Trichobilharzia ocellata: influence of infection on the interaction between the dorsal body hormone, a female gonadotropic hormone, and the follicle cells in the gonad of the intermediate snail host Lymnaea stagnalis

Activation of adenylate cyclase (AC)-cAMP system in follicle cells of Lymnaea stagnalis by the gonadotropic dorsal body hormone (DBH) is inhibited by schistosomin, an agent present in hemolymph of snails infected with Trichobilharzia ocellata. AC activation was determined enzyme cytochemically. This conclusion is based on the observation that the percentage of oocytes with AC-positive follicle cells in gonads incubated in the presence of schistosomin, i.e., in serum of infected snails (IS) with DBH, is significantly lower than that in gonads incubated in the absence of schistosomin, i.e., in serum of noninfected snails (NS) with DBH. Follicle cells in gonads preincubated in the absence of schistosomin, i.e., in NS, and subsequently incubated with freshly dissolved DBH showed a considerably lower response to DBH than those in not preincubated gonads. This indicates that the number of receptors for DBH on follicle cells had decreased during preincubation. The response to DBH also appeared to decrease when the hormone was preincubated in NS. This indicates that the activity of DBH decreases during preincubation. These data make it impossible to answer the question of whether or not schistosomin acts as an antagonist of DBH at the receptor level.     

Trichobilharzia ocellata: interference with endocrine control of female reproduction of Lymnaea stagnalis

Calfluxin (CaFl), one of the gonadotropic hormones of Lymnaea stagnalis, stimulates the influx of Ca2+ into the mitochondria of the cells of the albumen gland, one of the accessory sex organs of the snail. This effect is suppressed in glands of noninfected snails by an agent (schistosomin) present in the hemolymph of snails infected by Trichobilharzia ocellata as shown in in vitro experiments. The agent is present from 6 weeks postinfection onward. Ca2+ deposits in the mitochondria were demonstrated with the ultracytochemical antimonate precipitation technique. The percentage of Ca2+-positive mitochondria was taken as a measure for the effects of CaFl. This percentage appeared to be greatly reduced when glands were incubated in serum of infected snails (Sinf). The data showed that Ringer incubations can serve as controls for experiments with serum: no differences were found between Ringer incubations and incubations in either fresh or frozen serum of noninfected snails. Schistosomin was not affected by freezing, which enables cold storage of Sinf. The dose-response relationship of schistosomin shows that at a 1:2 dilution of Sinf with Ringer the response to CaFl was reduced more than 50%. Schistosomin is heat-stable and Pronase-labile, which indicates that it has a peptide nature. Probably schistosomin(s) is responsible for the reduction/cessation of fecundity in trematode-infected snails.     

Schistosomin, an antagonist of calfluxin

The percentage of Ca2+ positive mitochondria in the cells of the albumen gland of Lymnaea stagnalis was used as a measure for the effect of the gonadotropic hormone calfluxin (CaFl) and for the interaction between schistosomin, an agent present in the hemolymph of snails infected with the schistosome Trichobilharzia ocellata, and CaFl. From 3 weeks postexposure on glands of infected snails showed a lower response to CaFl than glands of noninfected snails. The response decreased with infection time. Apparently the glands are already affected at an early stage of parasitosis. Albumen glands of noninfected snails preincubated in serum of infected snails and subsequently incubated with CaFl gave a lower response to CaFl than control glands. The response of preincubated glands appeared to increase when the glands were rinsed in Ringer during increasing periods of time (up to 1 hr) before being incubated with CaFl. This indicates that schistosomin binds relatively tightly to albumen glands, probably to a receptor complex. Rinsing (1-2 hr) did not influence the (low) response to CaFl of glands of infected snails, whereas the response of glands of noninfected snails increased considerably after a 1- or 2-hr period of rinsing. The results probably indicate that only a low number of receptors for CaFl is still present in glands of infected snails. In glands exposed first to schistosomin and then to CaFl, the inhibition of the effect of CaFl was stronger than in glands exposed to schistosomin and CaFl at the same time. The response to CaFl appeared to increase when a higher concentration of CaFl was used. These data indicate that schistosomin and CaFl interact at the level of receptor complex(es) in the gland. It is argued that both substances not necessarily act at the level of one and the same receptor complex.     

Interaction of photoperiod and nutritive state in female reproduction of Lymnaea stagnalis

Summary

A study was made of the interaction of the photoperiod and the availability of food in influencing egg laying of the freshwater snail Lymnaea stagnalis. At a long day photoperiod (LD = 16 h light/8 h dark) egg laying of fed snails had increased compared with that of fed animals kept at a medium day photoperiod (MD = 12 h light/12 h dark). In MD snails oviposition ceased within a week of the beginning of a starvation period. This is most probably due to a reduction in the activities of the neuroendocrine caudodorsal cells which secrete an ovulation hormone. In contrast, in starved LD snails a low rate of ovipository activity continued, indicating that a lowered frequency of caudodorsal cell release cycles occurred under these conditions. The decreased mean size (number of eggs) of the egg masses in starved LD snails indicates that the activities of the endocrine dorsal bodies, which control vitellogenesis and synthetic activities in the female accessory sex organs, had decreased.

All MD snails survived, but nearly all LD snails died during the course of the experiment. Determinations of the mantle glycogen stores of LD snails suggest that the high mortality of LD snails is due to exhaustion of the animal's energy reserves.     

Isolation of schistosomin, a neuropeptide which antagonizes gonadotropic hormones in a freshwater snail

The molecular mechanisms underlying parasite-induced inhibitory effects on host reproduction were studied in the freshwater snail, Lymnaea stagnalis, infected with the schistosome parasite Trichobilharzia ocellata. This combination is used as a model system for host-parasite interactions involved in schistosomiasis transmission. The female gonadotropic snail neuropeptide, calfluxin, was labelled with fluorescein isothiocyanate (FITC) and used as a ligand in receptor-binding studies on membranes of its target organ, the albumen gland. The binding of calfluxin to its receptor-guanyl-nucleotide-binding-protein (G-protein) complex was inhibited in vitro in the presence of haemolymph of schistosome-infected snails. This inhibition appeared to be established by a peptidergic factor called schistosomin. The receptor assay was used to identify schistosomin from haemolymph during subsequent purification and characterization steps. The peptide could also be purified from the central nervous systems of non-infected snails, indicating that it is produced by the snail itself and released into the haemolymph as a result of infection. Analysis by plasma-desorption mass spectrometry revealed that purified schistosomin has a molecular mass of 8780 Da.     

Immuno-electron microscopy of formation,degradation and exocytosis of the ovulation neurohormone in Lymnaea stagnalis

Summary The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20–36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive.The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules.     

Effects of the serotonin receptor ligand methiothepin on reproductive behavior of the freshwater snail Biomphalaria glabrata: reduction of egg laying and induction of penile erection

The biogenic monoamine serotonin (5-HT) has been reported to enhance egg laying in the freshwater gastropod Biomphalaria glabrata, an intermediate host for human blood flukes. Methiothepin, a vertebrate 5-HT(1/2) receptor ligand which binds with high affinity to a 5-HT receptor (5-HTlym) in Lymnaea stagnalis was tested for its ability to block egg laying in B. glabrata as a possible target for snail control. A single 30-min application of methiothepin (1 microM) was sufficient to prevent egg laying for over 1 week and did so in a dose-dependent fashion. Furthermore, single applications of methiothepin (1 microM and 10 microM) induced penile erection in a high percentage of snails tested. Latency to erection was long (at least 8 hr), but the duration of erection was long-lasting (up to 48 hr). Despite the erections, methiothepin-treated snails failed to achieve copulations. The pharmacological effect of methiothepin on both male and female reproductive processes is similar to that produced in other molluscs, and points to the gene for the 5-HT receptor mediating or modulating both or either processes as a potential target of snail control strategies. J. Exp. Zool. 289:202-207, 2001.     

The effects of starvation and refeeding on egg laying and the synthetic activity of the albumen gland in Bulinus truncatus, a snail vector of urinary schistosomiasis

1. The effects of starvation (for 1, 2, 3, 6, 9 and 12 days, respectively) and refeeding (12 days starvation and 1, 2 and 3 days refeeding, respectively) on egg laying and albumen gland activity in the freshwater snail, Bulinus truncatus were studied. 2. The egg laying of starving snails rapidly decreased and ceased by day 6 of starvation. Egg laying was restored 24 hr after refeeding. The recorded decrease in albumen gland wet weight was proportional to the starvation periods. The DNA contents of the glands of the different experimental groups was not statistically different from the controls. 3. Albumen gland synthetic activity expressed as 14C-glucose incorporation into galactogen/microgram DNA and 3H-amino acids into total protein was determined. The glands showed an abrupt decrease in synthetic activity after 1 day of starvation and gradually decreased further until days 9-12. The decrease in activity of the glands was more rapid than that of egg laying. Upon refeeding, the activity of the glands recovered rapidly, simultaneous with the increase in wet weight and egg laying. 4. In conclusion, there is a correlation between egg laying and the in vitro activity of albumen glands. The results show a short-term effect of starvation on the fecundity of the snails. Such studies could be useful in field studies as well as snail control by applying molluscicides under optimal conditions.     

Interaction of Photoperiod,Grouping and Isolation in Female Reproduction of Lymnaea stagnalis

Summary

A study was made of the effects of the photoperiod on female reproductive activities in grouped and isolated specimens of the hermaphrodite freshwater snail Lymnaea stagnalis. Isolation of snails rapidly increased (< 3 days) egg production. The effect was long-lasting, and was probably caused by deprivation of copulation. Changes of photoperiod had similar effects in isolated and grouped snails. Compared to the effects of isolation, the photoperiodic effects were relatively slow in appearing (> 14 days). In comparison with a medium day (MD) (12 hr light/12 hr dark), a long day (LD; 16 hr light/8 hr dark) stimulated egg production whereas a short day (SD) (8 hr light/16 hr dark) lowered egg production. The LD effects were clearer in grouped than in isolated snails, but SD effects were clearer in isolated than in grouped snails. It is argued that the effects of grouping, isolation and photoperiod on egg production are mediated by the neuroendocrine system. It is suggested that the photoperiod plays an important role in the control of the egg laying season. Mating is probably important in the control of fecundity in relation to population density.     

The effect of mating on female reproduction across hermaphroditic freshwater snails

Sexual conflicts often arise between mating partners because each sex tries to maximize its own reproductive success. One major male strategy to influence a partner's resource allocation is the transfer of accessory gland proteins. This has been shown to occur in simultaneous hermaphrodites as well as in organisms with separate sexes. Although accessory gland proteins affect the investment of resources in both male and female function, we here specifically focus on female investment. In the great pond snail, Lymnaea stagnalis, previous studies found that the accessory gland protein ovipostatin reduced female fecundity by suppressing egg laying in the partner in the short term (days). To investigate whether this reduction in egg laying is a commonly found effect of mating in freshwater snails, we compared egg output for evidence of suppression in isolated and paired snails of eight pulmonate species. Furthermore, we determined whether the suppression of egg laying caused a shift in resource allocation to the eggs. We found that in five of the eight species egg laying was suppressed, with fewer and lighter egg masses being laid when they had access to a mating partner. In mated pairs of L. stagnalis and Biomphalaria alexandrina, allocation of resources to the eggs was altered in opposite ways: individuals of L. stagnalis laid fewer but larger and heavier eggs; individuals of B. alexandrina laid smaller and lighter eggs, with no change in egg numbers. Such changes in the female function are most likely the result of combined effects of receiving accessory gland proteins, and the cost of mating in both male and female roles. Thus, effects of the maternal environment, including the receipt of accessory gland proteins, on offspring investment are not restricted to species with separate sexes.     

Changes in molluscan neurosecretory cells during reproductive cessation: cause or effect?

The pond snail Lymnaea stagnalis has a maximum life span of about 22 months. At the age of about 250 days animals start to decrease egg laying activity and at about 500 days most animals ceased egg laying activity. At the age of cessation of egg laying the neurosecretory caudodorsal cells (CDCs) which control egg laying in Lymnaea exhibit reduced branching patterns. At this stage the cells still exhibit their physiological properties. CDCs still contain biologically active peptides and in the isolated CNS they still exhibit an afterdischarge upon electrical stimulation. Probably in the intact animal cessation of egg laying occurs because the CDCs are not activated anymore by natural egg laying inducing stimuli. In very old animals CDCs exhibit signs of degeneration indicating that cell death occur. After an extended period of no egg laying of Lymnaea physiological changes occur in the CDCs. CDCs from animals after an extended period of no egg laying failed to exhibit an afterdischarge. In such CDCs chemical and electrical coupling among the CDCs are reduced. Morphologically reduced CDCs predominantly fail to exhibit an afterdischarge. However, there are minimally branched CDCs that still could give an afterdischarge. Probably morphological reduction is not the only factor that defines afterdischarge failure. At present we suggest the following sequence of changes. 1. Morphological reduction of CDC branching patterns. 2. Cessation of egg laying. 3. Physiological changes in the CDCs resulting in afterdischarge failure. 4. Further morphological and physiological deterioration of CDCs.     

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.     

Gene expression in early development of Lymnaea stagnalis

Pond snails (Lymnaea stagnalis) were irradiated with doses of 2760 r or 5520 r in order to induce lethal factors in the germ cells. The descendants of these irradiated snails were individually reared to adulthood, when by self-fertilization they produced egg masses. The occurrence of lethal factors was studied in these egg masses and the stage at which they manifested themselves was determined. No developmental disturbance was found during cleavage, but gastrulation was affected. This indicates that gastrulation in Lymnaea is controlled by the genome of the embryo. The stage at which most genes interfere with development is the early trochophore stage. At later stages the number of genes, which for the first time are active in development, declines. Since the late veliger stage was never affected, all genes essential for development, are apparently functioning before this stage.     

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.     

The effect of temperature,darkness, starvation and various food types on growth,survival and reproduction of Helisoma duryi,Biomphalaria alexandrina and Bulinus truncatus (Gastropoda: Planorbidae)

Helisoma duryi has been proposed as a biological control agent in schistosomiasis due to its superiority in laboratory competition experiments with various species of the intermediate host snails. Therefore it was considered important to evaluate the response of this snail species and the intermediate host species, Biomphalaria alexandrina and Bulinus truncatus, to various physical, chemical and biological factors under laboratory conditions in order to obtain information on the similarities in the ecological niches of these species. The factors considered in the present paper are: temperature, darkness, starvation and food. All three species had optimal growth and egg laying at 26–28 °C. Only H. duryi survived for a longer period at 33°C and it was capable of starting egg laying at this temperature although the onset was delayed. However, low temperature (18°C) caused a relatively larger decrease in egg laying of H. duryi than in the other two species. Growth and egg laying was reduced for H. duryi and B. truncatus kept under darkness and B. alexandrina could not tolerate maintenance under darkness. A few days of starvation of juvenile snails had no effect on later growth and egg laying capacity of the survivors, although mortality in B. truncatus was increased. B. alexandrina had a lower tolerance to starvation than the other two species. Egg laying of snails fed only one of the three laboratory food types decreased for all three species in the order: Vov-vov (dog food in dry pellets), Tetramin (fish food) and lettuce. Combinations of lettuce and one or more proteinaceous food types gave optimal growth and egg laying for all three species.     

A photoperiod gene regulates vitellogenesis in Lymnaea peregra (Mollusca: Gastropoda: Pulmonata)

Summary

Populations of the fresh-water snail Lymnaea peregra from different localities vary in their capacity to lay eggs, responding either to long day or to short day photoperiods. These responses are genetically determined by a single gene locus with the long day allele dominant over the short day allele. Although previtellogenic stages of oogenesis are not affected by an inhibitory photoperiod, vitellogenesis is essentially shut down. The volumes of the dorsal bodies, which are endocrine organs that mediate vitellogenesis, are significantly smaller in snails maintained in an inhibitory photoperiod. Implantation of cerebral ganglia with attached dorsal bodies, both from egg laying animals and from dormant animals that have the potential to lay eggs in the postoperative photoperiod, into snails that were inhibited by insufficient photoperiod resulted in a significant increase in vitellogenesis in the ovotestes of the hosts. An increase in host dorsal body volume was also observed. The photoperiod gene appears to control some aspect of the cerebral ganglion that activates the dorsal body to produce a hormone that stimulates vitellogenesis in the ovotestes. Photoperiodic control of reproduction may be a factor contributing to the diverse life history strategies observed in this species.     

Correlation between steroid sex hormones, egg laying capacity and cercarial shedding in Biomphalaria alexandrina snails after treatment with Haplophyllum tuberculatum

Schistosomiasis is considered the second most pre-valiant worldwide parasitic disease ranked next to malaria. It has significant economic and public health consequences in many developing countries. Several ways have been practiced in order to bring the disease under an adequate control through the breakage of the life cycle of the parasite. Snail control could be regarded as a rapid and efficient of reducing or eliminating transmission and remains among the methods of choice for schistosomiasis control. The aim of this work is to evaluate the role of Haplophyllum tuberculatum (family Rutaceae) as a plant molluscicide. The mortality rate of Biomphalaria alexandrina snails were monitored after treatment with three extracts of the plant aerial parts; petroleum ether, chloroform and ethanol. Chloroform extract that recorded the most potent effect was further evaluated through measuring the toxicity pattern against B. alexandrina snails, egg laying capacity, cercarial shedding, phenol oxidase enzyme and the levels of steroid sex hormones. Histopathological examination of hepatopancreas and ovotestis of treated snails were also done for result confirmation. Treatment of snails by chloroform extract recorded reduction in egg laying capacity, decrease in cercarial shedding, diminution in phenol oxidase enzyme, disturbance in steroid sex hormones and sever alternation of the histopathological picture of snails tissue. In conclusion, H. tuberculatum recorded molluscicidal potency against B. alexandrina snails. Further studies are needed for its environmental applications.     

The suitability of several aquatic snails as intermediate hosts for Angiostrongylus cantonensis.

Sixteen species of aquatic snails of four families were tested by quantitative technique under standardized conditions for their suitability as intermediate hosts for Angiostrongylus cantonensis. These species were the planorbid snails Biomphalaria glabrata, Biomphalaria alexandrina, Planorbis planorbis, Planorbis intermixtus, Bulinus truncatus, Bulinus contortus, Bulinus africanus, Bulinus tropicus and Helisoma sp.; the lymnaeid snails Lymnaea natalensis, Lymnaea tomentosa, Lymnaea stagnalis, and Stagnicola elodes; the physid snail Physa acuta (an Egyptian and a German strain) and the ampullariid snails Marisa cornuarietis and Lanistes carinatus. All these snail species proved to be susceptible to infection with A. cantonensis, and first stage larvae reached the infective third stage in all of them. However, the rate and intensity of infection varied with different species. B. glabrata was the most susceptible snail species with a 100% infection rate and an average percentage recovery of third stage larvae of 26.1. This was followed by S. elodes and B. africanus, with a 100% infection rate and an average percentage recovery of third stage larvae of 15.6 and 14.6 respectively. The rest of snail species proved to be less susceptible. For comparative evaluation of the suitability of the various snail species as intermediate hosts of A. cantonensis a "Capacity Index" was determined. This index should provide a useful method for the evaluation of the suitability of various snails as intermediate hosts of nematode parasites under standardized conditions in the laboratory.     

Ecological modulation of environmental stress: interactions between ultraviolet radiation, epibiotic snail embryos, plants and herbivores

1. The distribution of egg masses of the freshwater snails Lymnaea stagnalis and Planorbarius corneus on the undersides of water lily leaves (e.g. Nuphar lutea) is related to the prevalence of the leaf-mining beetle Galerucella nymphaeae. 2. When given the choice, Planorbarius significantly avoids leaves that were infested by the mining beetle. Conversely, Lymnaea did not discriminate against mined leaves. 3. Intact Nuphar leaves block over 95% of incident ultraviolet radiation. Yet, ultraviolet transmission reaches almost 100% under beetle mining scars. These are several times wider than snail embryos. 4. When exposed to natural sunlight, Lymnaea embryos proved to be resistant to ambient ultraviolet, while Planorbarius embryos were rapidly killed. Thus, one selective advantage of Planorbarius discrimination against mined leaves when depositing its eggs could be the avoidance of ultraviolet radiation passing through mining scars. 5. Other mining-related modifications of the leaves, reduced area, decreased longevity, altered aufwuchs (i.e. biofilm and epibionts) are discussed but seem less relevant for the oviposition preference of Planorbarius. 6. The discriminatory behaviour of this snail species was triggered by water-borne cues emitted by the damaged leaf, not by the eggs or larvae of the beetle. 7. This study illustrates how environmental stress on a given species, ultraviolet radiation in this case, can be ecologically buffered (shading by Nuphar) or enhanced (reduction of Nuphar shading through beetle mining) by associated species. It highlights how the impact of a given stress depends on the identity of the target species as well as on the identity and role of other species in the community.     

The Effects of Isolation,Grouping and Population Density on Fecundity of Bulinus truncatus

Summary

Bulinus truncatus is the intermediate host of Schistosoma haematobium, a trematode parasite causing bladder bilharziasis in man. In the struggle against this disease, control of snail populations is an important goal and expansion of our knowledge of the reproductive activity of Bulinus is relevant in this regard.

In this study the effects of various factors on the fecundity of Bulinus were investigated in specimens kept under continuous water refreshment conditions. The results are considered to be more indicative of the natural situation than results obtained under the current use of discontinuous water refreshment.

Isolated snails produce considerably higher numbers of egg masses and eggs than grouped snails. The effect of grouping on isolated snails, and the reverse situation, is very rapid, being fully effective within one week. The increase in fecundity of isolated snails is most probably caused by the absence of copulatory activity, since transfer of semen is known to curtail egg laying in freshwater pulmonate snails. Egg mass production was shown to decrease with increasing population density. This may be caused either by the difference in space available or by the possible difference in copulatory activity. Isolated snails showed a faster body growth than grouped snails.

The possible role of hormones involved in establishing the above effects and the consequences of the results for snail control programs are discussed.