Structure-Function of Serotonin in Bivalve Molluscs

It has been observed that 5-HT excites the heart nerves in hard clam and regulates con- traction and relaxation of the anterior byssus retractor muscle in the blue mussel. It is now known that 5-HT regulates several neurobehavioral systems such as mood, appetite, sleep, learning, and memory. It also plays critical roles in the physiological functions of peripheral organs involved in stress, growth, and reproduction in the animal kingdom. The present study reviews conserved 5-HT biosynthesis and its localization in the ner- vous system, and its physiological contribution to regulate reproduction in bivalves. In the cytosol of neurons, tryptophan hydroxylase catalyzes hydroxylation of l -tryptophan to 5-hydroxytryptophan, which is converted to 5-HT by aromatic l -amino acid decarboxylase. A 5-HT transporter and a monoamine oxidase reuptakes and metabolizes 5-HT to control the amount of released 5-HT in the nervous system and peripheral organs. Perikarya and fibers of 5-HT neurons are mostly located in the cortices and neuropil of ganglia, respectively, and innervate the gonad. However, distribution and 5-HT content differ among species and sexes and undergo seasonal variations associated with gonadal development. The present review pays a special attention to future research perspectives to better understand 5-HT regulation of reproduction in bivalves. destruction of 5-HT also exists which is mediated by monoamine oxidase (MOA) located in the outer membrane of mitochondria (Mt). The MOA catalyzes the oxidative deaminative of 5-HT to 5-hydroxy-3-indolacetaldehyde (5-HIAL) that is metabolized into 5-hydroxy-3- indolacetic acid (5-HIAA) by aldehyde dehydrogenase (ALDH). Released 5-HT binds to its receptor(s) on the surface of a postsynaptic cell or postsynaptic neuron (not shown in the figure) to trigger intracellular signaling required for a cellular response, e.g., stimulation of oocyte and sperm maturation. The 5-HT receptors are mainly G-protein coupled receptor (5-HT 1, 2, 4, 6, 5, and 7 receptors), which induce adenylate cyclase (AC) or phospholipase C signaling (PLC). However, the 5-HT 3 receptor is a ligand-gated ion channel and regulates ionic influx.


Introduction
5-Hydroxytryptamine called serotonin (5-HT) is a transmitter substance of the nervous system in animal kingdom. From its first discovery in the 1940s, many laboratories have been directing their studies toward understanding the biology of 5-HT and its physiological functions on various biological systems especially on mammals as model organism [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. However, 5-HT has been also identified in bivalves from the period of its first discovery and earlier studies on these animals have led to convince the neurobiologist that it acts as a neurotransmitter.
A brief bibliography of 5-HT discovery and its physiological functions is provided in Table 1. Rapport et al. [16] was the first who isolated a vasoconstrictor substance from the blood serum in a crystalline form and tentatively identified it as 5-HT in a creatinine sulfate complex [17].

Year
Scientists Contribution to discovery of identification, localization, and characterization of 5-HT
Isolation of a substance from the blood serum that constricts blood vessels and contracts isolated intestinal strips [16] 1948 Rapport et al.
The substance contains an indole ring [42] 1949 Rapport Identification of chemical structure of 5-HT as a creatinine sulfate complex [17] 1951 Hamlin and Fisher Synthesis of 5-HT [18] 1953 Twarog and Page Identification of 5-HT in the extract of the brain of mammals (dog, rat, and rabbit) [19] 1953 Gaddum Assigning a role for 5-HT in normal cerebral function in mammals [43] 1953 Welsh 5-HT, in contrast to acetylcholine, excites the heart nerves in hard clam (Bivalvia, Mollusca) originating from visceral ganglion [21] 1954 Amin et al. Localization of 5-HT in the central nervous system (brain) of mammals (dog) [20] 1954

Wooley and Shaw
Human schizophrenia might be due to 5-HT deficiency [44] 1954 Twarog 5-HT-mediated relaxation of byssus retractor muscle in the blue mussel (Bivalvia, Mollusca) is antagonist of acetylcholine contracting the muscle [22] 1956 Hoyle and Lowy 5-HT is a putative neurotransmitter controlling contraction and relaxation of the anterior byssus retractor muscle in the blue mussel [23] 1957 Brodie and Shore Assigning 5-HT function as a neurotransmitter [24] 1957 Welsh Identification of 5-HT in the extract of nervous system of various bivalve mollusks [25] 1962 Falck et al. Development of Falck-Hillarp method to visualize monoaminecontaining cells as intense yellow-green fluorescence [45] 1964

Dahlström and Fuxe
Identification of 5-HT cell bodies in the pons and midbrain, from where they project with their axons to the forebrain, medulla, and spinal cord [46] 1968 Sweeney Identification and localization of 5-HT in whole body extract and in the nervous system of blue mussel using Falck-Hillarp's method [47] Within next 5 years, 5-HT has been synthesized [18], identified in the extract of mammalian brain [19], and localized in the brain of mammals [20]. Along with these studies on mammals, Welsh [21], Twarog [22], and Hoyle and Lowy [23] demonstrated that 5-HT excites the heart nerves in hard clam (Mercenaria mercenaria), and regulates contraction and relaxation of the anterior byssus retractor muscle in the blue mussel (Mytilus edulis) that both belong to Bivalvia, Mollusca. These observations resulted in identification of 5-HT as a neurotransmitter in the nervous system of mammals [24]. In the same year, Welsh [25] identified 5-HT in the nervous system of bivalves and demonstrated that 5-HT content in these animals is higher than other invertebrates and vertebrates [26]. Moreover, bivalves have served some advantages to be used as experimental model: (A) they are small which is a great opportunity to conduct serial examinations on the whole organism, (B) they have a simple nervous system, (C) the nervous system is relatively large in size and easy to dissect ganglia and connectives, and (D) the nervous system contains high amount of 5-HT.
Although 5-HT biosynthesis and its receptor structure have been reviewed in bivalves [39][40][41], there is a gap of review on physiological signaling of 5-HT in these animals. The present study reviews the biology of 5-HT in bivalves; particularly its contribution to reproduction. Biosynthesis pathway of 5-HT in the nervous system and cellular localization of 5-HT neurons in the nervous system are studied. Particular attention has then paid to 5-HT content and distribution of 5-HT neurons in the gonad. This study provides future perspectives that await investigation to better understand 5-HT network and signaling in bivalve reproduction. suggestion led to the discovery of an enzyme in mammalian kidney [51], later called aromatic L-amino acid decarboxylase (AADC) [52] that mainly decarboxylates 5-HTP to 5-HT [53]. In parallel, studies have shown that the extract of mammalian brain contains 5-HT [19], and administration of exogenous 5-HTP or tryptophan increases 5-HT level in the brain and peripheral organs [54,55]. A year later, Welsh and Moorhead [56] observed that homogenates of ganglia of hard clam are capable of synthesizing 5-HT from 5-HTP, in vitro. Further studies using the blue mussel (Mytilus edulis) demonstrated presence of precursors of 5-HT (either tryptophan or 5-HTP) [57][58][59], and decarboxylation of 5-HTP to 5-HT [60,61]. Thus, 5-HT biosynthesis in bivalves is similar to those of higher vertebrates. Although aforementioned studies have shown biosynthesis pathway of 5-HT and demonstrated that both nervous system and peripheral organs contain 5-HT; however, it was still unknown where the 5-HT biosynthesis takes place and how it gets transferred to other organs.
In 1960s, Bertaccini [62] and Gal et al. [63] demonstrated that the brain contains 5-HT even after partial or complete removal of 5-HT in the gastro-intestinal tissues and the brain produces 5-HT after intracerebral injection of radioactive labeled tryptophan. It is worth noting that it has previously been shown that the intestine contains large amount of 5-HT [64]. These studies provided the scientists with very important information that the brain independently synthesizes 5-HT from L-tryptophan, and suggested that exogenous 5-HT administration incorporates to 5-HT contents in the nervous system. Next studies resulted in molecular identity of two major enzymes in 5-HT biosynthesis pathway: tryptophan hydroxylase (TPH) and AADC [6,14,65,66] (Figure 1). In the cytosol of the nerve cells, TPH catalyzes hydroxylation of l-tryptophan to produce 5-HTP by incorporation of an atom of atmospheric oxygen into l-tryptophan and the other is reduced to water, in the presence of the cofactor agent, tetrahydrobiopterin. The pathway is rate-limiting step meaning that suppression of TPH activity results in stopping 5-HT biosynthesis. The AADC catalyzes conversion of 5-HTP to 5-HT which is not rate-limiting step. It has also been shown that the rate at which 5-HT is produced in the central nervous system highly depends on availability of tryptophan, tryptophan uptake into the brain, and dietary contents of tryptophan and other amino acids (such as tyrosine and phenylalanine) that compete with tryptophan uptake or transport carrier into the brain [8,14,67].
In the snail, it has been observed that certain nerves are capable of accumulating radioactive labeled 5-HT [68]. Using bivalves, Stefano and Aiello [69] observed that fluorescence intensity of 5-HT-immunoreactive (5-HT-IR) neurons increases in the blue mussel after administration of exogenous 5-HT. Thus, as in mammals, 5-HT biosynthesis in bivalve mollusks also takes place in the nervous system.
Further studies have shown that there are biological systems through which external amounts of the released 5-HT is regulated, as its rise may cause abnormal physiological functions or might be lethal for cells. Reuptake and metabolism of 5-HT are key determinants to remove and/or inactivate significant amount of released 5-HT, respectively. Metabolism of 5-HT is mediated by monoamine oxidase (MOA) located in the outer membrane of mitochondria, and catalyzes the oxidative deaminative of 5-HT to 5-hydroxy-3-indolacetaldehyde (5-HIAL), which is further metabolized into 5-hydroxy-3-indolacetic acid (5-HIAA) by an NAD + -dependent aldehyde dehydrogenase. In addition, an NADH-dependent aldehyde reductase or an NADPH-dependent alcohol-dehydrogenase converts 5-HIAL to 5-hydroxytryptophol (5-HTOL) [6,70] (Figure 1). In mollusks, small amount of MOA has been reported [71]. Boutet et al. [72] cloned MOA molecular structure in the Pacific oyster. Administration of MAO inhibitor leads to increase in the number and intensity of 5-HT-IR neurons in the blue mussel [69]. Thus, metabolism of 5-HT is active in bivalve mollusks. However, studies have demonstrated that 5-HT action at the synapse is mostly terminated by its reuptake across the presynaptic membrane [73][74][75][76][77]. Figure 1. Biosynthesis, metabolism and reuptake of 5-hydroxytryptamine (serotonin, 5-HT) in bivalves. In the cytosol of the 5-HT neurons, tryptophan hydroxylase (TPH) catalyzes hydroxylation of l-tryptophan to produce 5-hydroxytryptophan (5-HTP) that becomes converted to 5-HT by aromatic l-amino acid decarboxylase (AADC). Conversion of l-tryptophan to 5-HTP is rate-limiting step meaning that suppression of TPH activity results in stopping 5-HT biosynthesis, however AADC-catalyzed conversion of 5-HTP to 5-HT is not rate-limiting pathway. The 5-HT vesicles are transferred to axon terminal and released to synaptic cleft. Reuptake and metabolism of 5-HT are key determinants to inactivate significant amount of the released 5-HT. In mollusks including bivalves, 5-HT reuptake from synaptic cleft is more than the enzymatic destruction. It is an ionic-coupled system and mediated by a serotonin transporter (SERT) that transports 5-HT from synaptic cleft to the presynaptic 5-HT neuron. However, enzymatic destruction of 5-HT also exists which is mediated by monoamine oxidase (MOA) located in the outer membrane of mitochondria (Mt). The MOA catalyzes the oxidative deaminative of 5-HT to 5-hydroxy-3-indolacetaldehyde (5-HIAL) that is metabolized into 5-hydroxy-3indolacetic acid (5-HIAA) by aldehyde dehydrogenase (ALDH). Released 5-HT binds to its receptor(s) on the surface of a postsynaptic cell or postsynaptic neuron (not shown in the figure) to trigger intracellular signaling required for a cellular response, e.g., stimulation of oocyte and sperm maturation. The 5-HT receptors are mainly G-protein coupled receptor (5-HT 1, 2, 4, 6, 5, and 7 receptors), which induce adenylate cyclase (AC) or phospholipase C signaling (PLC). However, the 5-HT 3 receptor is a ligand-gated ion channel and regulates ionic influx.
The 5-HT reuptake is also similar between mollusks and mammals. It is an ionic-coupled pathway mediated by a serotonin transporter (SERT) that transport 5-HT from synaptic cleft to the presynaptic neuron [9,12,78]. SERT first binds a Na + ion, followed by 5-HT, and then a Clion in the synaptic cleft and transport to presynaptic neuron. After releasing 5-HT, K + efflux is involved in the translocation mechanism of SERT. This is an energy dependent process and a Na + /K + ATPase maintains the extracellular Na + concentration as well as the intracellular K + concentration [79]. This mechanism results in the inactivation of 5-HT by removing it from the synaptic cleft. Studies have also shown that a 5-HT reuptake inhibitor (SRI) interferes with SERT function to inhibit or suppress 5-HT reuptake [80,81].
The neuronal cell bodies "perikarya" are located at the cortices and the axonal processes lie at central core called "neuropil".
The pairs of CG lie on the sides of esophagus and are connected by a cerebral commissure in bivalves. In oyster species, CG are less developed and positioned at the sharp angle anterior to the labial palp, gills, and digestive gland [83]. In mussel and clam species, CG are located anterior to the digestive gland, and beneath the anterior adductor muscle [82,84]. In freshwater pearl mussel (Hyriopsis bialata), CG are fused [87]. In scallop species, the foot is positioned anterior to CG, and adductor muscle and digestive gland are located posterior to CG [82,86].
Each CG consists of an anterior lobe and a posterior lobe [88]. The CG innervate the palps, anterior adductor muscle, and parts of mantle [83,84,86].
In most bivalves, the pairs of PG lie on the foot and are connected by a pedal commissure [84][85][86]. However, PG are absent in oyster species [83]. In soft-shell clam (Mya arenaria), the PG are fused [89]. In freshwater pearl mussel, PG are positioned in the visceral mass [87]. The PG innervate the foot [84,86].
The paired VG are located on the ventral side of the adductor muscle, usually posterior to foot. In most bivalves, ganglia of VG are fused into a single organ [83,[89][90][91]. In scallop species, VG consist of five lobes; two anterior lobes, a posterior lobe, and two lateral lobes [88,90]. There is an accessory ganglion that locates at the point of the lateral lobes. The CG and VG are joined by a pair of cerebral-visceral connective that pass through the digestive gland or gonad. The VG innervate various organs, including gonads, gills, hearts, sensory organs, posterior adductor muscle, and parts of mantle [83,84,86].

Anatomy and annual cycle of neurosecretory cells in bivalves
Rawitz [92] seems to be first who isolated pear-or club-shaped neurons from the European flat oyster (Ostrea edulis). The neurons are classified into unipolar, bipolar, and multipolar neurons (Figure 3) [93]. Illanes-Bucher [94] classified the neurosecretory cells into A1, A2, A3, and A4 in the blue mussel. The A1-type neurons are small (6-15 μm), unipolar, and nucleus is located opposite to the axonal cone. The A2-type nerve cells are large (20-30 μm), multipolar, and nucleus is eccentric. The A3-type nerve cells are large (20-25 μm), unipolar, and nucleus is eccentric. The A4-type nerve cells are medium in size (12-15 μm), apparently unipolar, and contain numerous vacuoles surrounded by neurosecretory granules. Blake [95] observed that the neurosecretory cycle of neurons in the CG of the Bay scallop (Argopecten irradians) appeared identical to that of the VG. The neurosecretory cells also undergo distinct annual cycle [96][97][98][99]. Seasonal changes in the activity of neurosecretory cells are also associated with gonadal development, and the cells release their products at maturity stage [96]. Moreover, number of active neurosecretory cells positively correlates with progress of the gonad development in the Bay scallop [95], clam (Katelysia opima) [100], blue mussel [101], and greenlipped mussel (Perna canaliculus) [102].

Identification and cellular localization of 5-HT
Cellular localization of 5-HT neurons and its quantitative bioassay in the nervous system and gonads provide us with highly satisfactory knowledge to elucidate ontogeny and developmental biology of 5-HT biosynthesis, release, and reuptake, and to understand 5-HT regulation of reproduction in bivalves.

5-HT in the nervous system of bivalves
Welsh [25] was the first who identified 5-HT in the nervous system of the hard clam using a paper chromatography method. Then, Welsh and Moorhead [26,56,107] used a spectrofluorometric method to measure 5-HT in over 60 species from 11 different phyla that includes 7 bivalve species ( Table 2) [108]. They reported that (A) the nervous system of bivalves contains much higher 5-HT than that of other invertebrates. In the phylum Annelida, 5-HT is measured 0.1-10.4 μg/g wet in the nerve cords. In the phylum of Arthropoda, 5-HT is measured from <1.0 μg/g wet in the nerve cords, ventral ganglia, and green ganglia. In vertebrates, 5-HT is measured 0.3-2.6 μg/g wet in different parts of cat brain [109]. (B) Content of 5-HT is higher in the nervous system than the peripheral organs. (C) Content of 5-HT differs among various parts of the nervous system. It is higher in the ganglia than the connective nerves. In addition, they observed that 5-HT content is slightly lower in VG than those of CG and PG (10 vs. 15 μg/g wet) in the blue mussel. (D) The blood does not contain 5-HT. The authors suggested that 5-HT is produced in the nervous system: in cell bodies or synaptic region of neurons. Following development of cellular and molecular methods, 5-HT has been localized in the nervous system and gonad of several bivalve species (Table 3). Firstly, Falck-Hillarp's method has been used to localize 5-HT in fingernail clam (Sphaerium sulcatum) [47], blue mussel [69], and Yesso scallop [88]. In this method, histological sections are exposed to gaseous formaldehyde or glyoxylic acid to visualize monoamine containing neurons [45,121,122].
In all examined bivalve species, 5-HT-IR neurons are observed in CG, PG, and VG (Table 3). However, the Falck-Hillarp's method is not always useful as 5-HT fluorescence tends to faint rapidly. In addition, catecholamines neurons show similar intensity to that of 5-HT neurons at high concentrations [123]. In 1978, Steinbusch et al. [124] developed a rat monoclonal antibody against a 5-HT-bovine serum albumin conjugate to localize 5-HT in nervous system. Further studies have used monoclonal or polyclonal antibody against 5-HT to localize 5-HT-IR neurons in the nervous system and peripheral organs of bivalves ( Table 3). The advantage of immunohistochemistry method using antibodies against 5-HT is to describe morphology of 5-HT neurons, and to localize 5-HT distribution within different parts of nervous system, precisely. The 5-HT-containing neurons are mostly unipolar, although their sizes may differ among species (Table 3). Using an electron microscopy, it has been observed that 5-HT-IR neurons are often in close connection with each other, but without indication of gap junctions or other specialized junctions. The neurons possess numbers of granular vesicles (100-180 nm in Mediterranean mussel) containing 5-HT that concentrated at the cell periphery [104,125]. It has confirmed that 5-HT-IR fibers are the axon or axon terminals of 5-HT containing neurons that transport 5-HT to peripheral organs. Within the nervous system, 5-HT-IR fibers seem to be synaptic region, an area where release and reuptake of 5-HT occur.
In general, studies on bivalves show that 5-HT-IR neurons are mostly located in the cortices, and 5-HT-IR fibers are located in the neuropil of CG, PG, and VG ( Table 3). In Yesso scallop, 5-HT-IR neurons are located in the cortices of the right side of the left lobe and in the left side of the right lobe in anterior lobe (AL) of CG, while they are located throughout their cortices in PG and the posterior lobe (PL) of CG [105] (Figure 3). In the great scallop [90], distribution of 5-HT-IR neurons in the posterior lobe of CG slightly differs compared to Yesso scallop. In VG, 5-HT-IR neurons are restrictively scattered in the accessory lobe of scallop species [90,105,115] or at the roots of branchial nerves in clams [89]. Large numbers of 5-HT-IR fibers have also been observed in the cerebral-pedal, and cerebral-visceral-pedal connectives [90,103], suggesting that 5-HT transports from CG to VG [69,89,90,105]. Comprehensive overview of    Table 3. Cellular localization of 5-hydroxytryptamine (serotonin 5-HT) in the nervous system and gonad of bivalve mollusks. cellular localization of 5-HT indicates that localization and distribution of 5-HT-IR neurons may differ among subclasses of bivalve, for instance between Heterodonta (genus Mya, Ruditapes, and Venus) and Pteriomorphia (genus Pecten, Patinopecten, and Mytilus) ( Table 3). It might be due to differences in location of various parts of nervous system in the body to innervate peripheral organs.
Using histochemistry or immunohistochemistry methods, studies have shown that a few 5-HT-IR neurons are located in the cortex and neuropil of VG compared to those of the CG or PG, for instances in the blue mussel [47,69,128], Mediterranean mussel (Mytilus galloprovincialis) [103], great scallop [90], Atlantic deep-sea scallop (Placopecten magellanicus) [115], and soft-shell clam [89]. Matsutani and Nomura [105] reported no 5-HT-IR neurons in the VG of the Yesso scallop. Although VG contain a few 5-HT-IR neurons, they are usually rich in 5-HT-IR fibers. These studies confirm the Welsh and Moorhead's observation that 5-HT content differs among various parts of the nervous system.
Studies used spectrofluorometric method [26,47,56,[118][119][120] or electrochemical detection coupled with a high-performance liquid chromatography (HPLC-EC) to study 5-HT content in the nervous system of bivalves [90,110,114,115,117] ( Table 2). Results confirm aforementioned differences in 5-HT content among various parts of the nervous system, for instance it is higher in the CG than the VG of gaper clam (Tresus capax) and bent-nose clam (Macoma nasuta) [117]. In addition, the metabolite of 5-HT (5-HIAA) is detected in the nervous system of the brown mussel (Perna perna) [110] and Atlantic deep-sea scallop [114], suggesting that metabolism of 5-HT takes place in the nervous system.
Welsh and Moorhead [56] observed that in vitro 5-HT synthesis by the nerve tissues undergoes a seasonal variation and suggested seasonal variation of amine oxidase. Further studies have shown that 5-HT content in the nervous system undergoes seasonal variation along with gonadal development in bivalves ( Table 2). Content of 5-HT increases in the nervous system from early gonadal development to maturity stage in the brown mussel [110] and decreases following spawning in Peruvian scallop (Argopecten purpuratus) [113]. York and Twarog [120] reported that 5-HT in the PG of blue mussel is higher in April than March. It has also observed that 5-HT content in the whole nervous system of the blue mussel increases from April to October [118]. As the blue mussel spawns from late spring to late summer [129,130], these data suggest that 5-HT content increases during spawning. 5-HT content also correlates with the content of its metabolite (5-HIAA), suggesting that metabolism of 5-HT is in parallel to its biosynthesis in the nervous system [110].

5-HT in the gonad of bivalves
Localization of 5-HT in the gonad has studied in a few species of bivalves ( Table 3). Using method of Falck-Hillarp, Sweeney [47] and Matsutani and Nomura [88,105] observed the 5-HT-IR fibers in the gonoduct and epithelium around gonad in the Fingernail clam and Yesso scallop, respectively, and suggested that the 5-HT-IR fibers originate from CVC to innervate the gonad. Further studies using antibodies against 5-HT confirmed existence of 5-HT-IR fibers in the gonad of Yesso scallop [105], great scallop [90], Atlantic deep-sea scallop [115], surf clam [112], warty venus [91], and soft-shell clam [106]. These studies clearly indicated that the nervous system innervation of the gonads is mostly emerged from VG or derived from CVC. The 5-HT-IR fibers surround periphery of collecting tubes and of gonadal lobules (acini) in males and females filled with sperm and oocytes, respectively (Figure 3).
As seasonal-dependent 5-HT content in the nervous system, distribution of 5-HT fibers also changes in the gonad throughout reproductive cycle [91,106,112,115] (Figure 3; Tables 2  and 3). Generally, the 5-HT-IR fibers are occasionally observed around the germinal acini, and extensively distributed around the collective tubes at early developmental stage. However, the 5-HT-IR fibers around the acini are more frequent at maturity stage [112]. After spawning, the 5-HT-IR fibers still exist around collecting tubes, and are abundant around gamete empty acini.
Using spectrofluorometric or HPLC-EC method, 5-HT content has been measured in the gonad of the Atlantic deep-sea scallop [114,115], surf clam [112], Pacific lion's paw scallop (Nodipecten subnodosus) [111], and brown mussel [110]. Matsutani [131] reported a tendency toward an increase and a decrease of 5-HT content in the testis and ovary of Japanese scallop (Chlamys farreri nipponensis) during spawning, respectively. It has shown that 5-HT content increases from early developmental stage of the gonad to maturity stage in males and females [110,111]. In surf clam, Masseau et al. [112] reported that changes in 5-HT content are uncertain in males during testicular development and after spawning. However, in females, 5-HT is high at early development stage, decreases at maturity stage and spawning, and then increases after spawning. They also reported that 5-HT content does not differ between males and females when they are compared at similar gonadal development stage. Klouche et al. [110] pooled the data of males and females in brown mussel, as there are no differences between sexes, and observed that 5-HT content increases toward maturation of gonad. In Peruvian scallop, 5-HT content decreases in the male and female portions of gonad following spawning [113,116]. Observed differences in 5-HT content among studies may represent inter-species differences associated with 5-HT regulation of reproduction that might also be different between sexes. Klouche et al. [110] reported that the gonadal content of 5-HT metabolite (5-HIAA) in brown mussel is high at early development and become decreased at maturity stage. As 5-HT content is high at maturity, these suggest that 5-HT-dependent reproduction associates with decreasing 5-HT inactivation mediated by its metabolism.
A few studies show 5-HT content in both nervous system and gonad, for instance in the Peruvian scallop [113,116] and brown mussel [110]. Results show higher 5-HT content in the nervous system than gonadal tissue as 5-HT content is lower in connective nerves than 5-HT neurons [26,56].
Croll et al. [115] observed that distribution of 5-HT-IR neurons and fibers is similar between juvenile and adult in the Atlantic deep-sea scallop or between sexes in the surf clam [112]. However, abundance or distribution of 5-HT neurons and 5-HT content may differ between sexes. Martínez and Rivera [116] observed that 5-HT content is higher in the male portion than female portion of the gonad of the Peruvian scallop. Expression of 5-HT-IR fibers or neurons has been seen to be higher in the VG of females than that of males [127]. These studies may suggest inter-sex difference in 5-HT biosynthesis or inter-sex difference in 5-HT regulatory function of reproduction.

Conclusion and future research perspectives
The essential components of 5-HT biosynthetic pathway are highly conserved in the animal kingdom. The 5-HT biosynthesis from the essential amino acid L-tryptophan is catalyzed by TPH, which convert L-tryptophan to 5-HTP, and by AADC, which convert 5-HTP to 5-HT. All precursors of 5-HT are identified in the nervous system of bivalves. In mammals, there are two isoforms of TPH (TPH1 and TPH2), which are predominantly expressed in the peripheral organs and in the nervous system, respectively. However, TPH1 is the primary form and expresses earlier in neural development [132,133]. Molecular sequence of the gene encoding AADC has also been identified and localized in mammals [134,135]. It has a non-specific tissue distribution and is expressed in wide range of cell types [66]. In bivalves, molecular identity, localization, and characterization of TPH and AADC are unknown. These studies will provide us with satisfactory information to better understand ontogeny of 5-HT neurons in the nervous system and to elucidate developmental biology of 5-HT regulation of reproduction.
It has been seen that the first 5-HT-IR neurons appearing within the nervous system correspond to the location of the CG and apical ganglion (AG) during the late trochophore stage: 30-32 h postfertilization in blue mussel [136], 24 h postfertilization in surf clam [137], and 27 h postfertilization in the Bay mussel (Mytilus trossulus) [138]. Kreiling et al. [137] reported that the 5-HT-IR neurons appear in VG of surf clam at 48 h postfertilization. Following 72 h postfertilization, the 5-HT-IR neurons emerging from the CG and AG extend their processes to the VG, through which connections of the 5-HT-IR neurons between CG/AG and VG are formed at 96 h postfertilization. During the embryonic development, the size of the 5-HT area in the CG/AG and VG increases from 24 h to 96 h postfertilization, which is associated with an increase in 5-HT content. Cann-Moisan et al. [139] reported that 5-HT content undergoes variation throughout the larval and postlarval stages. It rises from 2 d to 27 d postfertilization (15-50 pg/μg of protein, respectively); however, it decreases to less than 1 pg/μg of protein after 55 d postfertilization. These indicate that 5-HT neurons form at the embryonic stage, and 5-HT content increases from embryonic development to metamorphosis, and decreases after metamorphosis. Voronezhskaya et al. [138] observed that 5-HT-IR neurons innervate the peripheral organs in the postmetamorphic stage, suggesting that 5-HT biosynthesis undergoes developmental variation. This might be related to the availability of the 5-HT precursors or inactivation mechanisms of 5-HT. However, further studies are required to investigate development of 5-HT fibers in the gonad through developmental stage.
As animals lost the ability to synthesize tryptophan, there possess developed biological mechanisms through which animals obtain tryptophan from their diets. Thus, 5-HT biosynthesis highly depends on dietary factors including availability of tryptophan and competitive uptake or transport of tryptophan with other amino acids (such as tyrosine and phenylalanine) into the 5-HT neurons. Studying nutritional effects on 5-HT biosynthesis will lead to better understanding of physiological relationships between seasonal variation in 5-HT content and gonadal development. In addition, it can help us to investigate the impacts of parental nutrition on gamete maturation and fertility in bivalves. These studies can provide us with knowledge to better understand 5-HT controls of feeding behaviors such as appetite and satiety, which have been demonstrated in mammals [140].
Mechanisms of 5-HT inactivation in the nervous system and peripheral organs of bivalves are poorly understood. It requires molecular identity, localization, and characterization of SERT and MOA. In this regard, several types of SERT and MOA inhibitors are available [80,114,141] that provide us with useful tools to elucidate molecular signaling that control 5-HT reuptake and metabolism. A few studies show that selective 5-HT reuptake inhibitors modulate 5-HT-induced spawning in bivalves. Fong [142] and Fong et al. [143,144] reported spawning of Zebra mussel treated with selective 5-HT reuptake inhibitors (fluvoxamine, fluoxetine, zimelidine, and paroxetine). Both males and females are capable of releasing their gametes after treatment with fluvoxamine at 10 −7 and 10 −6 M, respectively. Following treatment with fluoxetine, 100% of males have spawned at 10 −4 to 10 −5 M, however spawning has induced in 50-60% of females at 10 −5 M. Zimelidine induces spawning in 100 and 60-70% of males and females at 10 −4 M. Paroxetine induces spawning in 50 and 20% of males and females at 10 −6 and 10 −5 M, respectively. Considering spawning of males and females at 10 −3 M 5-HT, these results indicate that selective 5-HT reuptake inhibitors stimulate spawning in Zebra mussel at concentrations lower than that of 5-HT. Further examinations have revealed that mianserin and cyproheptadine interfere with fluvoxamine-, fluoxetine-, and zimelidine-induced spawning [144] suggesting that antagonists of 5-HT 2 receptor block stimulatory function of selective 5-HT reuptake inhibitors in spawning. Inhibition of 5-HT reuptake may increase the synaptic 5-HT concentrations, which in turn activate postsynaptic 5-HT receptor to induce spawning. It is also possible that selective 5-HT reuptake inhibitors act as ligands at postsynaptic receptor rather than inhibition of SERT. Overall, these studies suggest that 5-HT transport plays a key role in reproduction; however, the mechanisms of action are largely unknown.
So far, histochemistry and immunohistochemistry methods have been employed to localize the 5-HT neurons and fibers, and spectrofluorometric and HPLC-EC methods have been used to identify 5-HT content in the nervous system and gonad of various bivalve species. Successful implication of various mammalian monoclonal or polyclonal antibodies indicates that 5-HT structure is highly conserved through evolution across the animal kingdom. However, mechanisms through which 5-HT acts on a biological system may differ. The present review shows that 5-HT content highly differs in the nervous system and gonad of bivalve species. The inter-species differences in 5-HT content might be related to capability of nervous system to synthesize 5-HT, differences in 5-HT inactivation or 5-HT transport from nervous system to the gonad. In the latter case, 5-HT content in the gonad may correspond to 5-HT concentration that requires to stimulate spawning. The present review shows that 5-HT concentration to induce spawning highly differs between sexes, and among species. It is worth to note that tissue sampling, extraction procedure, and analytical method affect the results of 5-HT content. In addition, 5-HT content undergoes seasonal variation and change following spawning.