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Neuronal Development in the Larvae of the Invasive Biofouler Dreissena polymorpha (Mollusca: Bivalvia), with Special Attention to Sensory Elements and Swimming Behavior.

Introduction

Invasive dreissenid bivalves impart a strong ecological and economic threat in ecosystems (Aldridge et ai, 2008). They have invaded a variety of habitats outside their native range and expanded rapidly in both European and North American lakes and rivers (Claudi and Gerald, 1994; Bobeldyk et ai, 2005). With their rapid invasion, dreissenids can quickly colonize new areas, causing serious fouling problems by altering the stability of the original ecosystem in different ways, including the suppression of native bivalve species (Mackie, 1991; O'Neill. 1997; Lovell et ai, 2006). The zebra mussel (Dreissena polymorplta), which is native in the Caspian Sea region, appeared in Hungary in the early 1930s in Lake Balaton, the largest shallow lake in Europe (Entz and Sebesryen, 1942); and within a few years it completely invaded the lake and became the mling bivalve in it.

Early developmental stages are generally accepted as a critical and determining period of the mussel (bivalve) life cycle (He etal, 2015). Zebra mussel life history is similar to that of marine bivalves and differs from that of the indigenous freshwater molluscan fauna. Namely, it begins with fertilization and spiral cleavage but is then followed by extracapsular development of free-swimming trochophore larvae, appearing 6 hours after fertilization and existing for a total of 32 hours before entering the next veliger larval stage (Sprang. 1993; Ackerman et al, 1994).

Bivalve larvae display a broad repertoire of behavioral patterns mediated by early elements of the nervous system, first by peripheral sensory cells (Kempf et al, 1997). Since the common ancestor of all Trochozoa likely possessed a trochophore larva, the group has been the longtime focus of extensive comparative anatomical, systematic, evolutionary, and phylogenetic studies (Naef, 1928: Beklemishev. 1969: Ruppert and Barnes, 1994; Haszprunar etal. 1995; Nielsen, 1995. 1998). Recently, efforts have been made to understand the neuronal development in the trochophore stage of different molluscan species (Wanninger and Haszprunar, 2003; Kristof and Klussmann-Kolb, 2005; Voronezhskaya et al, 2008). Studies on molluscan larvae have revealed that they already possess a rather well-developed nervous system, including, in general, the cerebral and pedal ganglia and, most importantly, the apical organ (AO), a specific sensory structure that exists until the end of embryogenesis (Barlow and Truman, 1992; Kempf et al, 1992, 1997; Voronezhskaya and Elekes, 1993; Hay-Schmidt, 1995; Croll, 2000; Dickinson et al, 2000; Page, 2002; Voronezhskaya et al, 2002, 2003; Hadfield, 2011). The AO seems to participate in detecting environmental cues, including unfavorable conditions, and so initiates behavioral changes in the animals (Hadfield et al, 2000; Voronezhskaya et al, 2004; Hadfield, 2011; Glebov et al, 2014). Whether early sensory elements other than AO are also present and involved in the regulation of the embryonic development and behavior of molluscs has not yet been examined. In contrast to gastropod and poly-placophoran molluscs, a few studies have been aimed at the early neuronal development of bivalves (Da Costa et al, 2008; Aranda-Burgos et al, 2014; He et al. 2015). Analyzing the early developmental stages may also be important from a comparative aspect, because bivalves are considered to be more closely related phylogenetically to gastropods than to polyplacophorans (Taylor, 1996).

Morphological characteristics of the developing nervous system of molluscs, mostly gastropods, at the level of the individual neuronal elements, have been described by using antibodies raised against various neurotransmitters and neuromodulators, combined with the application of different immu-nohistochemical (IHC) techniques (Croll and Voronezhskaya, 1996; Elekes et al, 1996; Diefenbach etal, 1998; Voronezhs-kayaeftf/., 1999; Croll, 2000, 2006; Hegedus et al, 2004). Serotonin (5-HT) and FMRFamide (FMRFa) occupy a prominent place among the signal molecules that are involved in a number of regulatory processes at both cellular and systems levels in adult and developing molluscs (see, e.g., Walker, 1986; Walker et al, 1996; Croll. 2000; Hay-Schmidt, 2000; Lopez-Vera et al, 2008; and, e.g.. Diefenbach et al, 1991; Vitellario-Zucarello et al, 1991; Voronezhskaya and Elekes. 1996, 2003; Marais and Carew, 1997; Page and Parties, 2000; Leise et al, 2001; Croll and Dickinson, 2004; Newcombe et al, 2006). Serotonin has already been detected in molluscan oocytes (Buznikov. 2007; Ivashkin et al. 2012, 2015), and both 5-HT-immunoreactive (IR) and FMRFa-IR neurons have been found to appear as early as in trochophore larvae (Croll. 2000; Voronezhskaya and Croll, 2016). Early trochophore FMFRa-IR neurons have been suggested to be pioneer cells that form a scaffold along which the central ganglia develop later (Croll and Voronezhskaya, 1995, 1996).

The aim of our study was to analyze the early, trochophore, and veliger larval development of the nervous system of the biofouling bivalve Dreissena polymorph[R] (Dreissenidae, Bivalvia, Mollusca). We paid special attention to sensory elements and their transmitter content, combined with physiological (swimming) assays, in order to understand their organizational principles. These principles may thus determine larval behavior, and may be possibly involved in the successful invasive capability of this species. Namely, the proper and early sensation and processing of environmental cues may be important factors that lead to the species' quick and broad dispersal and settlement. To follow the neurodevelopmental events in Dreissena larvae, we applied multiple fluorescence IHC labeling of 5-HT- and FMRFa-containing sensory elements. Then, in the physiological assays, either combined with 5-HTergic pharmacological interventions or by mimicking altered environmental conditions through increased salinity, we followed the upward swimming response of 48-hour post-fertilization (hpf) veliger larvae.

Materials and Methods

Animals

Adult specimens of the zebra mussel, Dreissena poly-morpha (Pallas, 1771), were collected in Lake Balaton (Hungary) during late May-June 2014-2016. The 12-15-mm-long specimens were gently removed from rock substrates by cutting their byssus threads. Animals were then held in running aerated lake water at 20-22 [degrees]C. Prior to the spawning procedure, each mussel was quickly cleaned with 70% ethanol. Spawning was induced by a combination of thermal shock (Dyachuk and Odintsova, 2009) and subsequent incubation with fluoxetine (Fong, 1998). Fifty animals were placed in a small beaker containing 0.5 liters of filtered Lake Balaton water (FW) and were kept at 10 [degrees]C for 12 h. Thereafter, mussels were placed individually in vessels filled with 10 ml of FW containing 5 [micro]mol [1.sup.-1] fluoxetine at 23 [degrees]C. To maintain a constant temperature for 15-30 min, the vessels were placed in a large petri dish filled with 23 [degrees]C water. Males started to spawn after about 30 min, whereas females began after 1 h of incubation. Spawning individuals were immediately moved into 50-ml vessels filled with fresh FW.

Eggs originating from several females were collected after 30 min of spawning and placed in a 800-ml beaker containing 100 ml of FW. Then five or six drops of FW containing sperm obtained from one male individual were added and stirred gently. After 20 min of fertilization, the upper 50 ml of water was removed, and 500 ml of fresh FW was carefully added to the beaker. The cleavage process and subsequent larval development were followed under an Axioplan compound light microscope (Carl Zeiss, Oberkochen, Germany).

Fertilized eggs were maintained in 800-ml beakers at 22 [degrees]C to develop further. After 14-16 h, the hatched larvae were collected from the upper one-third of the water column and transferred into fresh FW containing 50 [micro]mol [l.sup.-1] neomycin (neomycin sulfate, Sigma, St. Louis, MO), to prevent bacterial contamination during subsequent larval development. Larvae developed further in 500 ml of FW under constant agitation of the water column by an air jet directed to the water surface.

To examine the development of the nervous system, we collected samples at the following stages: blastula (16 hpf): early, middle, and late trochophore (18 and 22 hpf, 28 hpf, and 32-36 hpf. respectively): and early, middle, and late veliger (40-48 hpf, 60 hpf, and 96 hpf, respectively).

Immunochemistiy

Larvae were placed in 7% MgCL diluted in FW for 30 min at 10 [degrees]C. Animals were then fixed in 4% paraformaldehyde diluted in 0.1 mol [1.sup.-1] phosphate-buffered saline (PBS, pH 7.4) for 6 h at 4 [degrees]C and rinsed five times in 0.1 mol [1.sup.-1] PBS overnight. Then larvae were put into PBS containing 0.1 mol l[1.sup.-1] ethylenediaminetetraacetic acid (EDTA) for 30 min at room temperature, to remove calcium from the developing shells. After thorough washing, samples were incubated with polyclonal anti-5-HT or anti-FMRFamide primary antibodies raised in rabbit (ImmunoStar, Hudson, WI, dilution 1:2000), in both instances together with monoclonal mouse anti-acetylated [alpha]-tubulin (Sigma, dilution 1:3000). All antibodies were diluted in 0.1 mol [1.sup.-1] PBS containing 1% Triton X-100 and 10% normal goat serum (PBS-TX-NGS). Incubation with primary antibodies lasted 24-48 h at 4 [degrees]C. After rinsing, the specimens were incubated overnight at 4 [degrees]C in a mixture of 1:1000 dilution of Alexa Fluor 488 goat anti-rabbit immunoglobulin G (for both 5-HT and FMRFamide detection; Molecular Probes, Eugene, OR) and Alexa Fluor 633 goat anti-mouse (for tubulin detection; Molecular Probes), both diluted in 0.1 mol [1.sup.-1] PBS-TX. Specimens were then washed three times in PBS, each for 15 min; stained with 0.1 [micro]g [ml.sup.-1] 4',6-diamidino-2-phenylindole (DAPI) (Sigma) diluted in PBS, in order to visualize the nuclei; washed again in PBS for 15 min; and, finally, immersed in 80% glycerol diluted with PBS. The specimens in the small vials were placed in a dark box and left in a refrigerator for three or four weeks for clearing, and then they were mounted on glass slides. At least 50 embryos of each stage were examined for both 5-HT and FMRFa immunostaining. The specificity of the antibodies used in our study has been documented previously in various molluscs (Hernadi et al., 1989; Kemenes et al. 1989; Voronezhskaya and Elekes. 1996; Moroz et al, 1997), including bivalves (Dickinson et al, 2000; Voronezhskaya et al, 2008; Dyachuk and Odintsova, 2009). Negative controls obtained by omitting the primary antibodies revealed no specific immunostaining.

All samples were viewed by using either an epifluorescent Keyence BZ-9000 microscope (Nikon, Minoto, Tokyo, Japan) or a Leica TCS SP8 confocal laser scanning microscope (Leica, Wetzlar, Germany), with appropriate wavelength filter configuration settings. The necessary number of optical sections (20-60) with a 0.5-0.7-/im step size were made to capture all of the visualized details. Subsequent reconstructions of full-depth images using Nikon or Leica software were applied. The schemes, graphs, and photos were combined into plates, using Adobe Photoshop CS (San Jose, CA). Only the brightness and contrast of the images were adjusted, if necessary.

Immunochemically detected fluorescence intensity has been shown to correlate well with the transmitter content of the nerve cell (Voronezhskaya et al. 2004; Ivashkin et al, 2015). In our experiments, 48-hpf veliger larvae were processed for immunostaining protocol and then used for quantification of relative fluorescence intensity. All samples were investigated under a Leica TCS SP8 confocal laser scanning microscope, using standard configuration settings. The fluorescence intensity of 5-HT-IR and FMRFa-IR elements was determined for individual embryos (n = 20 for each data point). Each element was manually bordered, and average pixel intensity was detected inside the border in arbitrary fluorescence units (a.u.). Measurements and analysis were performed by using FIJI software (Schindelin et al, 2012). Statistical analysis and graphs were made using GraphPad Prism (LaJolla, CA). The obtained data were compared for control and experimental groups by using nonparametric Mann-Whitney U tests.

Swimming activity assay

The experimental setup was assembled with two plastic syringes (each of 3-ml volume, with 10-mm diameter and 50-mm length), placed onto each other by their narrow ends and tightly connected via a narrow plastic tube. The upper syringe was filled with FW. Two milliliters of incubation solution containing 48-hpf veliger larvae taken from the respective experimental group (about 200-300 animals [ml.sup.-1]) was transferred into the lower syringe. The upper and lower syringes were then tightly connected to each other by plastic tubing, and the whole experimental setup was positioned vertically in a quiet, nonvibrating, shaded place for 30 min at 22 [degrees]C. After 30 min, the upper and lower syringes were gently detached, and FW containing larvae from the upper and lower syringes was collected in 3-ml test tubes. The larvae were then fixed with 4% paraformaldehyde (PFA) diluted in 0.1 mol F PBS and were counted under a stereo dissecting microscope. Data were compared for control and experimental groups by using nonparametric Mann-Whitney U tests.

To test the effect of para-chlorophenilalanine (pCPA, Sigma), a 5-HT synthesis inhibitor, 48-hpf veligers were maintained in 50 ml of FW containing [10.sup.-5] mol [I.sup.-1] pCPA for 6 h at 22 [degrees]C. To test the effect of 5-hydroxytryptophan (5-HTP, Sigma), a 5-HT synthesis precursor, 48-hpf veligers were maintained in 50 ml of FW containing [10.sup.-5] mol [1.sup.1] 5-HTP for 1 h at 22 [degrees]C. After incubations, the 48-hpf larvae were processed for the swimming assay, followed by quantitative immunochemical analysis. To test the response of the larvae to increased salinity, 2 g of NaCl (2%0) was added to 1 liter of FW, and larvae were then cultivated from 24 hpf to 48 hpf. The tested 2%0 salinity is within the highest concentration measured in Lake Balaton (Somlyodi etal, 1983). After incubations, the larvae were processed for the swimming assay, followed by quantitative immunochemical analysis.

Results

Development of the 5-HT immunoreactive system

The first 5-HT-IR neuron appeared in the blastula stage (16 hpf). It was located at the center of the upper part, was about 5 [micro]m X 8 [micro]m in size, and bore two long cilia (Figs, la, d, 2). Two hours later (18 hpf), in early trochophore larva, the 5-HT-IR cell displayed a flask-shaped form, with a short, thick dendrite at the apical part projecting toward the surface (Figs, lb, 2). In addition, a second 5-HT-IR neuron of similar morphology was seen closely adjacent to the first one (Fig. lb, inset). In 22-hpf larvae, a small, rudimentary neuropil was also visible near the two cells, indicating the developing AO (Figs, lc, 2). At the same trochophore stage, a 5-HT-IR unpaired bipolar posterior neuron (PN) appeared asymmetrically at the left-side posterior extreme of the larval body (Fig. lc). No connection was detected between the anterior and posterior 5-HT-IR neurons at this stage. From the early trochophore stage (18 hpf), the ciliated prototroch started to be transformed into the velum (22 hpf), the main larval swimming organ (Fig. lc).

At the early veliger stage (40 hpf), the 5-HT-IR neurons at the apical extreme of the body displayed an elongated flask-shaped form, with a short and thick dendrite bearing two long cilia, by which they reached the surface at the apical region (Figs. 1 d, 2). A third labeled flask-shaped nonciliated neuron appeared dorsally to the first two apical cells, and two more without sensory dendrites were located laterally adjacent to the flask-shaped AO neurons (Figs, le, f, 2). At 48 hpf, a thick, bright neurite projected from the midline of the AO to the anterior adductor muscle (Figs, li, 2).

At 40-48 hpf, a robust distal dendrite of the PN, with two long cilia connected to the dendritic knob, was constantly detected (Figs, lg-j, 2). On the opposite end of the PN, two thin varicose axon processes projected symmetrically on the left and right sides to the AO (Figs, lg-i, 2). The PN thereafter retained this anatomy throughout, until the end of the investigated larval development (96 hpf), that is, possessing axon processes reaching the anterior part of the body and a thick, short ciliated dendrite projecting distally to the caudal surface of the animal (Fig. 2). In addition, from 40 hpf, two more tubulin-IR cells were seen adjacent to the PN (Fig. lj, k, insets). These three cells seemed to be part of a possible posterior sensory organ, probably functioning with 5-HT and other neurotransmitters.

By 40 hpf of the early veliger stage, the digestive system was already formed, and the stomach was clearly visible. At this time, a 5-HT-IR cell with a typical pear-like form appeared in the stomach wall (Figs, lj, k, 2). Labeled processes originating from the stomach neuron could also be observed. By 40 hpf, the short, thick dendrite projected straight toward the stomach lumen. From the 48-hpf early veliger stage, a long axon process, bearing varicosities along its entire length, ran anteriorly to the AO, following the stomach curvature (Fig. lj, j'). By the late (96-hpf) veliger stage, the AO comprised a compact neuronal structure, including a neuropil: together with the other two sensory elements (the PN and the stomach cell) bearing long axon processes, they seem to represent the larval sensory system of Dreissena (Fig. 2). The main larval swimming organ, the velum, displayed a significant increase in size during veliger development (40-96 hpf), although without receiving any 5-HT-IR innervation (Fig. 1 g, h, k).

Development of the FMRFamide-immunoreactive system

The very first FMRFamide-fR (FMRFa-IR) neuron appeared in the late trochophore stage (32 hpf) at the anterior extreme of the larval body, at the site of the developing AO (Figs. 3a, 4). The solitary cell displayed a flask-shaped form, with a short, thick dendrite projecting to the surface, without bearing sensory cilia. The apical tuft of cilia was clearly identifiable nearby. By the early veliger stage (40 hpf), a total of seven Fa-IR cells were already present in the larval body, delineating the future organization of neuronal elements up to the end of the veliger stage (96 hpf) (Figs. 3b, 4). In the apical extreme of the body, one more labeled cell with weak fluorescence appeared that was connected directly to the surface, with its sensory dendrite bearing no cilia either. In addition, an FMRFa-IR neuron (the PN) at the posterior extreme of the body and four FMRFa-IR cells located laterally and symmetrically (two on the left side and two on the right side) could also be observed. Of the four laterals, two were situated more anteriorly and two more posteriorly. Consequently, they were called right and left lateral anterior cells and right and left posterior cells, respectively. These cells were connected by varicose processes to each other, starting to form a ring and later completing by association with the AO and the PN.

At 48 hpf, a third flask-shaped FMRFa-IR cell appeared in the AO, and a small local neuropil was also detected (Figs. 3c, d, 4). Three more FMRFa-IR neurons were observed at 48 hpf: one located in the stomach wall, displaying a short dendrite projecting to the stomach lumen, and two cells, one on each side, added to the lateral posterior cells (Figs. 3d, f, 4). This total population of labeled cells remained unchanged until the late veliger stage (96 hpf). Their processes, but not that of the stomach cell, also reached the adductor muscles (Figs. 3e, f.4).

Pharmacological manipulation of the larval serotonergic system and swimming activity

To reveal the possible connection between the 5-HT containing early larval neuronal elements and the swimming activity of Dreissena polymorpha, pharmacological manipulation of the 5-HT synthesis was applied in 48-hpf veligers, combined with quantitative immunofluorescence histochemistry and a swimming assay (Fig. 5).

In control experiments, 48-hpf larvae were found to display upward swimming in the water column. After 30 min, the larvae showed an equal distribution in the upper and lower chambers of the experimental setup (Fig. 5a). Application of [10.sup.-5] mol [1.sup.-1] pCPA. the blocker of tryptophane hydroxylase, significantly decreased the number of larvae reaching the upper chamber (Fig. 5a, b). In contrast, a 1-hour application of [10.sup.-5] mol [1.sup.-1] 5-HTP, which is a 5-HT synthesis precursor, significantly enhanced the number of larvae reaching the upper chamber (Fig. 5a, b). Altered fluorescence intensity in the investigated 5-HT-IR sensory elements could be detected by light microscopy (Fig. 5c, d). The relative fluorescence intensity increased by up to 50%-75% after incubation with 5-HTP and decreased by about 50% after pCPA treatment (Fig. 5e-g), showing a positive correlation with the changes detected in swimming activity after the pharmacological interventions.

Effect of increased salinity on the larval 5-HT and FMRFa content and swimming activity

As a possible environmental effect, we tested the influence of increased salinity on the 5-HT and FMRFa immunoreactivity of identified neuronal structures (Figs. 6, 7) and the swimming activity (Fig. 5a, b) of 48-hpf Dreissena larvae. After 24 h of incubation in FW containing 2 g [1.sup.-1] (2%0) NaCl, the swimming activity of the larvae increased significantly (Fig. 5a, b). Also, fluorescence intensity was changed in all selected 5-HT-IR (Fig. 6a) and FMRFa-IR (Fig. 7a) neurons, compared to the control. In the case of 5-HT-IR cells, this elevated fluorescence intensity could clearly be seen in the sensory and interneurons of both the AO and the PN (Fig. 6b). It was also supported by the quantification of the relative brightness of immunolabeling (Fig. 6c). The most sensitive element reacting to increased salinity was the PN, showing a 150% increase in fluorescence intensity compared to the control, followed by the apical intrinsic neurons, with about a 100% increase. Meanwhile, the apical sensory neurons and the neuropil region of the AO showed less than a 50% increase (Fig. 6c). In the case of FMRFa-IR larval neuronal elements, the response to increased salinity was different (Fig. 7b, c). The AO and lateral cells showed a significant increase of brightness intensity, compared to the control (Fig. 7c'), whereas FMRFa-IR fluorescence intensity in other neuronal structures, such as the AO neuropil and the PN, remained near the control value.

Discussion

The present study was devoted to the early (trochophore and veliger) neurodevelopmental events of an invasive bivalve species, the zebra mussel, Dreissena polymorpha, with special attention to 5-HT- and FMRFamide-containing cells, respectively, two pivotal signal molecules playing various roles in the regulatory processes of molluscs. Our findings provide, on the one hand, a detailed, until now unknown description of the larval neuronal development of Dreissena 5-HTergic and FMRFaergic sensory systems and, on the other, data on the possible involvement of these sensory systems in the swimming activity (possibly related to settlement competency) of this bivalve. The analysis of the effect of the pharmacological manipulation of the 5-HTergic system and enhanced salinity on the swimming behavior of the larvae, combined with quantitative IHC, points to a correlation between the appearance of a set of early sensory elements, their transmitter content, and the swimming activity of the bivalve larvae. This suggests that the role of these neurons in elaborating early strategies is possibly involved in the successful distribution of Dreissena, at the expense of other aquatic organisms. Studies on the neuronal development of different invertebrate Trochozoa species have shown that they possess a well-developed nervous system, which certainly detects and processes environmental stimuli (Lacalli, 1984; Hay-Schmidt, 1995; Kempf etal., 1997; Page. 2002; Voronezhskaya etal., 2004). According to earlier and recent data, the larval nervous system of bivalves already possesses, as in gastropods, an early transient sensory system, which is mainly concentrated in the AO (Ospovat et al., 1989; Koolakovsky and Phlyachinskaya. 1993; Croll et ai, 1997; Kreiling et al, 2001; Voronezhskaya et al, 2008; He et al, 2015).

Organization and morphological characteristics of the early larval nervous system in Dreissena

According to our IHC findings, in both the 5-HT-IR and FMRFa-IR systems visualized in larval Dreissena, three sensory "centers" appear during the 96-hour-long trochophore and veliger stages. The three different locations (apical, posterior, and stomach) of sensory cells remain the anatomical backbone of the larval organization of the Dreissena sensory system until the end of the veliger stage (96 hpf). Although the basic pattern of organization and development of both signaling systems is similar, significant differences in the timing of appearance and details of their neuromorphology could also be observed. As to the similarities, the flask-form 5-HT-IR and FMRFa-IR sensory cells appear first in the apical region, gradually forming the AO, which is followed by the emergence of the PN and stomach sensory neurons. The sensory neurons displayed typical bipolar morphology, with a robust sensory dendrite and a thin sensory axon, the latter sometimes bifurcating. The visualization of PN and stomach cells is an a priori observation that we believe has not been described before in molluscan trochophore and veliger larvae. A posterior sensory organ in a trochophore larva was identified earlier in a polychaete. Phyllodoce maculate (Nezlin and Voronezhskaya, 2003). The organ consisted of five bipolar sensory cells displaying similar morphology to the PN found in Dreissena, of which two were also 5-HT-immunopositive.

The differences between the 5-HT-IR and FMRFa-IR systems are as follows, (i) Nonsensory 5-HT-IR cells are added to the AO by the beginning of the veliger stage (not observed in the case of the FMRFa-IR AO), (ii) The tips of the sensory dendrite 5-HT-IR AO and the 5-HT-IR posterior neurons are supplied with cilia, whereas the FMRFa-IR sensory dendrites do not bear cilia. Although they possess no cilia, the role of FMRFa-IR sensory dendrites in perceiving environmental information is assumed (see Fig. 7). In the case of 5-HT-IR sensory neurons, the receptor molecules are most probably located on the cilia, whereas in FMRFa-IR elements they can be bound to the dendritic membrane, (iii) The 5-HT-IR sensory elements appear at an early thochophore stage (16 hpf), whereas the first FMRFa-IR cells can be observed only by the late trochophore and early veliger stages (32-40 hpf), followed at 40 hpf by another cell added to the AO, as well as by the posterior FMRFa-IR neuron and the first anterior and posterior lateral cells. In the early veliger stage, the complete 5-HT-IR system (totaling seven cells) is already present with most of its processes. However, from 48 hpf, both signaling systems are completely developed, which then can be observed until the end of the studied veliger stage (96 hpf). From the early (48 hpf) veliger stage, the 5-HT-IR nervous system contains altogether seven neurons: five in the AO, as well as the single PN and the stomach cell. At the same time, the 11 FMRFa-IR neurons are distributed as follows: 3 comprise the AO, and 6 are located laterally, in addition to the single PN and the stomach cell.

A very similar early 5-HTergic system has been described in bivalve trochozoa larva of Mytilus trossulus (Voronezhs-kaya et al, 2008), in which the first 5-HT-IR neuron appeared in the apical region of the trochophore. followed soon thereafter by four others, thus increasing the number of this type of cells to five. In contrast to Dreissena, however, the 5-HT-IR neurons were restricted to the apical region throughout the entire larval development. The larval organization of the 5-HTergic system is remarkably similar, even in phylogenetically distant gastropod species (Barlow and Truman, 1992; Kempf etal., 1997).

Other transmitters have also been shown in sensory neurons of developing molluscs. FMRFa-IR sensory cells were found in the AO of the trochophore larvae of the marine bivalve Mytilus (Voronezhskaya et ai, 2008), as well as in the AO of developing aplachophoran molluscan species (Redl et al., 2014). Tyrosine-hydroxylase-IR (dopamine-containing) sensory neurons appeared first in the AO and the foot of the trochophore larvae of the pond snail Lymnaea stagnalis and later in the tentacle and the lips during the veliger stage (Voronezhskaya etal, 1999). Also in Lymnaea, nitrogen monoxide-containing peripheral sensory neurons were found in the foot, mantle edge, and lips during the postmetamorphic period of embryogenesis (Serfozo et al, 1998).

Another important anatomical aspect, possibly from a functional point of view, is that while the apical cells do not send axons descending to the PN and/or the stomach neurons, the latter two in 5-HT immunolabeling and in the FMRFa-IR PN project to the AO, suggesting that they forward sensory information about the caudal (environmental) region and the feeding (satiation) state of the veliger to the AO. Therefore, in addition to its own sensory function, an integrative role of AO is also assumed, supported by the presence of a neuropil-like structure.

The AO, which is a pivotal sensory structure in free-swimming larvae also involved in the control of the velar prototrochal cilia function (Kempf et al, 1997), shows basic organization similarities in different planktonic and free-living gastropod species. In the course of ultrastructural investigations on Aplysia californica veligers, Marois and Carew (1997) have demonstrated five 5-HT-IR cells (one-third of all AO cells), among which three ampullary (sensory) neurons and two interneurons were found. In the AO of planktotrophic caenogastropod larvae, three to six, but in most cases five, 5-HT-IR sensory (ampullary and para-ampullary) cells and two lateral nonsensory cells were described (Page and Parries. 2000). Similarly, five 5-HT-IR cells, among them three sensory (ampullary) neurons and two interneurons, as well as a neuropil with extensions to the developing cerebral ganglia, were found in the AO of nudibranch larvae (Kempf et al, 1997). The organization of the Dreissena AO fits well in this general form of cellular arrangement described in other molluscs; hence, it is suggested that the additional presence of interneurons and a neuropil in the AO is a consequent feature and can be regarded as morphological evidence of local integrative processes.

An intriguing question concerns the appearance of the FMRFa-IR lateral cells, missing from the larval 5-HTergic system. According to our observations (see Fig. 4). the labeled cells are connected by long processes to the AO. In addition, they project to the adductor muscles, raising the possibility either of their direct regulatory role in early muscle function or of their involvement in the development of the muscle, thus acting as trophic factors. In chiton larvae, transient dorsolateral cells, although sensory, showing FMRFa immunoreactivity have been described previously (Haszprunar et ai. 2002: Voronezhskaya et ai. 2002).

The long axon processes of both the lateral cells and the sensory neurons appear to bear varicosities along their entire length until reaching their targets, the AO or the adductor muscles. The presence of varicosities may refer to the free release of 5-HT or FMRFa at varicosities, in this way exerting remote control (fine tuning) of (i) early physiological and/or behavioral events, such as velum cilia movement and related swimming activity, and (ii) the development and/or regulation of somatic events of the veliger Dreissena larvae. Both 5-HT and FMRFamide have been shown to be involved in remote control actions in a number of invertebrates, including molluscs, by varicose fibers located in the connective tissue covering the ganglia and/or peripheral nerves, as well as situated directly on their surface (see, e.g., Nassei, 1988, 1996; Elekes and Nassei, 1990; Elekes, 1991 ; Beltz, 1995). In Lymnaea embryos of the E40 stage, pharmacological manipulation of the 5-HTergic system by 5-HT agonists resulted in a significant rotational increase (Filla et al., 2009), whereas in early (E25) trochophore embryos of Helisoma trivolvis, a pair of identified 5-HTergic neurons, the future giant 5-HTegic cells in adults, were shown to send neuntes to surface ciliary cells responsible for rotation (see, e.g., Diefenbach et al, 1991; Kuang and Goldberg, 2001; Koss etal. 2003).

Functional-physiological considerations

In the present study we have made an attempt to deliver pharmacological, physiological, and quantitative immunoflourescence data for the possible involvement of 5-HTergic and FMRFaergic signaling systems in the regulation of the locomotion (swimming) behavior of larval Dreissena. Changes of 5-HT immunoreactivity in the sensory neurons and interneurons evoked by either stimulating or blocking the 5-HT synthesis were accompanied by the alteration of swimming behavior. This raises the question of the role of this monoamine in the regulation of Dreissena larvae swimming. Serotonin has long been demonstrated to play a role in a series of central and peripheral modulatory processes of gastropods (see, e.g., Walker, 1986; Walker et al, 1996; Gillette, 2006), including embryonic development and different behaviors, such as feeding and locomotion (rotation and gliding) (Helisoma trivolvis, Goldberg et al., 1994; Doran et al., 2004; Lymnaea stagnalis, Filla et al, 2009: Balog et al, 2012; IB, KE, RH, EEV, G. Balog, L. Hiripi, M. Y. Khaborova, G. Maasz, unpubl. obs.). In Lymnaea veliger larvae, no 5-HT immunolabeling could be detected following pCPA treatment, and pharmacological treatments affecting the 5-HTergic system (including specific receptors) influenced both early larval rotation and post-metamorphotic gliding, as well as the 5-HT-IR innervation of the buccal mass and related radula protrusion. In Helisoma embryos, the innervation of ciliary cells by an identified pair of 5-HTergic neurons (Koss et al, 2013) and the role of 5-HTergic receptors in ciliary beating, as well as the direct effect of 5-HT on the Ca content of ciliary cells, have been described (Doran etal, 2004). In an early study by Koshtoyants et al. (1961), the exogenous application of 5-HT was shown to accelerate ciliary beating in larval gastropods.

The perception and conect interpretation of inputs, including chemical stimuli, from the sunounding world are pivotal throughout the entire life period in the animal kingdom. They are equally important in sustainment (feeding, predation, escape) and reproduction. However, during early development they may also have a role in initiating events related to the finalization of somatic and regulatory processes. In this respect, free-living aquatic larvae are especially vulnerable when searching for the optimal settlement sites. Therefore, our experiments with increased salinity can be regarded as an approach in analyzing the possible effect of the change of environment on the sensory-motor system of the invasive Dreissena. According to our findings, both the 5-HT-IR and FMRFa-IR sets of neurons of the larval Dreissena nervous (sensory) system respond to increased salinity through a significantly higher intensity of immunofluorescence, compared to the control cells. This suggests the involvement of the anterior and caudal sensory centers in the perception of changes in the environment, in this case, higher salinity. The fact that the apical intemeurons, the AO neuropil, and the lateral cells also reacted may indicate the participation of nonsensory, integratory elements in processing the salinity effect. In the case of the PN and the lateral cells, an efferent transmission toward the developing adductor muscles is also assumed. In the larvae of freshwater snails Lymnaea and Helisoma. early FMRFa-IR neurons were shown to be involved in osmoregulation, and an elevated FMRFa level had a stimulating effect on 5-HT synthesis (Chaban and Voronezhskaya. 2008). Although increased salinity was found in our study to initiate a high intensity of swimming activity of the Dreissena veligers, recently it was also shown to be a limiting factor for the dispersal of both dreissenid and mytilid bivalves (van der Gaag et al, 2016). The marine bivalve Crenomytilus grayanus proved to be very sensitive to changes in salinity, which could block feeding and retard larval growth (Yaroslavtseva and Sergeeva, 2010). Moreover, it was shown that changes in salinity have an impact on the growth and survival of the larvae of another marine bivalve. Mytilus trossulus. influencing simultaneously the expression of 5-HT and FMRFa immunoreactivities (Vekhova et al, 2012). In the marine snail Ilyanassa obsoleta. acidic seawater was found to stimulate metamorphosis, a process also regulated by different transmitter systems, such as 5-HT, nitric oxide, and FMRFamide (Leise and Cahoon, 2012; Leise et al, 2014). When applying conditioned water to study conspecific adult-to-embryo information transmission, the role of 5-HTergic and dopaminergic AO neurons in retarding embryogenesis and related behaviors, such as rotation, was demonstrated in Helisoma and Lymnaea (Voronezhskaya et al, 2004).

The changes in the 5-HTergic and FMRFamidergic neuronal systems, accompanied by the altered swimming behavior of the Dreissena veligers observed in our study, both in the effect of pharmacological interventions and in increased salinity, may help, on the one hand, to better understand the development of the larval bivalve (molluscan) nervous system and, on the other, shed light on the possible neuronal background of behavioral responses to environmental factors. We assume that early detection of an environmental input--be it attractive or repellent--may decisively influence the proper preparation of the bivalve to select an optimal site for settlement.

Acknowledgments

This work was supported by grants from the Hungarian Scientific Research Fund (OTKA) to KE (Kl 11990) and from the Russian Foundation for Basic Research to LPN (14-04-00673) and Russian Science Foundation to EEV (17-14-01353). The skilful technical assistance of Mrs. Zsuzsanna N. Fekete and Mr. Boldizsar Balazs is greatly appreciated.

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IZ AB ELLA BATTONYAI (1,*, [dagger]), ELENA E. VORONEZHSKAYA (2,[dagger]), ALEXANDRA OBUKHOVA (2), REKA HORVATH (1), LEONID P. NEZLIN (2), AND KAROLY ELEKES (1)

(1) Department of Experimental Zoology, Balaton Limnological Institute, MT A Centre for Ecological Research, Hungarian Academy of Sciences, Klebeisberg Kuno u.3, H-8237 Tihany, Hungary; and (2) Institute of Developmental Biology, Russian Academy of Sciences, Vavilov Str. 26, Moscow 117808, Russia

Received 12 December 2017; Accepted 26 April 2018; Published online 18 June 2018.

(*) To whom correspondence should be addressed. E-mail: battonyai.izabella@okologia.mta.hu.

([dagger]) These authors contributed equally.

Correction: This article was reposted on June 22, 2018. to correct an error in the grant number.

Abbreviations: 5-HT. serotonin; 5-HTP. 5-hydroxytryptophan; AO. apical organ; DAPI. 4'.6-diamidino-2-phenylindole: EDTA. ethylenediaminetet-raacetic acid: FMRFa. FMRFamide; FW. filtered water: hpf. hours post-fertilization: 1HC. immunohistochemical: IR, immunoreactive: PBS. phosphate-buffered saline; PBS-TX-NGS. PBS containing \[degrees]Ic Triton X-100 and 10% normal goat serum; pCPA. para-chlorophenilalanine: PFA. paraformaldehyde: PN. posterior neuron.
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Author:Battonyai, Iz Ab Ella; Voronezhskaya, Elena E.; Obukhova, Alexandra; Horvath, Reka; Nezlin, Leonid P
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