Immunohistochemical techniques were used to characterise central neurons in the cerebral ganglia of both male and female Perna canaliculus. We used mollusc antibodies raised against neuropeptides and neurotransmitters known to control reproduction and spawning. Anti-ELH and anti-APGWamide showed very strong immunoreactivity in small type of neurons. Anti-5-HT and anti-DA immunoreactivity was mostly in large type of neurons. The labelled neurons are consistent with descriptions of neurosecretory cells implicated in the control of reproduction and spawning on the basis of earlier histological staining techniques used in this species. The use of selective immunological markers for peptides and amines appears to be a promising tool for further characterisation of neurosecretory cells, and to isolate and characterise neuropeptides and other biologically active materials involved in the control of reproduction in Perna canaliculus

Chakraborty, S.C.

Mahmud, S.

Mladenov, P.V.

Sheard, P.

Aquatic Commons

Bangladesh J. Fish. Res., 11(1), 2007: 105-112 
	 ©BFRI 
Neurons characterization in the cerebral ganglia of the 
green-lipped mussel, Perna canaliculus, using antibodies 
raised against neuropeptides and neurotransmitters involved 
in gastropod egg-laying behaviour and bivalve reproduction 
and spawning 
S. Mahmud*, P.V. Mladenov, P. Shear& and S C Chakraborty 2 
Department of Marine Science, University of Otago, P.O. Box 56, Dunedin, New Zealand 
' Department of Physiology, School of Medicine, University of Otago, P.O. Box 56, Dunedin, New Zealand 
'Department of Fisheries Technology, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh 
*Present address: Bangladesh Institute of Social Research, 15/A/1 Zigatala, Dhaka 1209, Bangladesh 
Abstract 
Immunohistochemical techniques were used to characterise central neurons in the 
cerebral ganglia of both male and female Perna canaliculus. We used mollusc antibodies 
raised against neuropeptides and neurotansmitters known to control reproduction and 
spawning. Anti-ELH and anti-APGWamide showed very strong immunoreactivity in 
small type of neurons. Anti-5-HT and anti-DA immunoreactivity was mostly in large 
type of neurons. The labelled neurons are consistent with descriptions of neurosecretory 
cells implicated in the control of reproduction and spawning on the basis of earlier 
histological staining techniques used in this species. The use of selective immunological 
markers for peptides and amines appears to be a promising tool for further 
characterisation of neurosecretory cells, and to isolate and characterise neuropeptides 
and other biologically active materials involved in the control of reproduction in Perna 
canaliculus. 
Key words: Perna canaliculus. Antibodies, Immunohistochemistry 
Introduction 
Demonstration of the existence of neurosecretory cells has been mainly through 
histological work. As far as mussels are concerned, a number of descriptive studies of 
the different types of neurosecretory cells have been published (Illanes-Bucher 1979). A 
number of more recent studies have been done using immunocytochemistry to 
characterise neurosecretory materials in the bivalve central nervous system (Stefano and 
Martin 1983, Croll et al. 1993, Kerkhoven et a/.1993). These studies indicate the 
presence of neurotransmitters and numerous neuropeptides in the nervous system of 
S. Mahmud et al. 
those bivalves. However, knowledge neurotransmitters and neuropeptides in the green-
lipped mussel lag far behind that of other bivalves and gastropod molluscs. 
The presence of APGWamide-like immunoreactivity has been demonstrated within 
central neurons of the scallop Pecten maximus Gegou et al. 1993). APGWamide is well 
studied in gastropod molluscs, where it appears to play an important role in the control 
of male reproduction. APGWamide may also function as a neurotransmitter within the 
central nervous system of Lymnaea stagnalis, inhibiting the activity of certain 
neuroendocrine cells (Croll et al. 1991) and co-ordinating the activity of different 
populations of penial motorneurons. Indeed, APGWamide is involved in myoactive and 
copulatory behaviour (Croll et al. 1991, De Lange et al. 1997), and it has effects on 
central neurons involved in control of egg-laying behaviour and metabolism (Croll et al. 
1991). APGWamide has been isolated from ganglia of the prosobranch Fusinus 
ferrugineus (Kuroki et al. 1990) and the ganglia of the African giant snail Achatina fulica 
(Lui et al. 1991). 
Further investigations indicate that the egg-laying preprohormone is relatively 
conserved across a wide range of molluscan classes (Nambu and Scheller 1986). Using 
this antibody, as well as in antibody raised against CDCH, it has also been shown that 
neurons in the bivalves Mytilus edulis, Mya arenaria and Placopecten magellanicus contain 
a similar vitellogenic factor (Croll et al. 1993). These selective immunological markers, 
therefore, suggest that related peptides may be involved in the egg laying of various 
gastropods and bivalve molluscs (Cummuns et al. 2000). Physiological functions in 
mussels are controlled by the central nervous system (Mathiei et al. 1990). 
To date, no study has attempted to locate and identify the neurons containing 
neurotransmitters or egg-laying hormones in the green-lipped mussel, P. canaliculus 
using immunohistochemistry. In the present study, these deficiencies are addressed by 
providing a detailed description of the distribution of serotonin (5-HT), dopamine 
(DA), APGWamide, and egg-laying hormone (ELH) within the cerebral ganglia of the 
green-lipped mussel, P. canaliculus. The immunocytochemical trials have been carried 
out to tentatively identify neurosecretory cells involved in reproduction in bivalve 
molluscs to examine whether the results can be generalised across the class for a better 
understanding of mussel reproduction and provide a basis for further research. 
Materials and methods 
Collection of mussels, fixation and dissection of ganglia 
The green-lipped mussels, Perna canaliculus, were collected from an exposed rocky 
shore at Purihurihu Point, near Blueskin Bay, in the South Island of New Zealand. 
Collection of ganglia of both sexes for immunohistochemistry was done shortly after 
transporting the mussels to the laboratory of the Department of Physiology at the 
University of Otago, Dunedin, New Zealand. The cerebral ganglia were collected from 
both sexes. Individual tissues were placed gently in the bottom of an aluminium foil 
boat containing pre-cooled Tissue-TekTM O.C.T. compound and then the foil boat was 
106 
Neurons characterization in green-lipped mussel, Perna canaliculus 
filled with O.C.T. compound. The tissue was snap frozen by partial immersion of the 
foil boat into isopentane cooled in liquid nitrogen. Individual tissues were preserved at 
—70°C for sectioning. 
Antibodies used for immuno-histochemistry 
Four antisera were used in this study, all produced in rabbits: (i) Anti-ELH was 
raised against a synthetic peptide representing the N-terminal fragment 
(ISINQDLKAITDML) from the egg laying hormone of Aplysia. This antibody was 
produced by G. T. Nagle and J. E. Blankenship (University of Texas Medical Branch), 
and its characterisation and specificity were described by Ram et al. (1998), (ii) Anti-
APGWamide (CHEMICON International, Inc. 28835 Single Oak Drive, Temecula, CA 
92590), (iii) Anti-Dopamine (CHEMICON International, Inc. 28835 Single Oak Drive, 
Temecula, CA 92590), and (iv) Anti-Serotonin was obtained from Dept. of Zoology, 
University of Otago, Dunedin, New Zealand. The unlabelled goat anti-rabbit secondary 
antibody was obtained from Cappel Research Products (Durham, North Carolina) and 
the peroxidase-antiperoxidase complex employing rabbit antibodies was obtained from 
Sigma Chemical Co. (Mississauga, Ontario). 
Immunocytochemistry protocol 
Serial sections (two sets - one for experimental and another for control) were cut at 
10 Am in a cryostat at —18°C and approximately 8-10 sections were mounted on each 
slide for immunohistochemistry. The dried sections were fixed for 10 minutes in freshly 
prepared 4% paraformaldehyde and were washed in PBS. Primary antiserum were then 
applied and left overnight at 4°C. Antiserum dilutions of between 1:400 and 1:100 were 
used in an immunodiluent (ID) solution of 2% normal goat serum (Sigma Chemical 
Co.) and 0.2% Triton X-100 (Sigma Chemical Co.) in PBS. 
Next day, secondary antibody was added to all slides after washing in PBS and was 
left for an hour at room temperature. The secondary antiserum was diluted 1:200 in ID. 
After another several washes in PBS the slides were kept for another one-hour 
incubation in peroxidase-antiperoxidase diluted 1:400 in ID. After incubation, slides 
were washed off again in PBS and were developed for • 2-3 minutes using 
diaminobenzidine (DAB)- hydrogen peroxide. Slides were dehydrated in graded 
ethanols washed in xylene, and mounted in DPX. One set of serial sections from each 
ganglion was processed as described above, with the elimination of the incubation in 
primary antibody as a negative control. Slides were viewed through an Olympus BX50 
Microscope and photographed digitally. 
Results 
The localisation of neurons containing different neuropeptides and 
neurotransmitters in the cerebral ganglia of the green-lipped mussel, Perna canaliculus, 
107 
S. Mahmud et al. 
were examined imuno-histochemically. The tentative locations of these immunoreactive 
neurons are shown in Fig. L 
Fig. 1. Schematic representations of anti-ELH immunoreactivity (black circles) and anti-5-HT 
immunoreactivity (white triangles) on the left side and anti-APGWamide immunoreactivity 
(white circles) and anti-DA immunoreactivity (black triangles) on the right side in the cerebral 
ganglia of Perna canaliculus. All descriptions are bilaterally symmetric in the ganglia. CC, cerebral 
commissure; CPC, cerebral-pedal connective; CVC, cerebral-visceral connective. 
The immunoreactive neurons and fibres throughout the cerebral ganglia labelled 
with anti-ELH, anti-APGWamide, anti-5-HT and anti-DA are presented in Fig. 2. 
Antibodies raised against anti-ELH labelled neurons and fibres throughout the cerebral 
ganglia of the females (Fig. 2A). The immunoreactivity was observed only in small cells. 
Numerous immunoreactive fibres were also present in the cerebral ganglia. The large 
neurons and their pigments/granules (Fig. 2B) revealed no immunoreactivity above the 
levels of control slides (Fig. 2C). The small cells also showed positive immunoreactivity 
and stained strongly with anti-APGWamide. The immunoreactive cells were mostly 
found along the cortex (Fig. 2D). Anti-APGWamide produced positive 
immunoreactivity in both sexes. Large numbers of immunoreactive fibres were found 
throughout the cerebral ganglia. 
108 
Neurons characterization in green-lipped mussel, Perna canaliculus 
Fig. 2. Immunoreactivity in the cerebral ganglia of Perna canaliculus. 
(A) Several small cells (long arrows) and nerve fibres (arrowheads) showing strong immunoreactivity to anti-
ELH in ripe female. Scale bar 20,am. 
(B) Several small cells (long arrows), nerve fibres (short arrows) showing positive immunoreactivity to anti-
APGWamide in spawned female. Scale bar 20µm. 
(C) A few large cells (long arrows) showing positive immunoreactivity to anti-5-HT in ripe female. Scale bar 
50 gm. 
(D) Large cells (long arrows) and nerve fibres (short arrows) showing positive immunoreactivity to anti-DA 
in a spawned female mussel. Scale bar 50µm. 
Mostly large cells, connective tissue sheath and numerous immunoreactive-fibres 
were revealed in the neuropile of the cerebral ganglia with antibodies raised against 
anti-5-HT. The immunoreactive cells were located near the periphery of the ganglia. 
Only a few of the large cells were found with strong immunoreactivity (Fig. 2E). A few 
small cells showed positive immunoreactivity with anti-5-HT. Mostly the peripheries of 
these cells were found with immuno-reaction. 
Discussion 
This study perhaps the first of its kind describes various monoamines and 
neuropeptides in the central nervous system of Perna canaliculus, which might be 
involved in controlling reproduction. Antibodies raised against ELH, APGWamide, 5- 
109 
S. Mahmud et al. 
HT and DA stained relatively large numbers of cell bodies, fibres, axons and connective 
sheaths in the cerebral ganglia of P. canaliculus. 
In the present study, some of the neurons in the cerebral ganglia were labelled with 
anti-ELH, anti-APGWamide, anti-5HT and anti DA (Figure 1). According to the size of 
the immunoreactive neurons in these ganglia, there were two distinct groups. Small cells 
were mostly located near the periphery of the ganglia with a few in the neuropile region. 
Large cells were mostly located between the peripheral edge of the ganglia and 
neuropile region. The small cells exhibited strong immunoreactivity with both anti-
ELH and anti-APGWamide in all three ganglia of P. canaliculus. The relationships 
between neurosecretory cells and gonad state were observed in the green-lipped mussel 
(Mahmud and Mladenov 2000). Similar patterns were observed in other studies in 
bivalves and gastropods. Neurosecretory staining in 'a' cell in Mytilus edulis was reported 
to correlate with the reproductive cycle (Mathieu and Van Minnen 1989). The studies in 
gastropods by Hahn (1990) in Haliotis discus hannai and by Upatham et al. (1998) in 
Haliotis asinina established that the secretion from certain cells in the ganglia of Haliotis 
spp. are correlated with vitellogenesis, gametogenesis or spawning. The injection of 
ganglionic homogenates caused spawning in green-lipped mussel. Therefore, the 
labelling of small cells by both anti-ELH and anti-APGWamide in the cerebral ganglia 
of the green-lipped mussel, Perna canaliculus, is strong evidence for the presence of 
ovulation and reproduction hormone(s). 
The anti-5-HT and anti-DA immunoreactive neurons were lightly stained and 
located in well-defined locations in the cerebral ganglia in P. canaliculus (Fig. 1). These 
neurons perhaps correspond to cell types 'C' and 'D' as shown in the previous study 
(Mahmud and Mladenov 1998). The labelled cell by both anti-5HT and anti-DA 
indicates the presence of neurotransmitters/monoamine(s) in these cells. The presence 
of 5-HT and DA-like substances has also been previously reported in M. edulis (Aramant 
et al., 1981; Mathieu and Van Minnen 1998). Although, the neurosecretory cell types 'C' 
and 'D' did not show any substantial changes in colour intensity with changes in gonad 
development and spawning (Mahmud and Mladenov 2000), the presence of 
neurotransmitters 5-HT and DA in these cells indicate that they might have other 
modulating or physiological functions in this species which need to be evaluated. 
The present study was based upon the hypothesis that peptides controlling 
reproduction might be evolutionarily conserved between gastropods and bivalves, it 
must also be considered that spawning and external fertilisation of bivalves are very 
different from in gastropods in terms of copulation and subsequent egg-laying 
behaviour (Croll et al. 1993). Therefore, even though related peptides might be involved 
in reproduction within both taxa, details of their distribution and mechanisms of 
actions are bound to vary. Their abundance should be investigated seasonally and 
correlated with stage of reproduction in order to determine which processes or 
mechanisms they are involved in. In the present study, samples from both mature and 
spawned mussels showed immuno-reactivity (Fig. 2). The sampling protocol (small 
number of samples) in the present study does not allow assigning such physiological 
roles to these cells. An elaborate investigation using immuno-histochemistry with 
110 
S. Mahmud et al. 
Kuroki, Y., T. Kanda, I. Kubota, Y. Fujisawa, T. Ikeda, A. Miura, Y. Minamitake and Y. 
Muneoko, 1990. A molluscan neuropeptide related to the crustacean hormone, RPCH. 
Biochem. Biophys. Res. Commun., 167: 273-279. 
Lui, G. J., D.E. Eantos and H. Takeuchi, 1991. APGW-amide as an inhibitory neurotransmitter of 
Achatina fulica Ferussac. Biochem. Biophys. Res. Commun., 177: 27-33. 
Mahmud, S. and P.V. Mladinov, 1998. Characterisation of neurosecretory cells in the greenshelled 
mussels, perna canaliculus. Abstract- Joint conference, Newzealand Marine Science Society, 
Australian Society for Phycology and Aquatic Botany, Denedin, Newzealand, p 138. 
Mathieu, M. and J.V. Minnen, 1989. Mise en evidence par immunocytochimie de cellules 
neurosecretrices peptidergiques dans les ganglions cerebroIdes de la moule Mytilus edulis. 
C.R. Acad. Sci. Paris, 308 (III): 489-494. 
Mathieu, M., I. Robins and F. Lenoir, 1990. A novel approach to the endocrine control of 
gametogenesis and the gemetogenesis related storage cycle of Mytilus edulis L. In: Advances in 
Invertebrate Reproduction . Vol. 5 (eds. M. Hoshi and 0. Yamashita), Elsvier Science 
Publishers (Biomedical Division): 239 — 245. 
Nambu, J.R. and R.H. Scheller, 1986. Egg-laying hormone genes of Aplysia: evolution of the ELH 
gene family. 31 Neuroscience, 6: 2026-2036. 
Ram, J.L., C.S. Gallardo, M.L. Ram and R.P. Croll, 1998. Reproduction-associated 
immunoreactive peptides in the nervous systems of prosobranch gastropods. Biol. Bull., 195: 
308- 318. 
Upatham, E.S., A. Thongkukiatkul, M. Kruatrachue, C. Wanichanon, Y.P. Chitramvong, S. 
Sahavacharin and P. Sobhon, 1998. Classification of neurosecretory cells, neurons, and 
neuroglia in the cerebral ganglia of Haliotis asinina Linnaeus by light microscopy. ,. Shellfish 
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(Manuscript received 16 July 2007) 
112 


Neurons characterization in the cerebral ganglia of the green-lipped mussel, Perna canaliculus, using antibodies raised against neuropeptides and neurotransmitters involved in gastropod egg-laying behaviour and bivalve reproduction and spawning

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Neurons characterization in the cerebral ganglia of the green-lipped mussel, Perna canaliculus, using antibodies raised against neuropeptides and neurotransmitters involved in gastropod egg-laying behaviour and bivalve reproduction and spawning

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