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Spiral cleavage is a mode of embryonic cell division found in species from several Phyla, including molluscs, annelids and flatworms. It reflects a tilting in the direction of spindle orientation and cell division at the 4 to 8-cell stage, which may be dextral or sinistral, and propagates into later organismal asymmetry. Genetic analysis in a small number of gastropod molluscs shows the direction of spiral cleavage is determined by maternal genotype, though whether this is also the case more generally for spiralians, and whether spiral cleavage at the 4-8 cell stage is preceded by earlier internal chirality in any spiralian species, is unknown. Here we study the early cleavage stages of two equal-cleaving spiralians, the dextral annelid Spirobranchus lamarcki and the sinistral mollusc Biomphalaria glabrata, using light sheet microscopy to image subcellular vesicles in live embryos and asking if chirality of movement is identifiable. We observe variability in the early cleavage of S. lamarcki , including a viable 3-cell stage. Image data are analysed by both particle tracking and particle image velocimetry. Neither finds evidence for chiral movement in 1-, 2-, 3-, or 4-cell embryos, nor do we detect consistent differences between the embryos of the dextral and sinistrai species. The methodological and evolutionary implications of this are discussed., (© 2019 Zoological Society of Japan.)

The snail Biomphalaria glabrata is the most important vector for Schistosoma mansoni. Control of this vector to prevent the spread of schistosomiasis is currently performed with the application of a niclosamide molluscicide, which is highly toxic to the environment. Screening of substances that show embryotoxic molluscicidal potential as well as have detrimental effects on cercariae is very relevant for the control of schistosomiasis, as the efficacy of prevention of the disease is increased if it acts as a molluscicide as well as on the cercariae of S. mansoni. The aim of this work was to evaluate the effect of potassium usnate derived from usnic acid on different stages of embryonic development of B. glabrata and on S. mansoni cercariae. After 24 h of exposure, potassium usnate showed embryotoxic activity across all embryonic stages. The values obtained from the LC 50 for the embryonic stages were the following: blastula 5.22 μg/mL, gastrula 3.21 μg/mL, trochophore 3.58 μg/mL, veliger 2.79, and hippo stage 2.52 μg/mL. Against S. mansoni cercariae, it had LC 90 and 100% mortality at concentrations of 2.5 and 5 μg/mL in 2 h of exposure. In conclusion, this is the first report of potassium usnate toxicity on the embryonic stages of B. glabrata and cercariae of S. mansoni, and this study shows the potassium usnate as a promising agent for the control of mansoni schistosomiasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The aquatic pulmonate snail Biomphalaria glabrata is a significant vector and laboratory host for the parasitic flatworm Schistosoma mansoni, an etiological agent for the neglected tropical disease schistosomiasis. Much is known regarding the host-parasite interactions of these two organisms, and the B. glabrata embryonic (Bge) cell line has been an invaluable resource in these studies. The B. glabrata BB02 genome sequence was recently released, but nothing is known of the sequence variation between this reference and the Bge cell genome, which has likely accumulated substantial genetic variation in the ~50 years since its isolation., Results: Here, we report the genome sequence of our laboratory subculture of the Bge cell line (designated Bge3), which we mapped to the B. glabrata BB02 reference genome. Single nucleotide variants (SNVs) were predicted and focus was given to those SNVs that are most likely to affect the structure or expression of protein-coding genes. Furthermore, we have highlighted and validated high-impact SNVs in genes that have often been studied using Bge cells as an in vitro model, and other genes that may have contributed to the immortalization of this cell line. We also resolved representative karyotypes for the Bge3 subculture, which revealed a mixed population exhibiting substantial aneuploidy, in line with previous reports from other Bge subcultures., Conclusions: The Bge3 genome differs from the B. glabrata BB02 reference genome in both sequence and structure, and these are likely to have significant biological effects. The availability of the Bge3 genome sequence, and an awareness of genomic differences with B. glabrata, will inform the design of experiments to understand gene function in this unique in vitro snail cell model. Additionally, this resource will aid in the development of new technologies and molecular approaches that promise to reveal more about this schistosomiasis-transmitting snail vector.

Purpose: Some phytochemicals have shown the potential of being radiomodifiers, especially phenolic compounds, such as lichenic secondary metabolites. To evaluate the phytochemical usnic acid as a radiomodifier, embryonic cells of molluscs have been used due to their ease of collection, high sensitivity to physical and chemical agents, well-known embryology and low cost for analysis., Materials and Methods: This study aimed to assess the radiosensitizing action of usnic acid on Biomphalaria glabrata embryos. Samples were irradiated with 4 Gy of gamma rays from a 60 Co source (dose rate 2.906 Gy/h). An acute toxicity test was performed using B. glabrata embryos in the blastula stage, in order to determine the toxicity of usnic acid and to establish the lethal Concentration for 50% (LC 50 ). Subsequently, the radiomodifing capacity of usnic acid was estimated using assays with B. glabrata embryos., Results: Irradiation increased the number of non-viable embryos compared to unirradiated controls. Additionally, it was observed that embryos exposed to a non-toxic concentration of usnic acid (0.6 µg/mL) before irradiation showed a further enhancement in non-viable embryos when compared with exposure to ionizing radiation alone., Conclusion: The results presented here indicate that usnic acid makes cells more sensitive to the damaging effects of radiation.

This study reports the molluscicidal activity of usnic acid isolated from Cladonia substellata Vanio (lichen) on embryos at various stages of development and in adult mollusks of Biomphalaria glabrata. The toxicity of usnic acid was also evaluated through Artemia salina larvae mortality. Usnic acid was extracted with diethyl ether, isolated, purified, and its structure confirmed by analyzing the spectra of proton nuclear magnetic resonance. LC 90 for 24 h of exposure were 1.62, 4.45, 5.36, and 4.49 μg mL -1 for blastula, gastrula, trocophore, and veliger embryonic stages, respectively, and 3.45 μg mL -1 for adult snails; LC 50 of usnic acid against A. salina was 2.46 μg mL -1 . LC 90 assessed 7 days after exposure was 2.56 μg mL -1 for adult mollusks. In conclusion, these findings demonstrate that under laboratory conditions usnic acid has teratogenic and molluscicide potential to control the aquatic snail B. glabrata and may prove to be a promising candidate in the search for new molluscicide agents, but further detailed studies on its molluscicidal effect and possible environmental effects are needed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The human blood fluke Schistosoma mansoni causes intestinal schistosomiasis, a widespread neglected tropical disease. Infection of freshwater snails Biomphalaria spp. is an essential step in the transmission of S. mansoni to humans, although the physiological interactions between the parasite and its obligate snail host that determine success or failure are still poorly understood. In the present study, the B. glabrata embryonic (Bge) cell line, a widely used in vitro model for hemocyte-like activity, was used to investigate membrane properties, and assess the impact of larval transformation proteins (LTP) on identified ion channels. Whole-cell patch clamp recordings from Bge cells demonstrated that a Zn2+-sensitive H+ channel serves as the dominant plasma membrane conductance. Moreover, treatment of Bge cells with Zn2+ significantly inhibited an otherwise robust production of reactive oxygen species (ROS), thus implicating H+ channels in the regulation of this immune function. A heat-sensitive component of LTP appears to target H+ channels, enhancing Bge cell H+ current over 2-fold. Both Bge cells and B. glabrata hemocytes express mRNA encoding a hydrogen voltage-gated channel 1 (HVCN1)-like protein, although its function in hemocytes remains to be determined. This study is the first to identify and characterize an H+ channel in non-neuronal cells of freshwater molluscs. Importantly, the involvement of these channels in ROS production and their modulation by LTP suggest that these channels may function in immune defense responses against larval S. mansoni.

The invertebrate cell line, Bge, from embryos of the snail Biomphalaria glabrata, remains to date the only established cell line from any species of the Phylum Mollusca. Since its establishment in 1976 by Eder Hansen, few studies have focused on profiling its cytometrics, growth characteristics or sensitivity to xenobiotics. Bge cells are reputed to be challenging to propagate and maintain. Therefore, even though this cell line is a noteworthy resource, it has not been studied widely. With growing interest in functional genomics, including genetic transformation, to elucidate molecular aspects of the snail intermediate hosts responsible for transmission of schistosomiasis, and aiming to enhance the convenience of maintenance of this molluscan cell line, we deployed the xCELLigene real time approach to study Bge cells. Doubling times for three isolates of Bge, termed CB, SL and UK, were longer than for mammalian cell lines - longer than 40 h in complete Bge medium supplemented with 7% fetal bovine serum at 25°C, ranging from ∼42 h to ∼157 h when 40,000 cells were seeded. To assess the potential of the cells for genetic transformation, antibiotic selection was explored. Bge cells were sensitive to the aminonucleoside antibiotic puromycin (from Streptomyces alboniger) from 5 μg/ml to 200 ng/ml, displaying a half maximal inhibitory concentration (IC50) of ∼1.91 μg/ml. Sensitivity to puromycin, and a relatively quick kill time (<48 h in 5 μg/ml) facilitated use of this antibiotic, together with the cognate resistance gene (puromycin N-acetyl-transferase) for selection of Bge cells transformed with the PAC gene (puroR). Bge cells transfected with a plasmid encoding puroR were partially rescued when cultured in the presence of 5 μg/ml of puromycin. These findings pave the way for the development of functional genomic tools applied to the host-parasite interaction during schistosomiasis and neglected tropical trematodiases at large., (Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The air-breathing snail Biomphalaria glabrata proliferates in stagnant freshwater, and nothing is known about the survival of eggs in intermittently (rather than perpetually) wet habitats. In the present study their egg masses matured, and juveniles subsequently eclosed and were mobile in a stable water film of transitory habitats simulated by two different simple test devices described here. The viability of eggs maintained in an unstable film however, was diminished. The maturation of egg masses in a water film or in water was significantly prevented by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. The efficiency depended on the fungal propagule and test environment. Hyphal bodies were more effective against egg masses than conidia. This appears to be a first report of activity of either entomopathogen against a mollusc. Both devices offer accurate and reproducible conditions to test both biological questions and the effects of substances or pathogens against B. glabrata egg masses in water films., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A protocol combining acute toxicity, developmental toxicity and mutagenicity analysis in freshwater snail Biomphalaria glabrata for application in ecotoxicological studies is described. For acute toxicity testing, LC50 and EC50 values were determined; dominant lethal mutations induction was the endpoint for mutagenicity analysis. Reference toxicant potassium dichromate (K2Cr2O7) was used to characterize B. glabrata sensitivity for toxicity and cyclophosphamide to mutagenicity testing purposes. Compared to other relevant freshwater species, B. glabrata showed high sensitivity: the lowest EC50 value was obtained with embryos at veliger stage (5.76mg/L). To assess the model applicability for environmental studies, influent and effluent water samples from a wastewater treatment plant were evaluated. Gastropod sensitivity was assessed in comparison to the standardized bioassay with Daphnia similis exposed to the same water samples. Sampling sites identified as toxic to daphnids were also detected by snails, showing a qualitatively similar sensitivity suggesting that B. glabrata is a suitable test species for freshwater monitoring. Holding procedures and protocols implemented for toxicity and developmental bioassays showed to be in compliance with international standards for intra-laboratory precision. Thereby, we are proposing this system for application in ecotoxicological studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions.

Plant molluscicides have been regarded as possible alternatives to the costly and environmentally hazardous molluscicides currently available. This study was undertaken to compare the developmental toxicity of a plant molluscicide (Euphorbia milii latex, LAT) with that of three synthetic molluscicidal compounds. Biomphalaria glabrata egg masses (0-15 h after spawning) were exposed to molluscicides for 96 h and thereafter examined up to the 14th day after spawning. Embryo deaths, abnormal embryo development (malformations) and the day of hatching were recorded. Although exhibiting a weak ovicidal effect, LAT markedly impaired the development of snail embryos at concentrations 1000 microg L(-1) and produced anomalies (EC(50)=2040 microg L(-1)) such as abnormal shells, hydropic embryos, cephalic and non-specific malformations. Embryolethal potencies of molluscicides were as follows: triphenyltin hydroxide (TPTH; LC(50)=0.30 microg L(-1))>niclosamide (NCL; LC(50)=70 microg L(-1))>copper sulphate (CuSO(4); LC(50)=2190 microg L(-1)) >>> LAT (LC(50)=34030 microg L(-1)). A few malformations were recorded in embryos exposed to concentrations of TPTH within the range of lethal concentrations, while almost no anomalies were noted among those treated with NCL or CuSO(4). A hatching delay (hatching on day 10 after spawning or later) was observed among LAT-exposed embryos. The effects of NCL, TPTH and CuSO4 on hatching were to some extent masked by their marked embryolethality. The no-observed effect concentrations (NOEC) for embryotoxicity were as follows: TPTH, 0.1 microg L(-1); NCL, 25.0 microg L(-1); CuSO(4), 500.0 microg L(-1) and LAT, 500.0 microg L(-1). Results from this study suggest that, although LAT was not acutely embryolethal after a short-term exposure, it markedly disrupted snail development. The marked embryotoxicity of E. milii possibly contributes to its effectiveness as a molluscicide., ((c) 2010 Elsevier Ltd. All rights reserved.)

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) are involved in the targeting of newly synthesized lysosomal enzymes and only MPR 300 also participates in the endocytosis of various exogenous ligands. The present study describes for the first time the MPR 300 dependent pathway of lysosomal enzyme sorting in the Biomphalaria glabrata embryonic (Bge) cells. Lysosomal enzymes (arylsulfatase A, beta-hexosaminidase and alpha-fucosidase) were identified by their enzymatic activities and by immunoprecipitation with specific antisera. Exposure of Bge cells to unio MPR 300 antiserum resulted in a dramatic loss of MPR 300 protein with a shortened half life of approximately 20 min as compared to control cells exposed to preimmune serum in which the half life of MPR 300 was of approximately 13 h. Loss of receptor proteins resulted in a significant misrouting of newly synthesized lysosomal enzymes and their secretion in cell culture medium as demonstrated by immunoprecipitation. The ability of Bge cells to uptake and internalize labeled arylsulfatase A, beta-hexosaminidase and alpha-fucosidase enzymes contained in cell secretion products also indicated the role of B. glabrata MPR 300 (CIMPR) protein in internalization and targeting of lysosomal enzymes. M6P dependent binding of lysosomal enzymes to MPR 300 was shown by confocal microscopy and coimmunoprecipitation experiments.

Previous studies have indicated that a molecule with cytokine activity, possibly an interleukin-1-like (IL-1) molecule, plays a role in the killing of larval stages of the blood fluke Schistosoma mansoni in the snail host Biomphalaria glabrata. The purpose of the present experiment was to test the effects of recombinant-human IL-1beta (rhIL-1beta) on embryonic B. glabrata (Bge) cell motility to determine whether the cells respond to the cytokine. Response was measured using a variation of a chemokinetic assay in which cells in culture were separated from variable concentrations of rhIL-1beta by a semi-permeable membrane containing pores to allow migration. A double staining technique was developed to ascertain cell movement across the membrane. The number of cells moving across the membrane significantly increased in a concentration-dependent manner relative to the presence of increasing amounts of rhIL-1beta below the membrane. The number of cells that moved across the membrane increased until a threshold was reached, after which migration decreased. Further, the rhIL-1beta-mediated increase in Bge cell migration across the membrane was abrogated by the addition of IL-1 receptor antagonist protein. These data indicate that Bge cells respond specifically to rhIL-1beta. As such, these data also indicate that Bge cells may serve as a useful model for elucidation of the role of cytokines or cytokine-like molecules in the snail/schistosome relationship.

Endosulfan (END) is an insecticide used in agriculture and as a wood preservative. Since END is practically insoluble in water, ethanol (ETOH) is often employed as a carrier solvent to spike it in the test medium in aquatic toxicity assays. In this study were investigated the effects of END and ETOH on the reproduction of the freshwater snail Biomphalaria tenagophila exposed over three successive generations. END (0, 0.001, 0.01, 0.1 mg L(-1)) was dissolved in the medium water using ETOH (up to 19.8 mg L(-1)) as carrier solvent. ETOH (19.8, 198, 1980 mg L(-1)) alone was tested as well. Adult snails (F(0)-generation) were exposed to END and ETOH for 8 weeks. The F(1)-generation continued to be exposed from embryo to reproductive maturity, while their descendants (F(2)) were exposed until day 10 after spawning. Effects on the fecundity (8-week production of eggs and egg-masses) of mature F(0) and F(1) snails were evaluated. Developmental toxicity was investigated in F(1) and F(2) embryos. END at the highest level tested (0.1 mg L(-1)) inhibited egg production by F(0) and F(1) snails. ETOH at levels 198 mg L(-1) also reduced fecundity of F(0) and F(1) an effect that was apparently aggravated by exposure over successive generations. END 0.1 mg L(-1) increased mortality and malformations and decreased hatching among F(1) embryos. ETOH drastically reduced the proportion of hatchings among F(2) embryos. The study-derived NOECs (no-observed-effect-concentrations) for END was 0.01 mg L(-1) (reduction in fecundity), and for ETOH were 19.8 mg L(-1) for reduction in fecundity and <19.8 mg L(-1) for developmental toxicity (hatching retardation).

Nonylphenols ethoxylates (NPEs) are surfactants used in a variety of products. They are found in domestic sewage, industrial effluents and as contaminants in water bodies. This study was undertaken to evaluate the effects of NPE with 9.5 ethoxylate units (NPE9.5; 0, 0.01, 0.1, 1.0 mg/L) on the reproduction of the snail Biomphalaria tenagophila. Adult snails (F0 generation) were exposed to NPE for 8 weeks. The F1 generation continued to be exposed from embryo to reproductive maturity while their descendants (F2) were exposed until day 10 after spawning. We determined the effects of NPE9.5 on the fecundity (8-week production of eggs and egg masses) of mature F0 and F1 snails. Developmental toxicity was investigated in F1 and F2 embryos. The two highest concentrations of NPE9.5 reduced the fecundity of F0. In the F1 generation, the lowest concentration enhanced the number of eggs laid per snail while the intermediate concentration had no effect and the highest one decreased the fecundity thereby suggesting a biphasic effect of NPE9.5. Study-derived NOECs (no-observed-effect-concentrations) for NPE were: fecundity, F0=10 microg/L, F1<10 microg/L; developmental toxicity, F1=100 microg/L, F2<10 microg/L. Results, therefore, indicated that B. tenagophila is highly vulnerable to NPE and that trans-generation exposure to NPE9.5 aggravates its reproductive toxicity.

In the present study, a tandem-repeat type galectin was characterized from an embryonic cell line (Bge) and circulating hemocytes of the snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni. The predicted B. glabrata galectin (BgGal) protein of 32 kDa possessed 2 carbohydrate recognition domains, each displaying 6 of 8 conserved amino acids involved in galactoside-binding activity. A recombinant BgGal (rBgGal) demonstrated hemagglutinating activity against rabbit erythrocytes, which was specifically inhibited by galactose-containing sugars (lacNAc/lac>galNAc/gal). Although native galectin was immunolocalized in the cytoplasm of Bge cells and the plasma membrane of a subset of snail hemocytes (60%), it was not detected in cell-free plasma by Western blot analysis. The findings that rBgGal selectively recognizes the schistosome-related sugar, lacNAc, and strongly binds to hemocytes and the tegument of S. mansoni sporocysts in a sugar-inhibitable fashion suggest that hemocyte-bound galectin may be serving as a pattern recognition receptor for this, or other pathogens possessing appropriate sugar ligands. Based on molecular and functional features, BgGal represents an authentic galectin, the first to be fully characterized in the medically-important molluscan Class Gastropoda.

Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of L-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host-parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval-host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (approximately 35-150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan-reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated glycoconjugates.

Dominant lethal effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated in the freshwater snail Biomphalaria glabrata. Wild-type snails were exposed during 10 days to 50, 75 and 100ppm of 2,4-D dimethylamine salt (2,4-D DMA) and paired with non-exposed albino snails 1, 11, 25 and 40 days after the exposure. The offspring of the non-exposed albino snails was scored for lethal malformations. One day after the exposure, a significant effect was observed at 75 and 100ppm without a dose-response relationship. After 11 days, the effect was observed only at the highest dose. After 25 days, significant increases in the dominant lethal effects occurred at 50 and 75ppm; effects were directly related to the doses. Background levels of lethal malformations were resumed after 40 days. Although the major and direct measure of dominant lethal mutations is the rate of lethal malformations in the heterozygous offspring of the albino snails, the sensitivity of the assay was substantially increased with the evaluation of all non-viable embryos, that are the sum of those with lethal malformations, identified or not as wild-type.

Following infection with Schistosoma mansoni larvae, haemocytes of resistant Biomphalaria glabrata snails execute a rapid defence during which they migrate towards and encapsulate the parasites. Such immediate and precise responses are thought to depend on signal transduction cascades though the signalling components involved remain largely unknown. It is proposed that mitogen-activated protein kinases may play a role in B. glabrata immune signalling, in particular p38 mitogen-activated protein kinases, which are known to be associated with stress and inflammatory signalling. Using degenerate PCR followed by Rapid Amplification of cDNA Ends a full-length p38 mitogen-activated protein kinase-like cDNA was cloned from both the B. glabrata embryonic (Bge) cell line (Bge-p38) and haemocytes (Bgh-p38). In addition, B. glabrata p38 mitogen-activated protein kinase activation was examined at the protein level in Western blot analyses using an antibody that specifically recognises activated/diphosphorylated p38 mitogen-activated protein kinase. Results showed that Bge cell p38 mitogen-activated protein kinase was activated/phosphorylated following 30 min incubation with anisomycin, an established p38 mitogen-activated protein kinase activator. Furthermore, p38 mitogen-activated protein kinase was also activated after only 5 min exposure to either the beta-glucan polymer laminarin or S. mansoni larval excretory-secretory products. In a comparative study, activated haemocyte p38 mitogen-activated protein kinase could also be detected using the anti-phosphorylated p38 antibody following cell treatment with anisomycin. However, in contrast with Bge cells, haemocyte p38 was not activated by either excretory-secretory products or laminarin treatments, suggesting fundamental differences in the role of p38 mitogen-activated protein kinase in signal transduction pathways between haemocytes and Bge cells.

The freshwater snail Biomphalaria glabrata possesses a diverse family of fibrinogen-related proteins (FREPs), hemolymph polypeptides that consist of one or two amino-terminal immunoglobulin superfamily (IgSF) domains and a carboxyl-terminal fibrinogen domain. Here, we show that the IgSF1 domain of the FREP3 subfamily is diversified at the genomic level at higher rates than those recorded for control genes. All sequence variants are derived from a small set of nine source sequences by point mutation and recombinatorial processes. Diverse FREP3 transcripts are also produced. We hypothesize a mechanism present in snails that is capable of diversifying molecules involved in internal defense.

Specific studies on schistosome gene functions require both access to the parasite stages, preferably the larvae, and to complete the life cycle. In the present study, we investigated whether short-term in vitro cultivation of sporocysts and surgical transplantation into snails could be combined to produce cercariae. Miracidia were maintained in vitro in the presence of Biomphalaria glabrata embryonic (Bge) cells or, alternatively, in Bge-cell-conditioned medium. The transformation of miracidia to mother sporocysts was observed in both cases. Two day-old sporocysts were transplanted into the cephalopedal sinus of recipient snails. Transplantation efficiencies varied between 16% and 43%, depending on the culture of the sporocysts in terms of the number of cercariae producing snails. Cercariae recovered from these snails were used to successfully infect hamsters, demonstrating that short term in vitro-generated sporocysts undergo normal cercariogenesis following transplantation. This combination of in vitro cultivation and transplantation may be useful for novel experimental approaches to investigate the genes involved in larval development or host-parasite molecular interactions.

Mutagens in the environment may represent a long-term risk for ecosystems. The reproductive potential of populations can be affected by alterations in the fecundity and offspring viability caused by germ cell mutations. Despite the ecological relevance of these effects, there are few studies on germ cell mutagenicity in natural populations. Biomphalaria glabrata was chosen for this study because of the scarcity of data on freshwater invertebrates and the ecological importance of this group. The aim of this study was to establish a germ cell mutagenicity test in B. glabrata by using a similar approach to that used in the dominant lethal test in rodents. Mitomycin C was used as a direct mutagen and cyclophosphamide as a mutagen that requires metabolic activation. Wild-type snails were exposed for 10 days to three concentrations of each agent and crossed with non-exposed albino snails at the end of the treatment. The total frequencies of malformations were analyzed in the offspring of wild-type snails; among the offspring of albino snails, only the heterozygous wild-type embryos were analyzed for malformations. Both agents induced germ cell mutations. The analysis of the offspring of the wild-type snails showed an effect of the exposure up to approximately 5 days after the end of the treatment with cyclophosphamide; the effect of mitomycin C was observed until 45 days after the end of the exposure. There was an increase in the frequencies of malformations in the wild-type offspring of the non-exposed albino snails crossed with the wild-type snails exposed to both agents. The dominant lethal test in B. glabrata proposed in this work is easy to perform, efficient, specific and sensitive in the evaluation of germ cell mutations induced by reference mutagens. The possibility of expanding its use to environmental biomonitoring studies seems very promising and worth trying.

Biomphalaria glabrataembryonic (Bge) cells have been shown to provide favourable environmental conditions for the development of Schistosoma mansoni sporocysts. We investigated the effect of Bge excretory-secretory products on metabolic activity and gene transcription in S. mansoni mother sporocysts. Using the differential-display technique, we identified several sporocyst transcripts regulated by exposure to Bge soluble components. Research in databases indicated that six of the eight differential products analysed were homologous to sequences already present in databases. Two transcripts appeared of interest for schistosome development since they could be associated with cell division and protein synthesis in developing sporocysts. Their up-regulation following contact with cell products was confirmed by semi-quantitative RT-PCR. The first fragment coded for a part of the chaperonin containing T-complex protein gamma subunit-like protein of S. mansoni (SmTCP 1-C). The second one represented a new S. mansoni expressed sequence tag encoding a protein homologous to various glutaminyl-tRNA synthetases (GlnRS). The full-length sequence of SmGlnRS was cloned from adult schistosomes and its primary sequence was compared to other GlnRS. The overexpression of SmTCP-1 and SmGlnRS could be correlated with the metabolic changes observed in Bge-exposed sporocysts.

Due to shared characteristics with snail haemocytes, the Biomphalaria glabrata embryonic (Bge) cell line has been used as a model in vitro system for the study of snail-trematode interactions. In this study, Bge cells were used to characterize the adherence of snail host cells to schistosome primary sporocysts, and to test the effect of carbohydrates as inhibitors of this behaviour. Bge cells bound to the surface of >90% of Schistosoma mansoni sporocysts and, based on a semi-quantitative cell adhesion scale of 1-4, exhibited a cell adhesion index (CAI) of 2.45. This cellular adhesion was significantly inhibited in the presence of selected carbohydrate-containing substances. Fucoidan was the most potent inhibitor, reducing Bge cell-binding prevalence to approximately 50% and the CAI to 1.6. Other inhibitory compounds included mannose-6-phosphate, heparin, dextran sulfate, and various forms of the polysaccharide carrageenan. Fluoresceinated-fucoidan was found to attach to Bge cells confirming their ability to associate with sugar moieties. These results were further supported by the specific binding of surface biotinylated Bge cell proteins to sporocyst tegumental glycoproteins ranging from 40 to 120 kDa. N-linked tegumental carbohydrates appeared to represent ligands for Bge cell proteins since N-glycosidase treatment of blotted tegumental glycoproteins completely abolished biotinylated Bge protein binding. We hypothesize that the involvement of lectins as potential host cellular receptors in snail cell-sporocyst interactions, and suggest that negatively charged (mainly sulfated) carbohydrate moieties may represent the schistosome surface ligand(s).

The embryonic development of the fresh-water snail Biomphalaria glabrata was examined under microgravity-conditions and compared with the ground control and standard embryos, putting special emphasis on the shell formation. The process of shell formation may be particularly sensitive to the change of gravitational forces. The project aimed at determining whether the processes of mineralization during the formation of the exoskeleton in the growing snail embryo take place normally under microgravity conditions. Twenty-four adult individuals of the tropical freshwater snail B. glabrata were maintained 9 days in the Closed Equilibrated Biological Aquatic System (CEBAS Minimodule) on Space Shuttle flight STS-89. The animals produced spawning packs throughout the duration of the mission so that embryos of all developmental stages were achieved. The embryos developed slightly slower in the CEBAS than under standard conditions, and in older embryos a decreased mineralization of the shell was detected. These phenomena, however, were observed in the flight module as well as in the ground control specimens and was not an effect caused by the microgravity conditions. Embryos of B. glabrata showed a correct morphogenesis under microgravity, no teratological effects were noticed, and the shell formation proceeded normally.

Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMA-induced expression of activated MAPK, suggesting that PKC-, Ras- and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek.

Tyrosine kinase receptors play a key role in the communication of cells with their environment. Growth hormone receptors, such as insulin receptors, are involved in the regulation of cell growth, differentiation and metabolism in multicellular organisms. Insulin-related peptides and members of the insulin receptor subfamily have been described in a wide variety of invertebrates, including freshwater molluscs. In this paper, we describe the metabolic effect of insulin on a mollusc cell line (Bge) derived from embryos of the snail Biomphalaria glabrata. Using a PCR strategy, we have cloned from Bge cells a cDNA encoding a protein (BgIR) homologous to, and exhibiting all of the typical features of insulin receptors. Northern blot analysis confirmed the expression of BgIR in B. glabrata snails and suggested its wide distribution in the snail body. Bge cells have been shown to provide the environmental conditions necessary for the in vitro development of the sporocysts of Schistosoma mansoni, a trematode parasite that uses B. glabrata as an intermediate host. The possible implication of BgIR in the activating and proliferating processes observed in Bge cells during their coculture with S. mansoni larvae is discussed.

The mineralized shell (consisting of calcium carbonate) of the tropical freshwater snail Biomphalaria glabrata was investigated with high resolution synchrotron X-ray powder diffractometry and X-ray absorption spectroscopy (EXAFS). Parts from different locations of the snail shell were taken from animals of different age grown under various keeping conditions. Additionally, eggs with ages of 60, 72, 120, and 140 hours were examined. Traces of aragonite were found as first crystalline phase in 120 h old eggs, however, Ca K-edge EXAFS indicated the presence of aragonitic structures already in the X-ray amorphous sample of 72 h age. The main component of the shell of adult animals was aragonite in all cases, but in some cases minor amounts of vaterite (below 1.5%) are formed. The content of vaterite is generally low in the oldest part of the shell (the center) and increases towards the mineralizing zone (the shell margin). In juvenile snails, almost no vaterite was detectable in any part of the shell.

In vitro cultivation of parasitic helminths, including the digenetic trematodes, has long been a valuable tool in medical and veterinary parasitology, permitting and/or facilitating the development of diagnostic reagents, chemotherapeutic agents, and vaccines and providing insights into naturally complex host-parasite interactions. In vitro cultivation of the intramolluscan stages of trematodes has been particularly challenging, given the ontogenic complexities involved in the production of multiple larval generations from germinal tissues through an asexual "budding" process. Recently, however, advanced larval development has been achieved by incorporating the Biomphalaria glabrata embryonic (Bge) cell line into cocultivation systems. Most notably, the entire intramolluscan cycle (from miracidium to cercaria) has been completed for the human blood fluke Schistosoma mansoni, while significant primary sporocyst development has been attained for several other digeneans including S. japonicum and Fascioloides magna. Here we review recent advances in the cultivation of several larval trematode species and discuss the potential use of this culture system for addressing fundamental questions of host-parasite compatibility., (Copyright 2000 Academic Press.)

We have cloned from Biomphalaria glabrata, the intermediate host of the helminth parasite Schistosoma mansoni, a 36-kDa apparent-molecular-mass molecule (BgSel) that shares sequence identity with selectins of the cell-adhesion-molecule superfamily. BgSel exhibited in its C-terminal part a putative C-type lectin domain similar to the selectin lectin domain. Using antibodies to the recombinant BgSel protein, we demonstrated the presence of BgSel in snail hemocytes as well as in the cell line derived from B. glabrata embryos (Bge). Anti-BgSel antibodies specifically recognized a 79-kDa component in Bge-cell-secreted products that was supposed to represent the native form of BgSel, as was confirmed after glycosidase treatment. Lectins are known to be implicated in recognition mechanisms participating in humoral and cellular immunity in molluscs. The possible role of BgSel in the interaction between sporocysts and Bge cells, particularly in the in vitro model of sporocyst development dependent on Bge cell factors, remains to be determined.

A Bge cell co-culture system, previously shown to support the in vitro production of daughter sporocysts from mother sporocysts of Schistosoma mansoni and S. japonicum, has proven capable of supporting the in vitro development of intramolluscan stages of the deer liver fluke, Fascioloides magna. Miracidia commenced transforming within 4 h of incubation with Bge cells, and had completely shed their epidermal plates within 18-24 h. Redial stages were visible inside in vitro-transformed mother sporocysts after 12-16 days of co-culture with Bge cells, and emerged as fully-developed larvae starting at 14-20 days post-cultivation. Rediae survived over 60 days of in vitro culture, and reached a maximum size of 150-170 microns. Although particulate material was visible in their cecae, rediae were not observed to actively feed on Bge cells. Bge cells did not attach to or encapsulate larval stages at any time throughout the incubation period. Unlike Schistosoma spp., in which a high percentage of miracidia spontaneously shed their ciliated epidermal plates and transformed into mother sporocysts in Chernin's balanced saline solution (CBSS), transformation of F. magna was dependent on Bge cell products. Less than 5% of F. magna miracidia transformed spontaneously in either CBSS or Bge medium with 10% fetal bovine serum (complete or C-Bge). However, incubation of miracidia in either Bge cell-conditioned C-Bge medium or a greater than 30 kDa fraction concentrated from conditioned CBSS increased transformation rates to 67 and 83%, respectively. This secretory Bge cell factor(s) appeared to be protein in nature since its activity was completely abrogated by heat or proteinase K treatments. Overall, these results demonstrate that Bge cells are required for stimulating in vitro miracidial transformation and supporting early larval development of a fasciolid trematode under culture conditions. This is the first report of in vitro development of rediae from miracidia for a digenetic trematode.

Studies were initiated to begin developing a genetic transformation system for cells derived from the freshwater gastropod, Biomphalaria glabrata, an intermediate host of the human blood fluke Schistosoma mansoni. Using a 70-kD heat-shock protein (HSP70) cDNA probe obtained from the B. glabrata embryonic (Bge) cell line, we cloned from Bge cells a complete HSP70 gene including a 1-kb genomic DNA fragment in its 5'-flanking region containing sequences indicative of a HSP promoter. Identified in the 5'-half (416 nucleotides) of this genomic fragment were TATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-shock element consensus sequence (Bge HSP70(0.5k) promoter). The 3'-half of this upstream flanking region was comprised of a 508-base intron located immediately 5' of the ATG start codon. To determine the functionality of the putative snail promoter sequence, Bge HSP promoter/luciferase (Luc) reporter gene constructs were introduced into Bge cells by N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP)-mediated transfection methods, and assayed for Luc activity 48 hr following a 1.5-hr heat-shock treatment (40 degrees C). Compared with control vectors or the Bge HSP70(0.5k/1.0k) promoter constructs at 26 degrees C, a 10- to 300-fold increase in Luc expression was obtained only in the Bge HSP70 promoter/Luc-transfected cells following heat-shock. Results of transfection experiments demonstrate that the Bge HSP70(0.5k) DNA segment contains appropriate promoter sequences for driving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characterization of an inducible promoter from a freshwater gastropod mollusc. Successful transient expression of a foreign reporter gene in Bge cells using a homologous, inducible promoter sequence now paves the way for development of methods for stable integration and expression of snail genes of interest into the Bge cell line.

Biomphalaria glabrata embryonic (Bge) cells have previously been shown to permit a successful cocultivation of Schistosoma mansoni and Schistosoma japonicum from miracidia to mother sporocysts (MS) and then to the production of daughter sporocysts (DS). To investigate further the properties of the Bge culturing system we used Echinostoma caproni under identical in vitro conditions. In vitro-derived miracidia were used either for experimental infections of B. glabrata snails, or for in vitro cultivation with Bge cells. Histological analysis showed that the development of MS in B. glabrata was similar to the previously described development in Biomphalaria pfeifferi in terms of final site of infection, development dynamics, growth dynamics, reproduction intensity, and life spans. Only short delays in migration dynamics were observed in B. glabrata. When cultivated under in vitro conditions, E. caproni MS could live for up to 17 wk in the presence of Bge cells, as compared with 2 wk in cell-free Bge medium. The presence of Bge cells also permitted significant growth of MS and development through complete embryogenesis of the next intramolluscan stage (embryos of 100-110 cells). However, degeneration of MS consistently occurred before production of this second generation. During the entire cultivation period, no visible contact was observed between MS and Bge cells, suggesting that development of MS was only triggered by soluble factors released by Bge cells.

In vitro cultivation of Schistosoma japonicum miracidia to the mother sporocyst (MS) and then to the daughter sporocyst (DS) stage was achieved using the Biomphalaria glabrata embryonic (Bge) cell line as a coculture system. When comparing the effect of Bge cell and MS density on MS development, it was apparent that Bge cell density had a highly significant effect on both MS viability and growth. Viability and growth rate of MS cultured under high cell density conditions (350 cells/mm2) were almost 2 times greater than those of MS cultured under conditions of low cell density (60 cells/mm2). Growth under high cell density conditions corresponded to a 20 to 30 times increase in MS estimated volume within the first 9 weeks of cultivation. Emergence of fully formed motile DS was first observed after 11 weeks of cocultivation. A few DS lived for 14 weeks after emergence and attained a size of 770 +/- 100 microns in length and 48 +/- 13 microns in width. In contrast to what was observed in Bge cell/Schistosoma mansoni cocultures, Bge cells did not encapsulate S. japonicum MS. Our results show that, although the cellular interactions between Bge cells and schistosomes MS display some level of specificity, Bge cells apparently secrete soluble factors that permit excellent survival and can trigger advanced in vitro development of S. japonicum.

The development of the statocyst of the freshwater snail Biomphalaria glabrata has been examined from embryo to adult. Special emphasis was put on the growth of the statoconia in the statocysts. In the statocysts of embryonic snails (90-120 h after oviposition) there is not a single statolith but an average of 40-50 statoconia per statocyst. The number of statoconia increases to 385-400 when the snails reach a shell diameter of 4 mm and remains relatively constant thereafter, irrespective of shell size. Small statoconia are found in supporting cells, which suggests that the statoconia are produced within these cells. The average diameter of statoconia and the total mass of statoconia increase with increasing shell diameter. The average number of large statoconia (diameter > 7 microm) per statocyst continues to increase from 2 to 10 mm animals while the number of small ones (diameter < 4 microm) initially rises and then decreases after 4 mm. These results demonstrate continuous growth of the statoconia in the cyst lumen of Biomphalaria. The single statoconia vibrate in a regular pattern in vivo, indicating beating of the statocyst cilia. The statoconia sink under the influence of gravity to load and stimulate receptor cells which are at the bottom. The length of cilia and the size of statocyst gradually increase as the animal grows. However, the increase in the volume of the statocyst is relatively small compared with the increase in body weight during normal development.

A dichloromethane extract of the seeds of the molluscicidal west African legume Millettia thonningii was tested for ovicidal activity toward Biomphalaria glabrata egg masses. The extract was found to be highly ovicidal at concentrations as low as 5 mg/L. Embryonic development of the snails in egg masses was monitored using photomicrographs from which embryonic diameters were estimated. Such measurements revealed that ovicidal effects were developmentally stage specific and normally induced a curtailment of development during the gastrula to trochophore transition.

In vitro production of Schistosoma mansoni daughter sporocysts (DS) from miracidium-derived mother sporocysts (MS) was achieved by synxenic larval cultivation with cells of the Biomphalaria glabrata embryonic (Bge) cell line. The in vitro growth and viability of MS cocultured with Bge cells or in Bge cell-conditioned medium were significantly extended beyond that of larvae cultured in fresh medium alone. However, complete DS development and emergence from MS were achieved only in the presence of Bge cells. Introduction of either miracidia or previously transformed MS onto Bge cell monolayers resulted in an initial attachment of cultured cells to sporocysts, followed by a gradual encapsulation of larvae by multiple layers of Bge cells. Sporocysts and their encapsulating cells eventually formed large cellular aggregates, within which MS increased 4-fold in size during the first 20 days of cultivation. The timing of in vitro DS development was somewhat variable; however, in general, early embryo formation, i.e., germ cell aggregates with surrounding primitive epithelium, was first detected at 15-20 days of culture, whereas motile, intra-MS daughter stages were seen at 25-30 days and thereafter. Mature, first generation DS, measuring 136 +/- 46 microns long by 22 +/- 6 microns wide, emerged from MS starting at approximately 30-45 days of initial cultivation. Although the basic morphology and size of emergent, in vitro-derived DS were comparable to those propagated in vivo, there was a large reduction in the in vitro reproductive capacity of the MS and a delay in DS culture development.(ABSTRACT TRUNCATED AT 250 WORDS)

Specimens of Biomphalaria occidentialis and Biomphalaria tenagophila were reared in S. Paulo, Brazil under laboratory conditions, with a view to know their biological characteristics. Oviposition and eggs/oviposition ratio were recorded over twelve months so as to obtain information on the number of egg-masses per animal and the number of eggs per egg-mass, as also the number of hatched eggs per egg-mass for each period of 30 days, i.e., the hatching-rate per period. The incubation period was about the same for both species, but the oviposition and egg oviposition ratio were greater in B. tenagophila.

The paper explains the basic conception of a "Closed Equilibrated Biological Aquatic System" (C.E.B.A.S.) incorporated in a Aquarack as a research tool for zoological, botanical and interdisciplinary research in space biology and describes the construction principle of laboratory prototype consisting of a modular habitat for aquatic animals a semi-biological/physical water recycling system and a computerized control unit. Further on the running scientific frame program in which as well five german and two U.S.-universities as the DLR Institute of Aerospace Medicine are involved is presented which includes the subtopics reproductive biology, genetics, embryology/teratology, neurobiology, vestibularis research, stress research general endocrinology, ethology, gerontology and skeleton research. Within this the choice of the experimental animals, the teleost Xiphophorus helleri (vertebrate model) and the pulmonate snail Biomphalaria glabrata (invertebrate model) is explained. Examples of results of the scientific program mainly derive from the hardware-related research and from the reproductive biology subproject with X. helleri.

During embryogenesis of the fresh water snail Biomphalaria glabrata (Say) (Pulmonata, Basommatophora) shell formation has been studied by light and electron microscopical techniques. The shell field invagination (SFI), the secretion of the first shell layers, the development of the shell-forming mantle edge gland and spindle formation have been investigated. During embryonic development at 28 degrees C environmental temperature, the shell field invaginates after 35 h. After 40 h the SFI is closed apically by cellular protrusions and scale-like precursors of the periostracum. The first electron translucent layer of the periostracum stems from electron dense vesicles of the cells which lie at the opening of the SFI. A second electron dense layer appears some hours afterwards. When the shell appears birefringent in the polarizing microscope (45 h of development) calcium can be detected in it using energy dispersive x-ray analysis. As calcification occurs the intercrystalline matrix appears under the periostracum and the SFI begins to open. In embryos of 60 h the mantle cavity appears at the left caudal side. When the mantle edge groove develops (65 h of development) lamellate units are added to the outer layer of the periostracum, but no distinct lamellar layer is formed in B. glabrata. In addition to the lamellar cell and the periostracum cell, a secretory cell can be observed in the developing groove. After 65 h of development, spindle formation starts and the shell begins to coil in a left hand spiral. After 5 days of development the embryos are ready to leave the egg capsules.

Experiments were carried out using aqueous extracts from leaves and flowers of Laurus nobilis on Biomphalaria glabrata. Treatments were performed on blastula stage (+/- 15 h after first cleavage) and on adult snails (11-18 mm). In both instances they were exposed for 24 h to different concentrations of the extracts on snails (200 to 2500 ppm) and embryos (20 to 300 ppm) at 25 +/- 1 degree C. The embryos were observed for a period of 20 days after treatment and the snails for 10 days. Results obtained with leaf aqueous extracts have shown a degree of toxicity on embryos starting at a concentration of 125 ppm, the flower extract being effective at 35 ppm. The malformation obtained with the different concentrations falls into the unespecific type category, however some cephalic and shell malformations were found in embryos treated with concentrations over 50 ppm (leaves) and 25 ppm (flowers). The LD90 on adult snails obtained by treatments with flower and leaf extract was observed at concentrations of 340 ppm and 1900 ppm respectively.

1. Egg masses of the Planorbid snail Biomphalaria glabrata contain 2-aminoethylphosphonic acid (AEP) in three different chemical environments, as determined by 31P nuclear magnetic resonance spectroscopy, giving signals at 20.9, 21.0 and 23.4 delta. 2. The signal at 21.0 delta decreased in intensity during embryonic development, whereas the other two did not change significantly. 3. The following relationship is suggested: Extraembryonic AEP--------Intraembryonic AEP--------Phosphates. 4. pH Titration behavior of macromolecularly-bound AEP and synthetic derivatives of AEP was examined and indicates that AEP is found in the egg masses linked to other molecules in the following ways: (a) R2-NH-CH2CH2-P(O)(OH)(OR1), (b) R3-NH-CH2-P(O)(OR2)(OR1), (c) NH2-CH2CH2P(O)(OH)(OR1).