 degrees C. Cercariae aged in ASW at 28 and 37.5 degrees C showed 50% survival at 16 and 10 h, respectively. Cercariae aged at different temperatures for various times were used to infect juvenile Helisoma trivolvis (Colorado strain) snails maintained in ASW at 23 degrees C. Index of infectivity was based on counting encysted metacercariae in the snails at 8 to 12 h post-infection. Cercariae aged at 23, 28 and 37.5 degrees C showed 50% encystment at 6, 8 and 4 h, respectively. Cercariae aged at 4 degrees C showed 50% encystment in 10 h and cercariae aged at 12 degrees C showed 50% encystment beyond 16 h. Cercariae showed maximal longevity and infectivity in snails when aged at 12 degrees C in ASW. For E. caproni, as in other digeneans, the infective period of cercariae is markedly shorter than the maximal life-span at any given temperature. Studies on in vitro encystment of E. caproni cercariae in Locke's solution:ASW (1:1) showed that encystment was optimal at 23 degrees C (78% encystment) and that it declined to 44% at 28 degrees C and became almost nil (0.02%) at 12 or 37.5 degrees C.