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Comparative aspects of free amino acids in the central nervous system of spiders
Comp. Biochem. Physiol., Vol. 67C, pp. 83 to 86
0306-4492/80/0901-0083502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
C O M P A R A T I V E ASPECTS O F FREE A M I N O A C I D S IN THE C E N T R A L N E R V O U S SYSTEM O F S P I D E R S W. MEYER*, H. M. POEHLINGt and V. NEUHOFFt *lnstitut ffir Zoologie, Tier~irztliche Hochschule Hannover, Bischofsholer Damm 15, D-3000 Hannover 1; and tMax-Planck-Institut fiir experimentelle Medizin, Forschungsstelle Neurochemie, Hermann-Rein-Str. 3, D-3400 G6ttingen, Federal Republic of Germany (Received 14 January 1980)
Abstract--1. The spectrum of free amino acids in the CNS of 6 species from 6 different spider families was analysed by microchromatography of dansyl derivatives. 2. We could identify 23 dansyl amino acids, the following ones being relatively abundant in all the species investigated: taurine, alanine, glycine, GABA, proline, glutamic acid, and aspartic acid. 3. The results obtained are compared with findings from other arthropod subphyla, and the role of amino acids as putative neurotransmitters in the arthropod CNS is discussed.
15 min at 4°C the supernatant was analysed for free amino acids. Amino acids were separated on 3 x 4cm micropolyamide sheets (A 1700 Schleicher u. SchiJll, Dassel F.R. Germany) as dansyl derivatives (1-dimethylaminonaphthalene-5-sulphonyl-chloride; Serva, Heidelberg F.R. Germany). Details of this method are described by Neubach et al. (1978), and Poehling et al. (1980). Dansyl-amino acid solution (0.5 1.0 pl) was applied with micropipettes on the corner of a polyamide sheet and developed in the first dimension with 2~o formic acid and in the second dimension with 20% acetic acid in toluol. Chromatograms were evaluated by fluorescence scanning photometry with the aid of a specially developed program for digital picture analysis (PDP-12/12 Computer Digital Equipment Co). Fluorescence intensity of each recognizable spot was recorded as %0 of the fluorescence intensity of internal standards (~-phenylglycine and sarcosine). Further details of chromatogram evaluation are described by Kronberg et al. (1978), Zimmer & Neuhoff (1977), and Zimmer et al. (1978). Data were only presented for clearly recognizable spots of dansyl-amino acids. Unknown dansylated substances are detectable but were not analysed. Only estimations were recorded for glutamine, asparagine, serine, and arginine because these amino acids could only be separated by hand correction of the computer analysis from the comigrating by-product of the dansylation reaction, sulfinic acid. This hand correction involves subjective errors.
INTRODUCTION
Until now free amino acids in the central nervous system of arthropods have received much attention only in insects and crustaceans (see for example Evans, 1973; Osborne & Neuhoff, 1974). Available evidence in these groups suggests that several amino acids are most likely candidates for neuro-transmitters (Gerschenfeld, 1973; Klemm, 1976; Walker, 1977). In contrast to these findings no information exists on the amino acids of the spider's nervous system. Therefore, the present study was designed to give a first, preliminary account of the spectrum of free amino acids as present in the nervous tissue of different spider families. The species used during this investigation exhibit varying habits, especially in relation to their different modes of orientation. This is also manifested by specific structural features of the brain (Meyer & Pospiech, 1977; Meyer & Idel, 1977). Our results may be of interest in connection with this, but mainly in view of possible putative neurotransmitters when compared with findings from the two other great arthropod subphyla.
MATERIALS AND METHODS
Spider species of the following 4 different families were captured during June and July 1979 in the vicinity of Hannover (Lower Saxony, West Germany): Tegenaria derhami (Scopoli) (Agelenidae), Araneus diadematus Clerck (Araneidae), Pardosa amentata (Clerck) (Lycosidae), Marpissa muscosa (Clerck) (Salticidae). Latrodeetus mactans tredecimguttatus Rossi (Theridiidae) was captured on the island Pag (Yugoslavia), and the orthognath species Aphonopelma eutylenurn (Chamberlin) (Theraphosidae) was purchased from a zoological supply company. The nervous system of adult animals of both sexes was removed from the cephalothorax in the cold, washed twice in buffered saline and transferred into 10#l capillaries (Brand Wertheim) or pyrex glass tubes (~b 1.5 mm) sealed at one end. One mg tissue was homogenized in 20 pl 0.05 M K 2 C O 3 , pH 9.0, using a dentist's drill for capillaries and Teflon pestels for pyrex tubes at 24,000 rpm, icecooled (Neuhoff, 1973). After centrifugation at 2000rpm for
RESULTS AND DISCUSSION
The results obtained during the course of this study are summarized in Table 1, a selected microchromatogram is presented in Fig. 1. In view of the data for 6 species from 6 different families we could identify 23 dansyl amino acids in the central nervous system of spiders. The total spectrum of free amino acids as revealed by microchromatography of dansyl derivatives generally shares common features with those described for the nervous system of insects and crustaceans (Pasantes et al., 1965; Ray, 1965; Evans, 1973; Osborne & Neuhoff, 1974). The following amino acids were relatively abundant in all the spider species investigated: taurine, alanine, 83
W. MEYER, H. M. POEHLINGand V. NEUHOFF
84
Table 1. Percentage composition of substances ( + S E M for 7 experiments) from the central nervous system separated as dansyl derivatives by microchromatography Spot No.
Dansylated substances
Aphonopelma eutylenum
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Isoleucine Leucine Histidine Phenylalanine Methionine Proline Valine GABA Alanine ETA Lysine Ornithine Tryptophan N-Tyrosine OH-Tyrosine Taurine Glycine Glutamic acid Aspartic acid Serine Glutamine Asparagine Arginine
0.71 1.06 3.01 0.47 0.40 13.93 2.39 12.32 17.56
+ ± + ± ± + + ± ±
0.11 0.05 1.06 0.02 0.02 0.73 0.17 0.51 1.34
0.96 0.17 0.97 1.29 1.26 11.65 7.39 11.74 12.78
± ± ± ± ± ± ± ± ± + + + +
0.02 0.02 0.07 0.00 0.03 0.20 0.06 0.27 0.67
Tegenaria derhami 0,61 ± 0.11 1.12 ± 0.15 0.31 + 0.04 0.59 ± 0.07 0.36 ± 0.06 9.01 ± 1.63 1.47 ± 0.30 22.25 + 2.48 21.09 ± 0.51 0.23 ± 0.09 1.10 + 0.411 0.27 ± 0.06 0.45 ± 0.05 0.38 ± 0.07 1.37 + 0.06 18.82 _+ 1,88 7.19 ± 0.78 7,97 _+ 0.99 5.26 + 0.67 + + + + +
+
Latrodectus react, tredecimgutt, 1.09 3,11 0.07 1.85 0.65 10.60 1.80 9.42 20.22 0.54 2.45 0.45 0.51
+ ± + ± ± ± ± ± + + + ± ±
0.21 0.43 0.02 0.38 0.06 0.35 0.24 0.24 0.92 0.10 0.54 0.15 0.07
1.08 25.00 14.74 5.79 0.63
± 0.28 _+ 2.06 ± 4.12 ± 1.48 ± 0.31 + + + + +
Araneus diadematus
Pardosa amentata
Marpissa rnuscosa
0.35 ± 0.02 0.69 + 0.02 0,30 + 0.11 0.43 ± 0.07 0.30 ± 0.15 4.02 + 0.94 0.80 + 0.07 17.41 ± 1.40 27.97 ± 0.96 0.39 ± 0.21 1.34 + 0.20 0.35 ± 0.17 0.26 ± 0.07 0.15 _+ 0.07 1.53 _+ 0.19 22.42 ± 0.75 13.01 ± 2.11 6.90 ± 0.51 1.06 ± 0.25 + + + + +
0.47 ± 0.19 0.77 _+ 0.31 0.41 + 0,15 /I.59 + 0.20 0.36 _t 0.08 5.57 ± 0.52 1.28 + 0.21 17.74 + 1.60 2459 + 1.31 0.54 +_ 0.15 0.82 + 0.32 0.19 ± 0.02 0.33 + 0.03 0.36 + 0.11 1,59 ± 0.12 22.89 ± 5.30 8.22 _t 1.111 9.81 ± 0.52 3.56 + 0.91 + + + + +
0.52 ± 0.13 0.73 ± 0.17 1.09 + 0,51 0.48 + 0.08 0.39 ± 0 0 3 5,56 ± 0.85 1,15 _+ 0.30 14.05 ± 2.35 18.17 ± 1.13 0.41 ± 0.18 1.22 + 0.10 1.04 + 0.27 0.30 + 0.02 0.26 ± 0.09 1.08 ± 0.29 34.20 ± 1.25 9.22 ± 1.08 6.82 ± 11.42 3.15 + 0.66 + + + + +
+ = very weakly present, + + = weakly present
A
)14
~21
( ~ 7 ~S2
( ~ 3 (~4 (~5
2.
9(~-']':dans'NH2
(~)Sl 1~1
(~)17
C~)12 C)15
( " ~ 16
B
f)
21
19] ,"
) "~lans.6H
./
~
~
23
So
Fig. 1. Selected microchromatogram (A) of dansylated substances extracted from the central nervous system of Tegenaria derhami (Scopoli). The spot numbers in the identification key (B) are explained in Table 1. ~ 1. direction 2% formic acid, ---~2. direction 20% acetic acid in toluol; S = starting points; $I = first standard (ct-phenylglycine), $2 = second standard (sarcosine), Sa = sulfinic acid.
85
Amino acids in spider CNS glycine, GABA, proline, glutamic acid, and partly also aspartic acid. The latter compound and proline were present in higher amounts only in the primitive orthognath spider (Aphonopelma eutylenum), but surprisingly low in the Mediterranean black widow spider (Latrodectus mactans tredecimguttatus). Compared with all the other species, the taurine contents of the nervous system of Aphonopelma were relatively low, while this substance was more concentrated in species representing a higher phylogenetic standard, like the specialized web-building (Theridiidae, Araneidae) and hunting spiders (Lycosidae, Salticidae). It is interesting to note that the 7 amino acids enumerated above are also accumulated in similar relative quantities in the nervous system of the two other great arthropod groups (Frontali, 1964; Osborne & Neuhoff, 1974; Evans, 1973). In relation to the quantitative or semi-quantitative evaluation of results obtained with dansylation methods, it must be admitted, however, that the different amino acids are not all dansylated in comparable equal amounts. This is especially true of glutamic acid and aspartic acid (see Poehling et al., 1980), i.e. the real relative amounts of these amino acids may be somewhat higher than perceptible from Table 1. Several of the amino acids occurring in higher concentrations in the spider CNS are thought to have transmitter functions in both, the vertebrate as well as the invertebrate nervous tissues (Pitman, 1971; Gerschenfeld, 1973; Krnjevic, 1974; Klemm, 1976; Walker, 1977). Evidence is strongest for GABA and glutamate, while there is only little information available about the possible involvement of taurine, glycine, aspartate, and proline in synaptic transmission in arthropods. As demonstrated for the vertebrate CNS, the function of GABA in insects was found to be exclusively inhibitory (Pitman, 1971; Roberts et al., 1976). Compared with GABA, glutamic acid is likewise present in the nervous system of all great arthropod groups (see for example Evans, 1973; Osborne & Neuhoff, 1974; Klemm, 1976; and the results for spiders as given in this study). This substance is now generally considered as putative transmitter in invertebrates, with essential importance for neuromuscular excitation in insects and crustaceans (Walker, 1977). But in contrast to vertebrates (Davidson, 1976; Cotman and Hamberger, 1978), evidence of specific physiological properties of glutamate in the CNS is still lacking or fragmentary in arthropods. The functions of glycine, proline and taurine in arthropod nervous tissues are largely speculative, but these compounds have been suggested to act as specialized transmitters or modulators in the mammalian CNS (Shank & Aprison, 1970; Werman, 1971 ; Krnjevic, 1974; Davidson, 1976; Huxtable & Barbeau, 1976). For example, taurine has received only little attention as putative neurotransmitter in arthropods for a certain time (Treherne, 1966; Klemm, 1976). We have shown that taurine occurs in considerable amounts in the spider's CNS, especially in the CNS of the salticid species investigated, that use their sight for orientation (34~o of total amino acids contents). It is also present in remarkable quantities in the nervous system of insects and crustaceans (Frontali, 1964; Ray, 1965; Evans, 1973). Probably taurine
is of inhibitory significance in the arthropod CNS, as has been assumed for vertebrates (Rassin & Gaull, 1978). In contrast to results from other arthropod groups (see for example Welsh & Moorhead, 1960; Murdock, 1971; Florey, 1967; Osborne & Neuhoff, 1974; Klemm, 1976) we were not able to prove the presence of serotonin in the nervous tissue of spiders. The only evidence of those biogenic monoamines that are regarded as possible neurotransmitters in invertebrates (Walker & Kerkut, 1978) were findings of catecholamines (especially dopamine) in the spider's CNS, though they do not occur in great quantities (Meyer & Jehnen, 1980). The results lately obtained on the presence of putative neurotransmitters in the nervous tissue of spiders strongly emphasize further investigations, because nothing is known about storage, release, uptake, and metabolism of these substances in the CNS of this interesting arthropod group.
REFERENCES
COTMAN C. W. & HAMBERGERA. (1968) Glutamate as a CNS neurotransmitter: properties of release, inactivation and biosynthesis. In Amino Acids as Chemical Transmitters (Edited by FONNUMF.), pp. 379-412. Plenum Press, New York. DAVlDSON N. (1976) Neurotransmitter Amino Acids. Academic Press, London. EVANSP. D. (1973) Amino acid distribution in the nervous system of the crab, Carcinus maenas (L.). J. Neurochem. 21, ll 17. FLOREYE. (1967) Neurotransmitters and modulators in the animal kingdom. Fedn Proc. Fedn Am. Soc. exp. Biol. 26, 1164-1178. FRONTAL!N. (1964) Brain glutamic acid decarboxylase and synthesis of 7-aminobutyric acid in vertebrate and invertebrate species. In Comparative Neurochemistry (Edited by RICHTERD.), pp. 185-192. Pergamon Press, Oxford. GERSCHENFELD H. M. (1973) Chemical transmission in invertebrate central nervous systems and neuromuscular junctions. Physiol. Revs 53, l 119. HUXTABLER. 8z BARBEAUA. (Eds) (1976) Taurine. Raven Press, New York. KLEMM N. (1976) Histochemistry of putative transmitter substances in the insect brain. Pro# Neurobiol. 7, 99-169. KRNJEVIC K. (1974) Chemical nature of synaptic transmission in vertebrates. Physiol. Revs 54, 418 540. KRONBERGH., ZIMMERH. G. & NEUHOFFV. (1978) Automatische Fluorometrie von Mikro-DfinnschichtChromatogrammen, Fresenius. Z. analyt. Chem. 290, 133. MEYER W. & IDEL K. (1977) The distribution of acetylcholinesterase in the central nervous system of jumping spiders and wolf spiders (Arachnida, Araneida: Salticidae et Lycosidae). J. comp. Neurol. 173, 717-744. MEYER W. & JEHNEN R. (1980) The distribution of monoamine oxidase and biogenic monoamines in the central nervous system of spiders (Arachnida: Araneida). J. Morphol. in press. MEYER W. & POSPIECHB. (1977) The distribution of acetylcholinesterase in the central nervous system of webbuilding spiders (Arachnida, Araneae). H istochemistry 51, 201 208. MURDOCK L. L. (1971) Catecholamines in arthropods: a review. Comp. 9en. Pharmac. 2, 254-274. NEUBACH C., SCHULZEE. ~; NEUHOFFV. (1978) Determination of amino acids using dansyl-chloride. Proc. Eur. Soc. Neurochem. 1 (Edited by NEUHOFFV.), 556.
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W. MEYER, H. M. POEHLING and V. NEUHOFF
NEUHOFE V. (1973) Micromethods in Molecular Biology. Springer-Verlag, Berlin. OSBORNE N. N. & NEUHOFF V. (1974) Amino acid and serotonin content in the nervous system, muscle and blood of the cockroach Periplaneta americana. Brain Res.
80, 251-264. PASANTES H., TAPIA R., ORTEGA B. & MASS1EU G. (1965) Free amino acids and activity of some pyridoxal phosphate-dependent enzymes in the nervous system of three Arthropoda species. Comp. Biochem. Physiol. 16, 523-529. PITMAN R. M. (1971) Transmitter substances in insects: a review. Comp. gen. Pharmac. 2, 347 371. POEHLING n . M., WYSS U. (~ NEUHOFF V. (1980) Microanalysis of free amino acids in the aseptic host-parasite system: Ficus carica-Xiphinema index (Nematoda). Physiol. Pl. Path. 16, 49-61. RASSIN D. K. (~ GAULL G. E. (1978) Taurine and other sulfur containing amino acids: Their function in the central nervous system. In Amino Acids as Chemical Transmitters (Edited by FONNUM F.), pp. 571-597. Plenum Press, New York. RAY J. W. (1965) The free amino acid pool of cockroach (Periplaneta americana) central nervous system. In The Physiology of the Insect Central Nervous System (Edited by TREHERNE J. E. & BEAMENT J. W. L.), pp. 31-38. Academic Press, New York. ROBERTS E., CHASE T. N. & TOWER D. B. (1976) GABA in
Nervous System Function. Kroc Foundation Ser. Vol. 5. Raven Press, New York. SHANK R. P. & APRISON M. H. (1970) The metabolism in vivo of glycine and serine in eight areas of the rat central nervous system. J. Neurochem. 17, 1461-1475. TREHERNE J. E. (1966) The Neurochemistry of Arthropods. Cambridge Univ. Press. WALKER R. J. (1977) Putative transmitters in invertebrates. Biochem. Soc. Trans. 5, 841 945. WALKER R. J. & KERKUT G. A. (1978) The first family (adrenaline, noradrenaline, dopamine, octopamine, tyramine, phenylethanolamine and phenylethylamine). Comp. Biochem. Physiol. 61C, 261-266. WELSH J. H. & MOORHEAD M. (1960) The quantitative distribution of 5-hydroxytryptamine in the invertebrates, especially in their nervous system. J. Neurochem. 6, 146 169. WERMAN R. (1972) Amino acids as central neurotransmitters. Res. Pubis Ass. Res. herr. ment. Dis. 50, 147 180. ZIMMER H. G. & NEUHOFF V. (1977) Quantitative Auswertung yon zweidimensionalen Mikro-DiinnschichtChromatogrammen. In Informatik Fachberichte, Vol. 8: Digitale Bildvearheituny (Edited by NAGEL H. H.), pp. 12 20. ZIMMER H. G., KRONBE~G H. & NEUHOFE V. (1978) Quantitative evaluation of chromatograms. Proc. 4th Int. Joint Conf. Pattern Recognition (Kyoto), 834 836.
0306-4492/80/0901-0083502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
C O M P A R A T I V E ASPECTS O F FREE A M I N O A C I D S IN THE C E N T R A L N E R V O U S SYSTEM O F S P I D E R S W. MEYER*, H. M. POEHLINGt and V. NEUHOFFt *lnstitut ffir Zoologie, Tier~irztliche Hochschule Hannover, Bischofsholer Damm 15, D-3000 Hannover 1; and tMax-Planck-Institut fiir experimentelle Medizin, Forschungsstelle Neurochemie, Hermann-Rein-Str. 3, D-3400 G6ttingen, Federal Republic of Germany (Received 14 January 1980)
Abstract--1. The spectrum of free amino acids in the CNS of 6 species from 6 different spider families was analysed by microchromatography of dansyl derivatives. 2. We could identify 23 dansyl amino acids, the following ones being relatively abundant in all the species investigated: taurine, alanine, glycine, GABA, proline, glutamic acid, and aspartic acid. 3. The results obtained are compared with findings from other arthropod subphyla, and the role of amino acids as putative neurotransmitters in the arthropod CNS is discussed.
15 min at 4°C the supernatant was analysed for free amino acids. Amino acids were separated on 3 x 4cm micropolyamide sheets (A 1700 Schleicher u. SchiJll, Dassel F.R. Germany) as dansyl derivatives (1-dimethylaminonaphthalene-5-sulphonyl-chloride; Serva, Heidelberg F.R. Germany). Details of this method are described by Neubach et al. (1978), and Poehling et al. (1980). Dansyl-amino acid solution (0.5 1.0 pl) was applied with micropipettes on the corner of a polyamide sheet and developed in the first dimension with 2~o formic acid and in the second dimension with 20% acetic acid in toluol. Chromatograms were evaluated by fluorescence scanning photometry with the aid of a specially developed program for digital picture analysis (PDP-12/12 Computer Digital Equipment Co). Fluorescence intensity of each recognizable spot was recorded as %0 of the fluorescence intensity of internal standards (~-phenylglycine and sarcosine). Further details of chromatogram evaluation are described by Kronberg et al. (1978), Zimmer & Neuhoff (1977), and Zimmer et al. (1978). Data were only presented for clearly recognizable spots of dansyl-amino acids. Unknown dansylated substances are detectable but were not analysed. Only estimations were recorded for glutamine, asparagine, serine, and arginine because these amino acids could only be separated by hand correction of the computer analysis from the comigrating by-product of the dansylation reaction, sulfinic acid. This hand correction involves subjective errors.
INTRODUCTION
Until now free amino acids in the central nervous system of arthropods have received much attention only in insects and crustaceans (see for example Evans, 1973; Osborne & Neuhoff, 1974). Available evidence in these groups suggests that several amino acids are most likely candidates for neuro-transmitters (Gerschenfeld, 1973; Klemm, 1976; Walker, 1977). In contrast to these findings no information exists on the amino acids of the spider's nervous system. Therefore, the present study was designed to give a first, preliminary account of the spectrum of free amino acids as present in the nervous tissue of different spider families. The species used during this investigation exhibit varying habits, especially in relation to their different modes of orientation. This is also manifested by specific structural features of the brain (Meyer & Pospiech, 1977; Meyer & Idel, 1977). Our results may be of interest in connection with this, but mainly in view of possible putative neurotransmitters when compared with findings from the two other great arthropod subphyla.
MATERIALS AND METHODS
Spider species of the following 4 different families were captured during June and July 1979 in the vicinity of Hannover (Lower Saxony, West Germany): Tegenaria derhami (Scopoli) (Agelenidae), Araneus diadematus Clerck (Araneidae), Pardosa amentata (Clerck) (Lycosidae), Marpissa muscosa (Clerck) (Salticidae). Latrodeetus mactans tredecimguttatus Rossi (Theridiidae) was captured on the island Pag (Yugoslavia), and the orthognath species Aphonopelma eutylenurn (Chamberlin) (Theraphosidae) was purchased from a zoological supply company. The nervous system of adult animals of both sexes was removed from the cephalothorax in the cold, washed twice in buffered saline and transferred into 10#l capillaries (Brand Wertheim) or pyrex glass tubes (~b 1.5 mm) sealed at one end. One mg tissue was homogenized in 20 pl 0.05 M K 2 C O 3 , pH 9.0, using a dentist's drill for capillaries and Teflon pestels for pyrex tubes at 24,000 rpm, icecooled (Neuhoff, 1973). After centrifugation at 2000rpm for
RESULTS AND DISCUSSION
The results obtained during the course of this study are summarized in Table 1, a selected microchromatogram is presented in Fig. 1. In view of the data for 6 species from 6 different families we could identify 23 dansyl amino acids in the central nervous system of spiders. The total spectrum of free amino acids as revealed by microchromatography of dansyl derivatives generally shares common features with those described for the nervous system of insects and crustaceans (Pasantes et al., 1965; Ray, 1965; Evans, 1973; Osborne & Neuhoff, 1974). The following amino acids were relatively abundant in all the spider species investigated: taurine, alanine, 83
W. MEYER, H. M. POEHLINGand V. NEUHOFF
84
Table 1. Percentage composition of substances ( + S E M for 7 experiments) from the central nervous system separated as dansyl derivatives by microchromatography Spot No.
Dansylated substances
Aphonopelma eutylenum
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Isoleucine Leucine Histidine Phenylalanine Methionine Proline Valine GABA Alanine ETA Lysine Ornithine Tryptophan N-Tyrosine OH-Tyrosine Taurine Glycine Glutamic acid Aspartic acid Serine Glutamine Asparagine Arginine
0.71 1.06 3.01 0.47 0.40 13.93 2.39 12.32 17.56
+ ± + ± ± + + ± ±
0.11 0.05 1.06 0.02 0.02 0.73 0.17 0.51 1.34
0.96 0.17 0.97 1.29 1.26 11.65 7.39 11.74 12.78
± ± ± ± ± ± ± ± ± + + + +
0.02 0.02 0.07 0.00 0.03 0.20 0.06 0.27 0.67
Tegenaria derhami 0,61 ± 0.11 1.12 ± 0.15 0.31 + 0.04 0.59 ± 0.07 0.36 ± 0.06 9.01 ± 1.63 1.47 ± 0.30 22.25 + 2.48 21.09 ± 0.51 0.23 ± 0.09 1.10 + 0.411 0.27 ± 0.06 0.45 ± 0.05 0.38 ± 0.07 1.37 + 0.06 18.82 _+ 1,88 7.19 ± 0.78 7,97 _+ 0.99 5.26 + 0.67 + + + + +
+
Latrodectus react, tredecimgutt, 1.09 3,11 0.07 1.85 0.65 10.60 1.80 9.42 20.22 0.54 2.45 0.45 0.51
+ ± + ± ± ± ± ± + + + ± ±
0.21 0.43 0.02 0.38 0.06 0.35 0.24 0.24 0.92 0.10 0.54 0.15 0.07
1.08 25.00 14.74 5.79 0.63
± 0.28 _+ 2.06 ± 4.12 ± 1.48 ± 0.31 + + + + +
Araneus diadematus
Pardosa amentata
Marpissa rnuscosa
0.35 ± 0.02 0.69 + 0.02 0,30 + 0.11 0.43 ± 0.07 0.30 ± 0.15 4.02 + 0.94 0.80 + 0.07 17.41 ± 1.40 27.97 ± 0.96 0.39 ± 0.21 1.34 + 0.20 0.35 ± 0.17 0.26 ± 0.07 0.15 _+ 0.07 1.53 _+ 0.19 22.42 ± 0.75 13.01 ± 2.11 6.90 ± 0.51 1.06 ± 0.25 + + + + +
0.47 ± 0.19 0.77 _+ 0.31 0.41 + 0,15 /I.59 + 0.20 0.36 _t 0.08 5.57 ± 0.52 1.28 + 0.21 17.74 + 1.60 2459 + 1.31 0.54 +_ 0.15 0.82 + 0.32 0.19 ± 0.02 0.33 + 0.03 0.36 + 0.11 1,59 ± 0.12 22.89 ± 5.30 8.22 _t 1.111 9.81 ± 0.52 3.56 + 0.91 + + + + +
0.52 ± 0.13 0.73 ± 0.17 1.09 + 0,51 0.48 + 0.08 0.39 ± 0 0 3 5,56 ± 0.85 1,15 _+ 0.30 14.05 ± 2.35 18.17 ± 1.13 0.41 ± 0.18 1.22 + 0.10 1.04 + 0.27 0.30 + 0.02 0.26 ± 0.09 1.08 ± 0.29 34.20 ± 1.25 9.22 ± 1.08 6.82 ± 11.42 3.15 + 0.66 + + + + +
+ = very weakly present, + + = weakly present
A
)14
~21
( ~ 7 ~S2
( ~ 3 (~4 (~5
2.
9(~-']':dans'NH2
(~)Sl 1~1
(~)17
C~)12 C)15
( " ~ 16
B
f)
21
19] ,"
) "~lans.6H
./
~
~
23
So
Fig. 1. Selected microchromatogram (A) of dansylated substances extracted from the central nervous system of Tegenaria derhami (Scopoli). The spot numbers in the identification key (B) are explained in Table 1. ~ 1. direction 2% formic acid, ---~2. direction 20% acetic acid in toluol; S = starting points; $I = first standard (ct-phenylglycine), $2 = second standard (sarcosine), Sa = sulfinic acid.
85
Amino acids in spider CNS glycine, GABA, proline, glutamic acid, and partly also aspartic acid. The latter compound and proline were present in higher amounts only in the primitive orthognath spider (Aphonopelma eutylenum), but surprisingly low in the Mediterranean black widow spider (Latrodectus mactans tredecimguttatus). Compared with all the other species, the taurine contents of the nervous system of Aphonopelma were relatively low, while this substance was more concentrated in species representing a higher phylogenetic standard, like the specialized web-building (Theridiidae, Araneidae) and hunting spiders (Lycosidae, Salticidae). It is interesting to note that the 7 amino acids enumerated above are also accumulated in similar relative quantities in the nervous system of the two other great arthropod groups (Frontali, 1964; Osborne & Neuhoff, 1974; Evans, 1973). In relation to the quantitative or semi-quantitative evaluation of results obtained with dansylation methods, it must be admitted, however, that the different amino acids are not all dansylated in comparable equal amounts. This is especially true of glutamic acid and aspartic acid (see Poehling et al., 1980), i.e. the real relative amounts of these amino acids may be somewhat higher than perceptible from Table 1. Several of the amino acids occurring in higher concentrations in the spider CNS are thought to have transmitter functions in both, the vertebrate as well as the invertebrate nervous tissues (Pitman, 1971; Gerschenfeld, 1973; Krnjevic, 1974; Klemm, 1976; Walker, 1977). Evidence is strongest for GABA and glutamate, while there is only little information available about the possible involvement of taurine, glycine, aspartate, and proline in synaptic transmission in arthropods. As demonstrated for the vertebrate CNS, the function of GABA in insects was found to be exclusively inhibitory (Pitman, 1971; Roberts et al., 1976). Compared with GABA, glutamic acid is likewise present in the nervous system of all great arthropod groups (see for example Evans, 1973; Osborne & Neuhoff, 1974; Klemm, 1976; and the results for spiders as given in this study). This substance is now generally considered as putative transmitter in invertebrates, with essential importance for neuromuscular excitation in insects and crustaceans (Walker, 1977). But in contrast to vertebrates (Davidson, 1976; Cotman and Hamberger, 1978), evidence of specific physiological properties of glutamate in the CNS is still lacking or fragmentary in arthropods. The functions of glycine, proline and taurine in arthropod nervous tissues are largely speculative, but these compounds have been suggested to act as specialized transmitters or modulators in the mammalian CNS (Shank & Aprison, 1970; Werman, 1971 ; Krnjevic, 1974; Davidson, 1976; Huxtable & Barbeau, 1976). For example, taurine has received only little attention as putative neurotransmitter in arthropods for a certain time (Treherne, 1966; Klemm, 1976). We have shown that taurine occurs in considerable amounts in the spider's CNS, especially in the CNS of the salticid species investigated, that use their sight for orientation (34~o of total amino acids contents). It is also present in remarkable quantities in the nervous system of insects and crustaceans (Frontali, 1964; Ray, 1965; Evans, 1973). Probably taurine
is of inhibitory significance in the arthropod CNS, as has been assumed for vertebrates (Rassin & Gaull, 1978). In contrast to results from other arthropod groups (see for example Welsh & Moorhead, 1960; Murdock, 1971; Florey, 1967; Osborne & Neuhoff, 1974; Klemm, 1976) we were not able to prove the presence of serotonin in the nervous tissue of spiders. The only evidence of those biogenic monoamines that are regarded as possible neurotransmitters in invertebrates (Walker & Kerkut, 1978) were findings of catecholamines (especially dopamine) in the spider's CNS, though they do not occur in great quantities (Meyer & Jehnen, 1980). The results lately obtained on the presence of putative neurotransmitters in the nervous tissue of spiders strongly emphasize further investigations, because nothing is known about storage, release, uptake, and metabolism of these substances in the CNS of this interesting arthropod group.
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