Norfluoxetine induces spawning and parturition in estuarine and freshwater bivalves.

Fluoxetine, a commonly prescribed antidepressant (Prozac), has been detected in sewage effluent. Its active metabolite norfluoxetine is more potent and has been detected in sewage influent and in fish tissues. We tested the effects of norfluoxetine on spawning and parturition in bivalves. Norfluoxetine induced significant spawning in zebra mussels and dark false mussels at concentrations as low as 5 microM. Norfluoxetine induced significant parturition in fingernail clams at 10 microM. Fluoxetine also induced spawning in dark false mussels at concentrations as low as 100 nM. Implications for environmental impacts of norfluoxetine and fluoxetine on native and exotic bivalves are discussed.

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

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

Cilia-driven rotational behavior in gastropod (Physa elliptica) embryos induced by serotonin and putative serotonin reuptake inhibitors (SSRIs).

We characterized the serotonin (5-hydroxytryptamine; 5-HT) receptor mediating cilia-driven rotational movement in embryos of the freshwater gastropod Physa elliptica. In addition, putative serotonin reuptake inhibitors (SSRIs), previously shown to induce other 5-HT-mediated processes in molluscs, were tested for their ability to induce rotation. As in previous studies with other freshwater gastropods, 5-HT induced a significant dose-dependent increase in rotation from 10(-6) to 10(-4) M. The 5-HT(1A) agonist 8-OH-DPAT produced a similar dose-dependent increase in rotation. However, the 5-HT(2) agonist alpha-CH3-serotonin evoked a significant rotational response only at the highest concentration of 10(-4) M. The 5-HT(2) receptor antagonist mianserin not only blocked 5-HT-induced rotation, it reduced rotation rate below that of baseline. However, two other antagonists, cyproheptadine (5-HT(2)) and propranolol (5-HT(1)), caused similar responses that consisted of an initial rotational surge followed by reduced rotation. Thus, these drugs appear to act as partial agonists. The putative SSRI fluvoxamine exhibited a significant dose-dependent increase in positive rotation as that seen with 5-HT. The SSRIs paroxetine and fluoxetine both caused an increase in rotation at 10(-6) and 10(-5) M but reduced rotation rate below that of baseline at 10(-4) M. These results agree with other studies on aquatic molluscs, suggest a 5-HT receptor with a mixed 5-HT(1)/5-HT(2) pharmacological profile and add to a now growing body of literature on the pharmacology of molluscan 5-HT receptors. In addition, all the tested putative SSRIs induced cilia-driven rotation in Physa embryos, indicating either the presence of 5-HT reuptake transporters or that these compounds act as 5-HT receptor ligands. J. Exp. Zool. 286:414-421, 2000.

Induction and potentiation of parturition in fingernail clams (Sphaerium striatinum) by selective serotonin re-uptake inhibitors (SSRIs).

Parturition in fingernail clams (Sphaerium spp.) can be induced by external application of serotonin and serotonergic ligands. Selective serotonin re-uptake inhibitors (SSRIs) increase neurotransmission at serotonergic synapses by blocking re-uptake transporters. We tested the efficacy of SSRIs (fluvoxamine, fluoxetine, paroxetine) at inducing parturition in the fingernail clam Sphaerium striatinum. Parturition was induced by fluvoxamine and paroxetine, but only potentiated by fluoxetine. Fluvoxamine was potent, significantly inducing parturition at concentrations from 10 nM to 100 microM compared with negative controls. Fluvoxamine also significantly potentiated a subthreshold (10 microM) concentration of serotonin. Paroxetine also induced parturition but was less potent. Only 10 microM paroxetine significantly induced parturition compared with controls. Fluoxetine (1-100 microM) did not induce any parturitions, but at 5 microM it potentiated parturition in subthreshold serotonin concentrations as low as 50 nM. These results suggest the presence of serotonin re-uptake transporters in bivalve molluscs and may provide a way of stimulating serotonergic mechanisms without using serotonin or its ligands.

Zebra Mussel Spawning Is Induced in Low Concentrations of Putative Serotonin Reuptake Inhibitors.

Serotonin (5-hydroxytryptamine, 5-HT) and its receptor ligands induce both oocyte maturation and spawning in zebra mussels (Dreissena polymorpha). The selective serotonin reuptake inhibitors (SSRIs) fluvoxamine ("Luvox"), fluoxetine ("Prozac"), and paroxetine ("Paxil") are commonly prescribed drugs for the treatment of depression in humans. They act to increase 5-HT neurotransmission by inhibiting reuptake transport proteins at synapses. I tested the efficacy of these drugs at inducing spawning in zebra mussels. All three compounds induced spawning in both sexes at concentrations lower than that for 5-HT itself. Fluvoxamine was particularly potent, inducing spawning in 100% of both sexes at 10-5 and 10-6 M. The concentration that induced a significant percentage of animals to spawn was as low as 10-9 M for males and and 10-7 M for females. The lowest concentration of fluvoxamine to induce spawning was 10-8 M for females (40%) and 10-10 M for males (20%). Gametes spawned in fluvoxamine (10-5 M and lower) were viable, and swimming trochophores were formed within 20 hours. Fluoxetine was also an effective spawning inducer, causing 100% of males to spawn at 5 x 10-6 M. The concentration of fluoxetine required to induce a significant percentage of spawning was as low as 5 x 10-8 M for males and 5 x 10-6 M for females. In both fluvoxamine and fluoxetine, more than 60% of the males spawned within the first hour of exposure. In contrast, paroxetine was a weak spawning inducer. At concentrations of 10-5 and 10-6 M it induced significant, but low (50% and 40%, respectively) percentages of males to spawn. Paroxetine did not induce significant spawning in females. Thus, fluvoxamine, fluoxetine, and paroxetine can induce spawning at low concentrations, and fluvoxamine is the most powerful spawning inducer in any bivalve. These may be useful agents for stimulating invertebrate serotonergic mechanisms without applying exogenous 5-HT, and they are potentially important in bivalve aquaculture. Moreover, these results suggest, for the first time, the presence of 5-HT reuptake transporters in bivalve molluscs.

Serotonergic Ligands Induce Spawning but not Oocyte Maturation in the Bivalve Mactra chinensis from Central Japan.

We examined the spawning sensitivity to serotonin and serotonergic ligands in the Japanese bivalve Mactra chinensis. Spawning was induced by both injected and externally applied scrotonin (5-hydroxytryptamine. 5-HT). The vertebrate 5-HT2 receptor agonist alpha-methyl 5-HT and the selective 5HT1A agonist 8-OH-DPAT were also effective at inducing spawning. However TFMPP (m-trifluoromethylphenylpiperazine, a verterbrate 5-HT1 receptor agonist) and 1-methyl-chlorophenyl biguanide (a vertebrate 5-HT3 agonist) were not effective spawning inducers. The 5-HT-induced spawning was blocked by mianserin (a vertebrate 5-HT2 antagonist). The rank order of potency of the agonists was: 5-HT > alpha-methyl 5-HT > 8-OH-DPAT > TFMPP > 1-methyl-chlorophenyl biguanide; these data support a growing body of literature invoking a mixed 5-HT1/5-HT2 pharmacological profile for serotonin receptors mediating reproductive processes in bivalves. However, neither 5-HT nor 8-OH-DPAT induced germinal vesicle breakdown (GVBD) in Mactra oocytes. Sperm induced GVBD in a high percentage of oocytes. This is the first report of a bivalve in which spawning, but not GVBD, can be induced by 5-HT. This result might be expected because Mactra spawns germinal vesicle oocytes that normally undergo GVBD upon fertilization, but is in contrast to the case of the closely related Spisula spp. in which serotonin induces both processes. The ability of 5-HT to induce spawning but not GVBD makes Mactra chinensis a model organism for studying spawning and meiotic mechanisms in bivalves.

The Effect of Salinity and Temperature on Spawning and Fertilization in the Zebra Mussel Dreissena polymorpha (Pallas) from North America.

Zebra mussels have dispersed from their original site of introduction in the Great Lakes into the Mississippi River, Hudson River, and other watersheds in which they will encroach upon brackish water estuaries. To investigate their likelihood of reproductive success in such estuaries, we investigated the conditions of temperature, salinity, and acclimation under which spawning and fertilization could occur. Reproductive function of mussels that were acclimated to salinities up to 7.0 parts per thousand (ppt) at 12°, 20°, and 27°C for 1 to 21 days was tested. Reproductive function of non-acclimated mussels that had been maintained in fresh-water aquaria was also tested in various salinities. Spawning was induced by serotonin, previously demonstrated to elicit spawning of fertile gametes in fresh water. Successful fertilization was indicated by oocyte cleavage after adding sperm. Nonacclimated mussels spawned in salinities of 1.75 and 3.5 ppt at 12°, 20°, and 27°C, but not at 7.0 ppt. Fertilization using gametes from non-acclimated mussels occurred only in fresh water and at 1.75 ppt. Acclimation for as little as 2 days enhanced spawning. Fertilization rate in a salinity of 3.5 ppt improved within 4 days of acclimation and continued at a high level for as long as 21 days of acclimation. Although animals acclimated for 4 days in 3.5 ppt spawned readily when tested in salinities as high as 7.0 ppt, almost no fertilization occurred in 7.0 ppt. The reduction in fertilization at increasing salinities may be due in part to reduced sperm motility. Unfertilized oocytes remain intact for hours in fresh water; however, in salinities as low as 0.7 ppt, unfertilized oocytes tended to rupture within 2 hours. These data show that although sudden increases in salinity produce an immediate decrease in the reproductive capacity of zebra mussels, acclimation to brackish water can occur, and zebra mussels may be able to reproduce in brackish water below 7.0 ppt.

In vivo and in vitro induction of germinal vesicle breakdown in a freshwater bivalve, the zebra mussel Dreissena polymorpha (Pallas).

Oocyte maturation and germinal vesicle breakdown (GVBD) was induced in zebra mussel (Dreissena polymorpha) oocytes by in vivo and in vitro application of serotonin (5-hydroxytryptamine, 5-HT), and in vitro application of 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, a 5-HT1A receptor agonist). Oocytes initiated GVBD approximately 30 minutes after exposure to 5-HT (10(-3) M) at 23 degrees C, and by 40-50 minutes after exposure, most oocytes lacked a germinal vesicle. An exposure time to 5-HT as brief as five to ten minutes was required to trigger the maturation process, which terminates in spawning of fertilizable oocytes in nearly all mussels. But, with an exposure time of less than five minutes, spawning was reduced by application of 10(-4) M methiothepin (a potent blocker of 5-HT-induced spawning in zebra mussels). Thus, the sequence of oocyte maturation events in zebra mussels was determined. Oocytes are arrested at the germinal vesicle stage (prophase I) within the ovary. 5-HT reinitiates the maturation process, including GVBD and spawning of metaphase I oocytes, which are further arrested until fertilization. To our knowledge this is the first demonstration of oocyte maturation induction by serotonergic ligands in a freshwater bivalve.

Inhibition and sex specific induction of spawning by serotonergic ligands in the zebra mussel Dreissena polymorpha (Pallas).

Serotonin (5-hydroxytryptamine, 5-HT) stimulates spawning in the zebra mussel (Dreissena polymorpha), a macrofouling European bivalve that has recently invaded North America. To develop methods of controlling zebra mussel spawning, two vertebrate serotonin antagonists, methiothepin and metergoline, known to bind with high affinity to snail 5-HT receptors, were tested for their ability to block 5-HT-induced spawning in zebra mussels. Methiothepin inhibited 5-HT-induced spawning at concentrations as low as 10(-6) M. Metergoline (10(-4) M) inhibited 5-HT-induced spawning; however, at lower concentrations (10(-8) to 10(-5) M), metergoline by itself significantly induced spawning in male, but not female zebra mussels. Metergoline (10(-5) M)-induced male spawning was inhibited by 10(-5) M methiothepin. Thus, methiothepin is the most effective inhibitor and metergoline the most powerful inducer of spawning yet tested in zebra mussels.

Correlated evolution of the cis-acting regulatory elements and developmental expression of the Drosophila Gld gene in seven species from the subgroup melanogaster.

The tissue-specific expression patterns of glucose dehydrogenase (GLD) exhibit a high degree of interspecific variation in the adult reproductive tract among the species in the genus Drosophila. We chose to focus on the evolution of GLD expression and the evolution of the Gld promoter in seven closely related species in the melanogaster subgroup as a means of elucidating the relationship of changes in cis-acting regulatory elements in the Gld promoter region with changes in tissue-specific expression. Although little variation in tissue-specific patterns of GLD was found in nonreproductive tissues during development, a surprisingly high level of variation was observed in the expression of GLD in both developing and mature reproductive organs. In some cases this variation is correlated with changes in sequence elements in the Gld promoter which were previously shown to direct tissue-specific expression in the reproductive tract. In particular D. teissieri adult males do not express GLD in their ejaculatory ducts, atypical of the melanogaster subgroup species. The Gld promoter region of D. teissieri specifically lacks all three of the TTAGA regulatory elements present in D. melanogaster. The TTAGA elements were previously shown to direct reporter gene expression to the ejaculatory duct. Together these data suggest the absence or presence of the TTAGA elements may be responsible for variation in the absence or presence of GLD in the ejaculatory duct among species.

Spawning in the zebra mussel (Dreissena polymorpha): activation by internal or external application of serotonin.

The zebra mussel, Dreissena polymorpha, was recently introduced accidentally into the Great Lakes and, due in part to its prodigious reproductive capacity, is spreading rapidly in temperate fresh waters of North America. The present studies examine some of the mechanisms that regulate spawning in this animal. In August and September 1990 and in May 1991 injection of serotonin (5-hydroxytryptamine, 5-HT) induced ripe male, but not female, zebra mussels to spawn. During mid-summer 1991, 5-HT induced spawning in both males and females, and 5-HT could produce spawning responses by either injection or external application. External pH over a broad range (6.0 to 9.1) had no effect on spawning, neither inhibiting induction of spawning by 5-HT nor significantly eliciting spawning itself. With external application, 10(-3) M and 10(-4) M 5-HT caused spawning, but 10(-5) M and 10(-6) M did not. Cyproheptadine, a 5-HT receptor antagonist, reduced the response of both males and females by more than half. Spawning in response to 5-HT was blocked at 4 degrees C, but not at 12 degrees C, 20 degrees C, or 27 degrees C. For male zebra mussels morphological criteria for judging gonadal maturity were well-correlated with probability of spawning in response to 5-HT. For females, the likelihood of spawning in response to 5-HT was not tightly coupled to morphological maturity of the gonad, with many morphologically ripe females failing to spawn and some apparently immature animals releasing oocytes. Prior spawning reduced subsequent responsiveness and intensity of spawning of animals to 5-HT. These experiments support a role for 5-HT in regulating reproduction in zebra mussels and help define conditions by which zebra mussel spawning may be stimulated or inhibited.

Evidence for a Programmed Circannual Life Cycle Modulated by Increasing Daylengths in Neanthes limnicola (Polychaeta: Nereidae) From Central California.

Timing of parturition, fecundity, and life span were determined in laboratory cultures of the semelparous, self-fertilizing, viviparous polychaete Neanthes limnicola. Worms were exposed to fixed daylengths (short--8h light: 16h dark; neutral--12h:12h; long--16h:8h), switched between different fixed daylengths, and switched from fixed daylengths to increasing or decreasing daylengths. Timing of parturition was synchronized when under neutral daylength, but became asynchronous under both short and long daylength, as well as when any of the fixed daylength was followed by decreasing daylengths. Worms under neutral daylength had the highest fecundities and shortest life spans, while those under long days had the lowest fecundities and longer life spans. When fixed daylength (short, neutral, long) was followed by increasing daylengths, timing of parturition was synchronized, fecundity was high, and life span shortened. These and earlier published experiments on the influence of seasonally changing photoperiods indicate that the life cycle of the estuarine N. limnicola is programmed to be completed in somewhat less than a year, and that seasonally changing photoperiods modulate it to determine the optimal time of parturition.