Camp. Biochem. Physiol. Vol. 86C. No. I, pp. 215-218. 1987 Printed in Great Britain

0306.4492187 $3.00 + 0.00 i’ I987 Pergamon Journals Ltd

THE ROLE OF THE NERVOUS SYSTEM IN

ALGAE-INDUCED GAMETE RELEASE BY

MYTILUS CALIFORNIANUS

JOHN R. SMITH

Department of Physiology and Pharmacology, School of Dentistry, Oregon Health Sciences University, 611 SW Campus Drive, Portland, OR 97201, USA. Telephone: (503) 22.5-8770

(Received 6 May 1986)

Abstract-l. Algae-induced spawning in M. californianus was associated with an increase in dopamine in the visceral ganglia and a decrease in serotonin in the pedal ganglia.

2. Lesioning studies indicate that nervous communication between the pedal and visceral ganglia plays a major role in gamete release.

INTRODUCTION

Spawning in marine bivalves may be influenced by internal and external factors. Considerable effort has been devoted to understanding the modulatory role of external factors such as temperature, wave action, food and mechanical stimuli (Orton, 1920; Nelson, 1928; Galtsoff, 1938, 1940; Young, 1945; Korringa, 1947; Bayne, 1965). These stimuli are generally be- lieved to trigger the release of gametes at the oppor- tune time or to condition the animal so that it is predisposed to other environmental factors. Internal factors influencing spawning in mussels have received less attention. It has been proposed that neuro- secretion from the cerebral ganglia modulates spawn- ing in mussels by inhibiting their receptiveness to external stimuli. This theory is based upon the seasonal differences in the histology of neuro- secretory cells in gravid vs spent mussels and upon the observation that the removal of the cerebral ganglia led to precocious spawning (Lubet, 1956, 1959, 1965, 1966; Bayne et al., 1976).

The r61e of the nervous system of Mytilus in spawning behavior has received little attention. Ganglionic ablation, in addition to eliminating neurosecretory cells, disrupts dopaminergic and sero- tonergic nerve pathways (Sweeney. 1963; Dahl, 1966; Stefano and Aiello, 1975; Stefano et al., 1976; Smith, 1982). Presumably, triggering stimuli rely upon these nerve pathways to integrate the environmental infor- mation needed to initiate a pattern of reproductive behavior. The purpose of these studies was to gain some basic information about the relationship be- tween gamete release and nerve activity in A4ytifu.v using neurochemical and surgical techniques.

MATERIAIS AND IMETHODS

The experimental animals in this series of tests were marine mussels, Mytilus caiifarnianus. All specimens were gathered from rocky intertidal regions of the mid-Oregon coast in the vicinity of Yaquina Bay. Size ranges were 3&50 and 6G-90 cm in shell length. The smaller mussels were used for neurochemical experiments and the larger for lesion studies. All animals were cleaned of large shell debris and

held in tanks supplied with filtered, U.V. light-treated sea- water (3 l/min) that was held at 12 i 1°C.

Neurochemical studies

Mussels were transferred from the holding tank to indi- vidual dishes to which was added 200 ml of a concentrated solution (1.74.5 million cells/ml; approx. pH 9.0) of the unicellular marine alga, Pseudoisochr~sisparadoxa. Cultures of this algae were grown according to the methods of Breese and Malouf (1975). This treatment has been shown to induce spawning in M. cul[fornianus after a lag time of anorox. 1.5 hr (Smith and Strehlow. 1983). Two different ganglia-sampling protocols were used. In tie first protocol, the bioassay was run for 5 hr and ganglia (pedal and cerebral) from spawned and unspawned animals were re- moved and processed at the end of the experiment. In the second protocol, unspawned animals were sampled immedi- ately after one of the animals spawned (usually about 1.5 hr). After the predetermined number of unspawned animals had been sampled, the remaining animals were allowed to spawn and sampled in turn. In order to ensure an adequate number of spawned animals, it was necessary on one occasion to transfer some of the mussels into fresh algae solution 3.5 hr after the start of the experiment. Significant variation due to procedural differences could not be identified, so all of the data were pooled for final analysis.

The various ganglia from each animal were manually homogenized with glass tissue grinders in 0.5 ml of a 95:5 solution of 0.2 N citric aciddibasic sodium phosphate buffer and spectrographic-grade methanol containing N- methyldopamine as an internal standard. This solution also served as the mobile phase in later chromatographic pro- cedures. The homogenates were centrifuged for 2 min at 7000~ in a cold room (2 C), and the supernatants were transferred to l-ml syringes and stored at -20°C. Twenty- microliter samples of each ganglionic extract were injected into an Altech high-performance, liquid-chromatography system (Beckman Instrument Co., Redmond, WA) consis- tmg of a 40 x 3.5mm LiChrosorb C-18 guard column (MC/B Manufacturing Chemists, Cincinnati, OH) and a 250 x 4.~ mm Ultrasphere C-18 separation column (Beck- man Instrument Co., Redmond, WA). The flow rate of the mobile phase was 1 mlimin, and all separations were done at room temperature (22 C). Dopamine (DA) and serotonin (5HT) levels were determined with an electrochemical de- tector (Bioanalytical Systems, West Lafayette, IN) by using a glassy-carbon electrode set at a potential of +0.6 V vs the reference electrode. The electronic controller was set at 5 nA/V; the recorder set at I V full scale. Identification of

215

216 JOHN R. SMITH

DA and 5HT was based upon retention times of standards, and quantification of amines was accomplished by analysis of peak heights of samples vs the internal standard (Saraswat et al., 1981). Student’s f-test was used for statis- tical evaluations. An alpha of 0.05 was the minimum criteria for statistical significance.

Lesion studies

The lesions described in the legend of Table 2 were performed in the following manner. The valves of the mussel were held open by inserting the broad end of a l.O-ml disposable pipet tip into the posterior end of the mussel. The other end of the pipet tip was then secured to a clamp so that the animal was held upright. A blunt probe was used to isolate the various lesion sites, and the nerve tracts were severed using iris scissors and forceps. This procedure required 24min depending upon the particular lesion. Afterwards the pipet tip was removed, and the animal was immediately returned to the holding tank. Sham operations consisted of small incisions made into connective tissue adjacent to the lesion site under study. Generally animals were given a 2- or 4-day recovery period.

At the end of the recovery period, the animals were exposed to high concentrations of algae (3.65.6 million cells/ml; pH 9.0) as previously described. and the ratio of spawned to unspawned animals was recorded at the end of the monitoring period. Greater than 97% of all spawnings occurred 4.5-9.0 hr after the addition of algae. In some of the experiments, additional algae was added several hours after the start of the experiments. Statistical analyses were based upon confidence intervals calculated for proportions. An alpha of 0.05 was used as criteria for significance.

RESULTS

Neurochemical studies

This series of experiments was initiated to identify possible neurochemical correlates to gamete release in mussels.

AS illustrated in Table 1, the releasing of gametes can be temporally associated with a change in neuro- transmitter levels in two ganglia. In the visceral ganglia of spawned mussels there was a significantly greater amount of dopamine than in the visceral ganglia of unspawned animals. In the pedal ganglia of spawned animals there was significantly less sero- tonin compared to unspawned animals. The values reported in Table 2 are pooled from both male and female mussels. Analysis of the data for sex bias failed to identify any consistent differences.

Table 2. The effects of various nerve lesions on algae-mduced spawning in M. culifornianus

I I

3 @ 3

2 2

4 4 5

Lesion site Spown1ng

ratio ”

I C-V 2 050 60

2 c-vu I I .oo 20

3 P 3 0.78 20

4 B 4 loo 20

5 C-VP/P 1.3 0 67 20

6 C -V=/V-v/P 2,395 000 20

7 v-v/c-v= 2,5A I.00 40

n Posterror

D Anterror.

b Unltaterat

The schematic diagram represents the three pairs of ganglia that comprise the nervous system of M. californianus. From top to bottom they are the cerebral, pedal and visceral ganglion pairs. In this series of experiments, the connectives joining these ganglia were severed at various sites designated by the numbers l-5. The lesion site(s) for each group of animals is listed next to the group number. Lesion site number 3 (in groups 3, 5 and 6) interrupts the pallial nerve, and site number 4 (in group 4) interrupts the branchial nerve as it emerges from the visceral ganglion. The animals in group 7 were lesioned at site 2 only on the left-hand side (unilateral). The terms posterior and anterior refer to the direction from the point at which the pedal nerve joins the major cerebrovisceral connective. Each group was matched with an equal number of sham-lesioned controls. Following recovery from surgery, each group of animals and their controls were immersed individually in high concentrations of the marine alga P.wudoisochr)si.~ paradoxa, and gamete release was monitored. The spawning ratio is the proportion of lesioned animals that spawned compared to the number of sham-lesioned animals that spawned.

Table I. Changes in neurotransmitter levels m eanelia followine aleae-Induced soawnine

Dopamlne* Serotonln~

VlSCeral Pedal Cerebral wscerat Pedal Cerebral gangtta gong11a gangila gangt1a gongtia gang11o

Spawned 834tll IA 317+2 7 247+3 4 148 41-22 4 759+67’ 42 If43

8” 25O 23’ 80 23” 230

Unspawned 450f72 35 Of2 I 25324 3 1105+130 936?55 39 3+_35

100 23O 23O 100 250 23O

A Plcomoks/ganglla pafr + SEM

A Sqlflcantly different from uspawned levels (PC0 05)

q Number of antmats tested

Mussels were individually immersed in high concentrations of the marine alga Ps~u~k~i.\o~~/rr~s~s parudmu and observed for ewdence of gamete release. Ganglion pairs were dlasected free and assayed for dopamine and serotonin Irvels. Animals that did not spawn were alw killed and ganglionic dopamlne and serotomn levels were determined in a slmdar fashion.

Nervous system in mussel spawning 217

Le.&m studies

The lesion studies (Table 2) were performed to gather information about the participation of the various ganglia in the gamete release response of mussels that are exposed to high concentrations of algae.

Interruption of the major nerve connective between the cerebral ganglia and the visceral ganglia {C-V} resulted in two different effects depending upon the actual site of the lesion. Cutting the connective anterior to the juncture of the pedal nerve (lesion No. 2) had no effect upon spawning frequency. When the lesion was made just anterior to the visceral ganglion (lesion No. l)+ the inhibition of spawning was statis- tically significant.

The inff uence of pa&al nerve lesioning by itself was insignificant. The combination of pallial lesions with C-V(A) lesions led to a slight decrease in spawning which was nat statistically significant. However, if the pallial nerve was cut along with the C-V(P) and the visceral connective (lesion No. 6), none of the animals spawned. This was the only lesion pattern that com- pletely prevented algal-induced spawning in mussels. The mussels treated in this manner displayed consid- erable gapping and variable algae filtering abilities. However, these eharaGte~sti~ were true of the C-V(A)/P group (lesion No. 5) as well, yet they were still capable of releasing gametes. The branchiaf nerves do not appear to play a significant role in gamete release since lesioning (lesion No. 4) had no effect on this response. Interruption of the visceral connective combined with unilateral C-V(P) lesion- ing (lesion No. 7) also had no influence upon the spawning response to algae. The lesioned animals that spawned released gametes from both sides.

The lesion experiments attempted to identify those nerve tracts that participate in induced spawning. The data suggest that the spawning activity utilizes more than one pathway, although interconnection between the pedal and visceral ganglia appears to play a dominant role. This is concluded by observing that C-V(A) lesions had no effect on spawning while C-V(P) lesions Ied to a 50% decrease in spawning. Thus, nerve connections between the pedal ganglia and the cerebral ganglia are not necessary for the immediate spawning response under consideration here, while communication between the pedal and visceral ganglia is crucial. Cerebral/visceral ganglia interaction also appears to contribute to the overall response since pa&al nerve lesions either alone or in combination with C-V(A) lesions did lead to a slight deerease in spawning, and pallial iesions combined with CV-V(P) lesions completely abolished spawn- ing. It has been suggested that the cerebral ganglia play a critical role in reproductive behavior in mus- sels through the production of an inhibitory neuro- secretion (Lube& 1956, 1959, 1966; Bayne et al., 1976). The effects of pa&a1 nerve lesioning upon neuros~retory activity in the cerebral ganglia were not studied. Since various stimuli are known to increase or decrease neurosecretory activity in these cells (Lubet and Pujol, 1963, 19651, pallial nerve

inte~ption may con~ivably have enhanced cerebral ganglia neurosecretory activity leading to an in- hibition of algal-induced spawning. Even if this were the case, this mechanism would still appear to make only a minor contribution to the overall spawning response observed in this study.

The neurochemical observations further support the important of the pedal and visceral ganglia in induced-spawning. In the visceral ganglia there was a signi~~ant rise in dopamine content associated with gamete release, while in the pedal ganglia this event was accompanied by a significant decrease in sero- tonin. These two changes may be related since there is good evidence to suggest that in bivalves ganglionic and tissue concentration of serotonin are inversely related to dopamine concentrations (Stefano et ai., 1976). The possible relationship between ganglioni~ neurotransmitter alterations and gamete release are speculative, at best, due to the rudimentary under- standing of the process of gamete release in ~ytilu~ and in other bivalves. Anatomical evidence suggests that the gonads of Myths are innervated by nerves originating from the viscera1 ganglia (Bayne ef af., 1976). Conceivably, excitatory serotonergic nerves arising from the pedal ganglia project to these go- nadal cell bodies in the visceral ganglia via the cerebrovisceral connective. The activity of these ex- citatory serotonergic nerves in the pedal ganglia may be under tonic, negative influence of dopaminergi~ nerves originating from the visceral ganglia and traveling to the pedal ganglia via the cerebrovisceral connective. This would be reasonable since it has been shown that C-V(P) transections in Mytilus edulis lead to an increase in cerebral ganglia seratonin and visceral ganglia dopamine (Stefano and Aiello, 1975). One scenario, then, for algal-induced spawning may be that sensory input which is activated by enviranmental stimuli (algae) depresses dopamine neurons in the visceral ganglia (evidenced by an increase in visceral dopamine content). This, in turn, causes disinhibition of excitatory pedal serotonergic neurons (evidenced by a decrease in pedal serotonin). These nerves then stimulate excitatory nerves in the visceral ganglia which results in gamete release ap- pear to be influenced by nerves originating from either the cerebral ganglia or from peripheral sensory endings. This is inferred from the inhibitory e&cts of pallial nerve lesioning. Based upon this theory, tran- section of the cerebrovisceral connective disrupts SHT/DA communication between the pedal and vis- ceral ganglia C-V(P), which would then be expected to have a major impact upon spawning ability. Furthermore, additional deficits in spawning re- sponse would be expected if the palliai nerve were cut since it carries messages to the visceral ganglia from the periphery and the cerebral ganglia.

The preceding speculation is based upon the as- sumption that the neurotransmitter differences seen in spawned and unspawned mussels represent an immediate response to the environmental stimuli (algae). It could be argued that the animals re- sponsive to algae exposure were more predisposed to spawn than the otbers and that the gangl~onic neuro- transmitter levels were a physiological reflection of this fact. However, the experimentat design used in these experiments provides support for the former

218 JOHN R. SMITH

inte~retation. The mussels were already naturally predisposed to spawn, i.e. control experiments with algae exposure routinely yielded 9&100% spawning. Thus, if the experiments were allowed to proceed uninterrupted, almost all of the animals would have spawned which generally was 1.5 hr after the addition of algae. Since most animals spawn between 1.5 and 3.5 hr after algae addition, the unspawned animals that were sampled were considered to be on the verge of spawning. It appears then that the neuro- transmitter changes observed occurred in very close temporal association with gamete release and were not extant prior to algae exposure.

Based upon the information gained in this series of experiments, it appears that the nervous system of Mytilus californianus plays a significant role in gamete-release behavior in both males and females. The changes in 5HT and DA in the pedal and visceral ganglia associated with this behavior suggest that these neurotransmitters and structures are especially important in algal-induced spawning in mussels.

Acknowledgements-Funds for this study were made avail- able through NIEHS Grant ES0 1926. The author also wishes to thank David Strehlow for his valuable assistance on this project.

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