Characterization, expression patterns of molt-inhibiting hormone gene of Macrobrachium nipponense and its roles in molting and growth.

The oriental river prawn, Macrobrachium nipponense, is an important commercial aquaculture resource in China. In order to overwinter, M. nipponense displays decreased physiological activity and less consumption of energy. Sudden warming would trigger molting and cause an extensive death, resulting in huge economic losses. Therefore, it is of great practical significance to study the molting mechanism of oriental river prawns. Molt-inhibiting hormone gene (MIH) plays a major role in regulating molting in crustaceans. In this study, a full length MIH cDNA of M. nipponense (Mn-MIH) was cloned from the eyestalk. The total length of the Mn-MIH was 925 bp, encoding a protein of 119 amino acids. Tissue distribution analysis showed that Mn-MIH was highly expressed in the eyestalk, and that it had relatively low expression in gill, ovary, and abdominal ganglion. Mn-MIH was detected in all developmental stages, and changed regularly in line with the molting cycle of the embryo and larva. Mn-MIH varied in response to the molting cycle, suggesting that Mn-MIH negatively regulates ecdysteroidogenesis. Mn-MIH inhibition by RNAi resulted in a significant acceleration of molting cycles in both males and females, confirming the inhibitory role of MIH in molting. After long-term RNAi males, but not females had significant weight gain, confirming that Mn-MIH plays an important role in growth of M. nipponense. Our work contributes to a better understanding of the role of Mn-MIH in crustacean molting and growth.

Differential effects of silencing crustacean hyperglycemic hormone gene expression on the metabolic profiles of the muscle and hepatopancreas in the crayfish Procambarus clarkii.

In order to functionally characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), gene expression of CHH in the crayfish (Procambarus clarkii) was knocked down by in vivo injection of CHH double-stranded RNA (dsRNA), followed by metabolomic analysis of 2 CHH target tissues (the muscle and hepatopancreas) using nuclear magnetic resonance spectroscopy. Compared to the levels in untreated and saline-injected (SAI) animals, levels of CHH transcript, but not those of molt-inhibiting hormone (a CHH-family peptide), in the eyestalk ganglia of CHH dsRNA-injected (DSI) animals were significantly decreased at 24, 48, and 72 hour post injection (hpi), with concomitant changes in levels of CHH peptide in the sinus gland (a neurohemal organ) and hemolymph. Green fluorescence protein (GFP) dsRNA failed to affect levels of CHH transcript in the eyestalk ganglia of GFP DSI animals. Number of metabolites whose levels were significantly changed by CHH dsRNA was 149 and 181 in the muscle and 24 and 12 in the hepatopancreas, at 24 and 48 hpi, respectively. Principal component analysis of these metabolites show that metabolic effects of silencing CHH gene expression were more pronounced in the muscle (with the cluster of CHH DSI group clearly being separated from that of SAI group at 24 hpi) than in the hepatopancreas. Moreover, pathway analysis of the metabolites closely related to carbohydrate and energy metabolism indicate that, for CHH DSI animals at 24 hpi, metabolic profile of the muscle was characterized by reduced synthesis of NAD+ and adenine ribonucleotides, diminished levels of ATP, lower rate of utilization of carbohydrates through glycolysis, and a partially rescued TCA cycle, whereas that of the hepatopancreas by unaffected levels of ATP, lower rate of utilization of carbohydrates, and increased levels of ketone bodies. The combined results of metabolic changes in response to silenced CHH gene expression reveal that metabolic functions of CHH on the muscle and hepatopancreas are more diverse than previously thought and are differential between the two tissues.

RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera.

Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs ß bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs ß and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs ß and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.

Hid arbitrates collective cell death in the Drosophila wing.

Elimination of cells and tissues by apoptosis is a highly conserved and tightly regulated process. In Drosophila, the entire wing epithelium is completely removed shortly after eclosion. The cells that make up this epithelium are collectively eliminated through a highly synchronized form of apoptotic cell death, involving canonical apoptosome genes. Here we present evidence that collective cell death does not require cell-cell contact and show that transcription of the IAP antagonist, head involution defective, is acutely induced in wing epithelial cells prior to this process. hid mRNAs accumulate to levels that exceed a component of the ribosome and likewise, Hid protein becomes highly abundant in these same cells. hid function is required for collective cell death, since loss of function mutants shows persisting wing epithelial cells and, furthermore, silencing of the hormone bursicon in the CNS produced collective cell death defective phenotypes manifested in the wing epithelium. Taken together, our observations suggest that acute induction of Hid primes wing epithelial cells for collective cell death and that Bursicon is a strong candidate to trigger this process, possibly by activating the abundant pool of Hid protein already present.

Ecdysteroids regulate the levels of Molt-Inhibiting Hormone (MIH) expression in the blue crab, Callinectes sapidus.

Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle.

Pharmacophore based approach to design inhibitors in crustaceans: an insight into the molt inhibition response to the receptor guanylyl cyclase.

The first set of competitive inhibitors of molt inhibiting hormone (MIH) has been developed using the effective approaches such as Hip-Hop, virtual screening and manual alterations. Moreover, the conserved residues at 71 and 72 positions in the molt inhibiting hormone is known to be significant for selective inhibition of ecdysteroidogenesis; thus, the information from mutation and solution structure were used to generate common pharmacophore features. The geometry of the final six-feature pharmacophore was also found to be consistent with the homology-modeled MIH structures from various other decapod crustaceans. The Hypo-1, comprising six features hypothesis was carefully selected as a best pharmacophore model for virtual screening created on the basis of rank score and cluster processes. The hypothesis was validated and the database was virtually screened using this 3D query and the compounds were then manually altered to enhance the fit value. The hits obtained were further filtered for drug-likeness, which is expressed as physicochemical properties that contribute to favorable ADME/Tox profiles to eliminate the molecules exhibit toxicity and poor pharmacokinetics. In conclusion, the higher fit values of CI-1 (4.6), CI-4 (4.9) and CI-7 (4.2) in conjunction with better pharmacokinetic profile made these molecules practically helpful tool to increase production by accelerating molt in crustaceans. The use of feeding sub-therapeutic dosages of these growth enhancers can be very effectively implemented and certainly turn out to be a vital part of emerging nutritional strategies for economically important crustacean livestock.

Formation of diapause cyst shell in brine shrimp, Artemia parthenogenetica, and its resistance role in environmental stresses.

Artemia has attracted much attention for its ability to produce encysted embryos wrapped in a protective shell when subject to extremely harsh environmental conditions. However, what the cyst shell is synthesized from and how the formative process is performed remains, as yet, largely unknown. Over 20 oviparous specifically expressed genes were identified through screening the subtracted cDNA library enriched between oviparous and ovoviviparous Artemia ovisacs. Among them, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. Lacking SGEG protein (by RNA interference) caused the cyst shell to become translucent and the chorion layer of the shell to become less compact and pultaceous and to show a marked decrease of iron composition within the shell. The RNA interference induced defective diapause cysts with a totally compromised resistibility to UV irradiation, extremely large temperature differences, osmotic pressure, dryness, and organic solvent stresses. In contrast, the natural cyst would provide adequate protection from all such factors. SGEG contains a 345-bp open reading frame, and its consequentially translated peptide consists of a 33-amino acid residue putative signal peptide and an 81-amino acid residue mature peptide. The results of Northern blotting and in situ hybridization indicate that the gene is specifically expressed in the cells of shell glands during the period of diapause cyst formation of oviparous Artemia. This investigation adds strong insight into the mechanism of cyst shell formation of Artemia and may be applicable to other areas of research in extremophile biology.

Molecular characterization of gonad-inhibiting hormone of Penaeus monodon and elucidation of its inhibitory role in vitellogenin expression by RNA interference.

One of the important peptide hormones that control reproduction in crustaceans is gonad-inhibiting hormone (GIH). GIH is known to modulate gonad maturation by inhibiting synthesis of vitellogenin (Vg), the precursor of yolk proteins. In this study, a cDNA encoding a GIH (Pem-GIH) from the eyestalk of Penaeus monodon was cloned using RT-PCR and RACE techniques. Pem-GIH cDNA is 861 bp in size with a single ORF of 288 bp. The deduced Pem-GIH consists of a 17-residue signal peptide and a mature peptide region of 79 amino acids with features typical of type II peptide hormones from the CHH family. Pem-GIH transcript was detected in eyestalk, brain, thoracic and abdominal nerve cords of adult P. monodon. The gonad-inhibiting activity of Pem-GIH was investigated using the RNA interference technique. Double-stranded RNA, corresponding to the mature Pem-GIH sequence, can trigger a decrease in Pem-GIH transcript levels both in eyestalk ganglia and abdominal nerve cord explant culture and in female P. monodon broodstock. The conspicuous increase in Vg transcript level in the ovary of GIH-knockdown shrimp suggests a negative influence for Pem-GIH on Vg gene expression, and thus implies its role as a gonad-inhibiting hormone. This is the first report to demonstrate the use of double-stranded RNA to elucidate the function of GIH in P. monodon.

Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus.

In a study of genes expressed differentially in the freshwater crayfish Pacifastacus leniusculus infected experimentally with the white spot syndrome virus (WSSV), one protein, known as antilipopolysaccharide factor (ALF), was chosen, among those whose transcript levels increased upon viral infection, for further studies. ALF RNA interference (RNAi) experiments in whole animals and in cell cultures indicated that ALF can protect against WSSV infection, since knockdown of ALF by RNAi specifically resulted in higher rates of viral propagation. In a cell culture of hematopoietic tissue (Hpt) from P. leniusculus, quantitative PCR showed that knockdown of ALF by RNAi resulted into WSSV levels that were about 10-fold higher than those treated with control double-stranded RNA (dsRNA). In addition, RNAi experiments with other crayfish genes that had been found to be up-regulated by a WSSV infection did not result in any changes of viral loads. Thus, the cell culture does not respond to dsRNA in a similar manner, as shown earlier for dsRNA injected into shrimp, which gave a higher degree of resistance to WSSV infection. If ALF transcription in whole animals was stimulated by the administration of UV-treated WSSV, a partial protection against a subsequent challenge with the active virus was conferred to the host. This is the first crustacean gene product identified with the capacity to interfere with replication of this important pathogen.

Evidence for endocrine disruption in invertebrates.

The issue of endocrine disruption (ED) in invertebrates has generated remarkably little interest in the past compared to research with aquatic vertebrates in this area. However, with more than 95% of all known species in the animal kingdom, invertebrates constitute a very important part of the global biodiversity with key species for the structure and function of aquatic and terrestrial ecosystems. Despite the fact that ED in invertebrates has been investigated on a smaller scale than in vertebrates, invertebrates provide some of the best documented examples for deleterious effects in wildlife populations following an exposure to endocrine-active substances. The article provides an overview of the diversity in endocrine systems of invertebrates. The principal susceptibility of invertebrates to endocrine-active compounds is demonstrated with the case studies of tributyltin effects in mollusks and of insect growth regulators, the latter as purposely synthesized endocrine disrupters. The additional evidence for ED in invertebrates from laboratory and field studies is summarized as an update and amendment of the EDIETA report from 1998. Finally, conclusions about the scale and implications of the observed effects are drawn and research needs are defined.

Potent antagonistic action of synthetic analogues of APGWGNamide, an antagonist of molluscan neuropeptide APGWamide.

Fifty-five kinds of analogues of APGWGNamide (Ala-Pro-Gly-Trp-Gly-Asn-NH2), which is an antagonist of molluscan neuropeptide APGWamide, were synthesized and their antagonistic activities were examined on two molluscan smooth muscles. Among all the analogues tested, on spontaneous contraction of the crop of the land snail, Euhadra congenita, APGWG(L-biphenylalanine, Bip)amide showed the most potent antagonistic activity and its potency was 50-100 times higher than that of APGWGNamide. Likewise, on phasic contraction of the anterior byssus retractor muscle (ABRM) of the sea mussel, Mytilus edulis, the effect of APGWG(D-homophenylalanine, dHfe) was the most potent and showed 5-10 times stronger activity than that of APGWGNamide. In the tolerance test to known exo- and endopeptidases or the crop tissue homogenate, APGWGNamide was not only easily degraded by a proline-specific endopeptidase but also by the homogenate. Two kinds of potent antagonists were thus developed: APGWG(Bip)amide and APGWG(dHfe)amide, which will be useful tools for investigation of the function of APGWamide in the snail and the mussel, respectively.

The roles of transcription and translation in mediating the effect of electrical afterdischarge on neurohormone synthesis in Aplysia bag cell neurons.

The molecular links between membrane excitability and neurohormone synthesis were investigated using a simple model system: the bag cell neurons of Aplysia. We tested the hypothesis that the electrical afterdischarge, which leads to depletion of egg-laying hormone (ELH) by triggering secretion, rapidly stimulates ELH synthesis to replenish bag cell stores of hormone. Newly synthesized peptides were radiolabeled, and ELH peptide was immunoprecipitated. Within 4 h of afterdischarge, there was a 100% increase in ELH synthesis compared with that in unstimulated controls. Northern blot analysis showed that ELH messenger RNA (mRNA) levels were not altered for at least 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by 4-h treatments that inhibited either transcription (with actinomycin D) or translation (with anisomycin). Further work revealed that ELH mRNA is stable, with a half-life exceeding 32 h. Notably, the stimulatory effect of afterdischarge on ELH synthesis was blocked in response to treatment with the transcription inhibitor actinomycin D. These results suggest that the afterdischarge-induced increase in ELH synthesis is mediated by an increase in the rate of translation of already existing ELH mRNA, and that transcription of a non-ELH gene(s) is required for this effect to occur.

FMRFamide-like and allatostatin-like immunoreactivity in the lateral heart nerve of Periplaneta americana: colocalization at the electron-microscopic level.

Both allatostatin immunoreactivity (AS-IR) and FMRFamide immunoreactivity (FMRFa-IR) have been demonstrated light-microscopically in the lateral heart nerve of Periplaneta americana. The identical labeling of some fibers suggests the coexistence of the two antigens. Electron-microscopically, six granule types in the peripheral part of the lateral heart nerve can be distinguished according to their size and density (types 1-6). These granule types can be subdivided immunocytochemically by means of a new mirror-section technique. Granules of types 4 and 5 always exclusively show FMRFa-IR. In the populations of fibers containing granules of types 1 and 6, axon profiles can be found that contain granules colocalizing FMRFa-IR and AS-IR. Other axon profiles of these populations only contain immunonegative granules of the same ultrastructure. Granules of type 2 can be differentiated immunocytochemically in three forms in the same section: In some fibers, they are nonreactive; in other fibers of the same section, they show FMRFa - IR, whereas in a third fiber type, granules show AS - IR. Finally, granules of type 3 can be observed with FMRFa-IR. In other fibers, they occur with the same ultrastructure but exhibit no immunoreactivity. Two soma types occur in the lateral heart nerve. Soma type I is characterized by the production of electron-dense granules that show FMRFa-IR. Type II is in close contact with various fibers, forming different types of axosomatic synapses, hitherto unknown in Insecta.

Complex avoidance behaviour and its neurochemical regulation in the land snail Cepaea nemoralis.

1. In hot plate experiments, the pulmonate land snail Cepaea nemoralis displays a biphasic passive/active avoidance behaviour composed of retraction and subsequent searching mediated by antagonistic muscular systems. The switch, between the behaviours, is under neuronal control. 2. Leu- and met-enkephalin, as well as FMRFamide-antiserum, attenuated the retraction response and potentiated the searching behaviour. Opposite effects were achieved by injection of antisera to the enkephalins. 3. Both retraction and searching behaviours were potentiated by 5-HT. Methysergide antagonized the effects of the enkephalins on the searching behaviour. 4. We conclude that endogenous opioids act antagonistic to FMRFamide in the neuronally controlled switch between passive and active avoidance behaviour.

Suppressing effects of neuroactive peptides on the inward current caused by achatin-I, an Achatina endogenous peptide.

1. Modulatory effects of the four molluscan neuroactive peptides. FMRFamide (Phe-Met-Arg-Phe-NH2), APGW-amide (Ala-Pro-Gly-Trp-NH2), oxytocin and [SER2]-Mytilus inhibitory peptide ([SER2]-MIP) (Gly-Ser-Pro-Met-Phe-Val-NH2) were examined on the inward current (Iin) caused by achatin-I (Gly-D-Phe-Ala-Asp), which has been isolated from the Achatina ganglia. 2. Two Achatina giant neurone types, v-RCDN (ventral-right cerebral distinct neurone) and PON (periodically oscillating neurone), were used. Achatin-I was applied locally to the neurone tested by brief pneumatic pressure ejection, and the other molluscan neuroactive peptides were perfused around the ganglia. 3. FMRFamide, perfused at 3 microM, suppressed markedly the Iin elicited by the achatin-I of both v-RCDN and PON. APGW-amide at 3 microM also suppressed the Iin of v-RCDN, but did not affect that of PON. Oxytocin at 1 microM suppressed the Iin of PON, but did not affect that of v-RCDN. [Ser2]-MIP at 3 microM did not affect the Iin of v-RCDN. 4. The dose-response curves of FMRFamide, APGW-amide and oxytocin, indicated that their respective suppressive effects on the Iin of achatin-I were dose-dependent, and that APGW-amide was slightly more potent than the other peptides. The dose (pressure duration)-response curves of achatin-I (1 kg/cm2, 10(-3) M, 5 min interval), obtained by varying the duration of the achatin-I pressure ejection, were measured in the presence and absence of each of the three peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of new brassinosteroids with potential activity as antiecdysteroids.

The synthesis of four new brassinosteroids with 2 beta,3 beta-diol functionality and A/B cis and A/B trans ring junction is reported. These brassinosteroids could present activity as antiecdysteroids.

Induction of an inactivation pathway for ecdysteroids in larvae of the cotton leafworm, Spodoptera littoralis.

Treatment of the last-instar larvae of the cotton leafworm (Spodoptera littoralis) with ecdysteroids (moulting hormones) results in the induction of an ecdysteroid-inactivation pathway. Administration of ecdysone, 20-hydroxyecdysone or an ecdysteroid agonist, RH 5849, leads to induction of an ecdysteroid 26-hydroxylase activity. This induction occurred in both early sixth-instar larvae and in older larvae which had been head-ligated to prevent the normal developmental increase in ecdysone 20-mono-oxygenase activity. The induction of 26-hydroxylase activity requires both RNA and protein synthesis, as demonstrated by experiments involving actinomycin D and cycloheximide. The 26-aldehyde derivative of ecdysone and ecdyson-26-oic acid were also formed from ecdysone in the RH 5849-induced systems. Formation of the aldehyde and the corresponding 26-oic acid (ecdysonoic acid) from 26-hydroxyecdysone was directly demonstrated in a cell-free system, thus establishing the following inactivation pathway: Ecdysteroid-->26-hydroxyecdysteroid-->ecdysteroid 26-aldehyde-->ecdysteroid 26-oic acid.

The occurrence of schistosomin, an antagonist of female gonadotropic hormones, is a general phenomenon in haemolymph of schistosome-infected freshwater snails.

In haemolymph of Lymnaea stagnalis, parasitized with the digenetic trematode parasite Trichobilharzia ocellata, a neuropeptide (schistosomin) occurs which antagonizes female gonadotropic hormones, e.g. calfluxin (CaFl). By means of an ultracytochemical hormone-assay, the CaFl assay, it was demonstrated that the occurrence of schistosomin is a general phenomenon in schistosome-infected freshwater snails. Haemolymph of the schistosomiasis-transmitting snail species Biomphalaria glabrata and B. pfeifferi, parasitized with Schistosoma mansoni, also appeared to contain an antagonizing factor, i.e. schistosomin. In contrast, in haemolymph of L. stagnalis parasitized with Diplostomum spathaceum (Diplostomatidae) no schistosomin could be found. This suggests that schistosomin may only occur in snails infected with parasites belonging to the Schistosomatidae. The effect of schistosomin is rather specific. Haemolymph of B. glabrata parasitized with S. mansoni had not the capacity to inhibit the response to CaFl in the target organs for CaFl, the albumen glands of L. stagnalis and Bulinus truncatus. The same holds true for haemolymph of infected L. stagnalis: it did not inhibit the CaFl response in glands of B. glabrata and B. truncatus and even not in those of a related species (L. ovata). Schistosomins in haemolymph of infected B. glabrata and B. pfeifferi, on the other hand, seem more related. Both appeared to inhibit the hormone response in glands of the two Biomphalaria species studied. The results indicate that schistosomin in haemolymph of schistosome-infected pulmonate snails, although functionally related, may differ structurally.

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.

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.