Neuropeptide schistosomin inhibits hormonally-induced ovulation in the freshwater snail Lymnaea stagnalis.

This study examines the interaction between the caudodorsal cell hormone (CDCH) and schistosomin, a peptide secreted by the central nervous system of the snail (Lymnaea stagnalis) infected with the avian schistosome Trichobilharzia ocellata. Non-infected snails were injected with synthetic as well as native CDCH in the absence or presence of purified schistosomin. The response to 2 pmol of synthetic CDCH was blocked for 90% by coinjection with 3.5 pmol of schistosomin. The ovulation-inducing activity of extracts of cerebral commissures (the storage area of native CDCH) was also blocked by schistosomin. The degree of inhibition (65%), however, was less than that observed with synthetic CDCH. These results show that schistosomin inhibits ovulation and egg laying in Lymnaea. This explains the decrease or absence of egg laying in schistosome-infected freshwater 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.     

Isolation, purification and characterization of an iron-binding protein from the horseshoe crab (Limulus polyphemus).

The presence of an iron-binding protein in the haemolymph of the horseshoe crab, Limulus polyphemus, was detected by gel filtration of 59Fe-labelled haemolymph. Lysis of amoebocytes did not change the amount of iron-binding protein in haemolymph samples. The protein was purified to homogeneity by ion-exchange chromatography. The molecular mass of the purified protein was estimated to be 282,000 +/- 10,000 Da by gel filtration and analytical ultracentrifugation. SDS/polyacrylamide-gel electrophoresis demonstrated that the protein is composed of ten subunits having a molecular mass of 28,000 +/- 2,000 Da. The purified, unlabelled protein efficiently sequestered 59Fe in the absence of haemolymph indicating that no other haemolymph factors are required for the incorporation of iron into the protein. No 59Fe was removed from the purified protein with EDTA or 2,2'-bipyridyl. Partial removal of 59Fe was achieved by dialysis with nitrilotriacetic acid or desferal. Analysis of the iron-loaded protein indicated that each subunit has the capacity to bind two iron atoms with high affinity. The isolation of an iron-binding protein from L. polyphemus supports the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins which serve as oxygen carriers.     

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.     

Amino acid sequence and biological activity of a gamma-conotoxin-like peptide from the worm-hunting snail Conus austini

A novel 31-residue toxin, named as7a, was isolated and characterized from the venom of Conus austini, a vermivorous cone snail collected in the western Gulf of Mexico. The complete amino acid sequence, TCKQKGEGCSLDVgammaCCSSSCKPGGPLFDFDC, was determined by automatic Edman sequencing after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and the sequence motif -gammaCCS-, which has only been found in the gamma-conotoxin family. The molecular mass of the native peptide was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which confirmed the chemical analyses and suggested a free C-terminus. The purified peptide elicited toxic effects in the freshwater snail Pomacea paludosa after intramuscular injection, but it had no effect when injected intracerebrally into mice. The structural similarity of peptide as7a to other gamma-conotoxins suggests that modulation of pacemaker channels could be responsible for its biological activity.     

九香虫血淋巴及其纯化蛋白抑菌活性的研究

对九香虫AsporgopuschinensisDallas血淋巴及其血淋巴蛋白质分离物的抗菌活性进行了研究,抗菌活性检测指示菌为大肠杆菌Escherichiacoli和金黄色葡萄球菌Staphilocalliesacereus。测定结果表明,九香虫血淋巴及其离心上清液都具有明显的抗菌活性。用凝胶过滤法从血淋巴蛋白分离提纯获得一种小分子肽,SDS PAGE电泳为单一带,分子量约为1～1 4 4kD。该小分子蛋白对大肠杆菌和金黄色葡萄球菌都有抑菌作用,与血淋巴对2种细菌的抗菌性一致,表明其是九香虫血淋巴中具抗菌作用的主要物质之一。     

Characterization of the venom of the vermivorous cone snail Conus fulgetrum

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.     

棕尾别麻蝇(Sarcophaga peregrina)幼虫与蛹血淋巴凝集素的纯化与特性

用亲和层析法纯化了棕尾别麻蝇幼虫和蛹血淋巴凝集素。以兔红细胞吸附幼虫血淋巴凝集素为抗原制备的抗体、球球蛋白和甲状腺蛋白等三种亲和层析吸附剂纯化得到的幼虫凝集素是相同的,其分子量73kD左右。用甲状腺球蛋白为亲和配基纯化的蛹血淋巴凝集素由二种亚基组成,其分子量分别为30和32kD。幼虫和蛹血淋巴凝集素活性的抑制糖明显不同：乳糖、岩藻糖和N－乙酰半乳糖胺对幼虫血淋巴凝集素活性有抑制作用;而甘露糖胺、半乳糖胺和葡萄糖胺则对蛹血淋巴集素有一定抑制。而且,用兔红细胞吸附幼虫血淋巴凝集素为抗原制备的抗血清对蛹的凝集素活性无交叉反应,表明这两种凝集素是不相同的。虽然本文所纯化的麻蝇蛹血淋巴凝集素的分子量和Komano等报道的麻蝇蛹以及幼虫体壁 伤害诱导的凝集素SPL相同,但其糖的抑制特性有明显差异。     

Acharan sulfate, the new glycosaminoglycan from Achatina fulica Bowdich 1822. Structural heterogeneity, metabolic labeling and localization in the body, mucus and the organic shell matrix.

Acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica, has a major disaccharide repeating unit of -->4)-2-acetyl,2-deoxy-alpha-d-glucopyranose(1-->4)-2-sulfo-alpha-l-idopyranosyluronic acid (1-->, making it structurally related to both heparin and heparan sulfate. It has been suggested that this glycosaminoglycan is polydisperse, with an average molecular mass of 29 kDa and known minor disaccharide sequence variants containing unsulfated iduronic acid. Acharan sulfate was found to be located in the body of this species using alcian blue staining and it was suggested to be the main constituent of the mucus. In the present work, we provide further information on the structure and compartmental distribution of acharan sulfate in the snail body. Different populations of acharan sulfate presenting charge and/or molecular mass heterogeneities were isolated from the whole body, as well as from mucus and from the organic shell matrix. A minor glycosaminoglycan fraction susceptible to degradation by nitrous acid was also purified from the snail body, suggesting the presence of N-sulfated glycosaminoglycan molecules. In addition, we demonstrate the in vivo metabolic labeling of acharan sulfate in the snail body after a meal supplemented with [35S]free sulfate. This simple approach might be applied to the study of acharan sulfate biosynthesis. Finally, we developed histochemical assays to localize acharan sulfate in the snail body by metachromatic staining and by histoautoradiography following metabolic radiolabeling with [35S]sulfate. Our results show that acharan sulfate is widely distributed among several organs.     

Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion

The mannose-specific snowdrop lectin (Galanthus nivalis agglutinin: GNA), when fed to insects, binds to the gut epithelium and passes into the haemolymph. The ability of GNA to act as a carrier protein to deliver an insecticidal spider venom neurotoxin (Segestria florentina toxin 1: SFI1) to the haemolymph of lepidopteran larvae was investigated. Constructs encoding SFI1 and an SFI1/GNA fusion protein were expressed in Pichia pastoris. The insecticidal activity of purified recombinant proteins on injection was found to be comparable to published values for SfI1 purified from spider venom [Toxicon 40 (2002) 125]. Whereas neither GNA nor SFI1 alone showed acute toxicity when fed to larvae of tomato moth (Lacanobia oleracea), feeding SFI1/GNA fusion at 2.5% of dietary proteins was insecticidal to first stadium larvae, causing 100% mortality after 6 days. The protein also showed a significant, dose dependent, toxicity towards fourth and fifth stadium larvae, with growth reduced by up to approximately 90% over a 4-day assay period compared to controls. Delivery of intact SFI1/GNA to the haemolymph in these insects was shown by western blotting; haemolymph samples from fusion-fed larvae contained a GNA-immunoreactive protein of the same molecular weight as the SFI1/GNA fusion. SFI1/GNA and similar fusion proteins offer a novel and effective approach for delivering haemolymph active toxins by oral administration, which could be used in crop protection by expression in transgenic plants.     

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.     

The effect of endogeneous proteinase inhibitors on the prophenoloxidase activating enzyme,a serine proteinase from crayfish haemocytes

Three proteinase inhibitors have so far been isolated and purified from crayfish haemolymph. One of these, isolated from crayfish plasma, namely a trypsin inhibitor with a molecular mass of 155 kDa was found to inhibit a serine proteinase, ppA, which is involved in the activation of prophenoloxidase, and is localized in the haemocytes. Another high molecular mass proteinase inhibitor, an α₂-macroglobulin from crayfish plasma, which is a dimer of 190 kDa-subunits, was only inhibitory towards ppA to a lesser extent. A 23 kDa subtilisin inhibitor, purified from haemocytes, did not have any effect on the serine proteinase.We suggest that mainly the trypsin inhibitor, but to some extent also the α₂-macroglobulin, are important in the regulation of the prophenoloxidase activating cascade, as they both inhibit ppA, which in its active form has been shown to mediate prophenoloxidase activation.     

Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin.     

Inhibition of evoked neurotransmitter release from rat hippocampus by a polypeptide toxin isolated from the marine snail Conus distans.

The active fraction, isolated and partially purified from the crude venom of the marine snail Conus distans, with a molecular mass of about 25 kDa, inhibits neurotransmitter release in rat hippocampus. This toxin (distans Toxin) inhibits the electrically evoked tritium labelled noradrenaline release from rat hippocampal slices in a dose and time dependent manner. The neurotransmitter release is mainly regulated by N-type of voltage sensitive Ca(2+)-channels. The distans toxin behaves as a partial antagonist of calcium in the buffer, possibly by competing with calcium for this type of voltage sensitive Ca(2+)-channels.     

Acid phosphatases of Metarhizium anisopliae during infection of the tobacco hornworm Manduca sexta

Three acid phosphatase (AcP) isozymes, pI 8.1, 8.0 and 7.8, were isolated, purified and partially characterised from optimised cultures of the entomopathogenic fungus Metarhizium anisopliae. The enzymes had similar molecular masses (approximately 44.0 kDa), and could degrade sugar phosphates found in the haemolymph of a host insect, the tobacco hornworm Manduca sexta. The AcP activity in haemolymph of mycosed insects increased significantly over controls, and some new isozymes were present. The infection-related isoforms were similar in molecular mass and pI to some of the in vitro AcP isozymes of M. anisopliae. Results of dot blot and Western blot analyses using anti-AcP antibodies suggested that at least one Metarhizium phosphatase isoform was present in haemolymph of infected caterpillars. Antibodies did not cross-react with immune (chemically stimulated) or non-immune haemolymph from Manduca sexta. Consistent with the appearance of highly active fungal phosphatase in caterpillar blood, free phosphate concentration increased dramatically during the late stages of infection to a level two to five times that of controls. Phosphate was limiting to growth of the fungus at the concentration found in control haemolymph and supplementation of phosphate significantly increased fungal growth in vitro in haemolymph. These results suggest that Metarhizium AcP may play a key role in providing phosphorus for fungal growth at the expense of the insect.     

Phosphofructokinase from mollusc muscle is activated by phosphorylation.

Phosphofructokinase was purified from muscle tissue of two different molluscs, edible snails, Helix pomatia (gastropoda), and mussels, Mytilus edulis (bivalvia). Under denaturing conditions, both enzymes had a molecular mass of 82 kDa. In the presence of ATP-Mg2+, the enzymes were rapidly phosphorylated in vitro by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase purified from snail muscle and also by the C subunit of protein kinase from bovine heart. The extent of phosphorylation was 0.6 and 0.5 phosphate residues per subunit for the snail and the mussel phosphofructokinase, respectively. Phosphorylation of both phosphofructokinases effected a decrease in ATP inhibition at neutral or slightly acidic pH values and increased the affinity for fructose 6-phosphate. The resulting activation in the presence of suboptimum fructose 6-phosphate concentrations was more distinct for the snail enzyme. In addition, phosphorylated phosphofructokinase from mussels exhibited a marked increase in Vmax when activated by either 5'-AMP or fructose 2,6-bisphosphate.     

Identification of an extracellular acid trehalase and its gene involved in fungal pathogenesis of Metarizium anisopliae

Trehalose is the main sugar in the haemolymph of insects and is a key nutrient source for an insect pathogenic fungus. Secretion of trehalose-hydrolysing enzymes may be a prerequisite for successful exploitation of this resource by the pathogen. An acid trehalase [EC 3.2.1.28] was purified to homogeneity from a culture of a locust-specific pathogen, Metarhizium anisopliae, and its properties were characterized. The gene (ATM1) of this acid trehalase was also isolated. The pure enzyme can efficiently hydrolyze haemolymph trehalose into glucose in vitro. The new acid trehalase appearing in the haemolymph of Locusta migratoria infected with M. anisopliae had the same pI and substrate specificity as the purified fungal acid trehalase, and the concentration of trehalose in the haemolymph decreased sharply after infection. RT-PCR also revealed the ATM1 gene's expression in the haemolymph of the infected insects. Our results indicated that the acid trehalase may serve as an "energy scavenger" and deplete blood trehalose during fungal pathogenesis.     

Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick,Amblyomma hebraeum

Ovaries of the ixodid tick, Amblyomma hebraeum Koch, grew rapidly after engorgment as a result of yolk uptake. At 26 °C, oviposition began by day 10 post-engorgement, plateaued on days 16-18, and ended by day 38. Vitellin (Vt) was partially purified from ovaries of day 10 engorged ticks by gel filtration and ion exchange chromatography. This Vt comprises seven major and several minor polypeptides. Two polypeptides (211 and 148 kD) from haemolymph of engorged female ticks corresponded to minor polypeptides of similar molecular weight in the ovary. The haemolymph titre of the 211 and 148 kD polypeptides increased up to the onset of oviposition. These polypeptides were absent in males and non-vitellogenic females (day 0 engorged or day 10 partially-fed females), and were thus designated as vitellogenin (Vg). Antibodies raised against haemolymph Vg211 and 148 recognized these polypeptides in partially purified Vt, as well as six of the seven major polypeptides. Using these antibodies we developed an indirect, competitive ELISA to quantify Vg. Rise in haemolymph Vg-concentration lagged slightly behind the rise in haemolymph ecdysteroid (ES)-concentration, and Vg-synthesis was stimulated by injections of 20E into non-vitellogenic females. These observations indicate that an ES is the vitellogenic hormone in A. hebraeum.     

Specific tyrosine phosphorylation induced in Schistosoma mansoni miracidia by haemolymph from schistosome susceptible, but not resistant, Biomphalaria glabrata

Molecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata - Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.     

Search for shared antigens in the schistosome-snail combination Trichobilharzia ocellata-Lymnaea stagnalis

Mother and daughter sporocysts of Tricholbilharzia ocellata, developing in the snail host Lymnaea stagnalis, were searched for substances with antigenic similarities to the snail's haemolymph. Antisera to cell-free snail haemolymph and fractions thereof were used in three different immunocytochemical staining methods, applied on sections of parasitized snails. Snail tissue was consistently stained; cercariae were stained, indicating that the applied methods were successful. Most sections through mother and daughter sporocysts were completely unstained. It is concluded that neither mother nor daughter sporocysts are masked by the antigens studied or substances mimicking these. The relevance of the present observations is discussed.