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Dopamine, N-Acetyldopamine and Serotonin Concentrations in the Visual System of Praying Mantis During Postembryonic Development
Comp. Biochem. Physiol. Vol. 116A, No. 4, pp. 379–386, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00362-3
Dopamine, N-Acetyldopamine and Serotonin Concentrations in the Visual System of Praying Mantis During Postembryonic Development Monika Germ Institut fu¨r Zoologie, Karl-Franzens-Universita¨t Graz, Abteilung Neurobiologie, Universita¨tsplatz 2, A-8010 Graz, Austria ABSTRACT. High-performance liquid chromatography with electrochemical detection was used to quantify the two biogenic amines dopamine (DA) and 5-hydroxytryptamine (5-HT, serotonin) as well as a metabolite of DA, N-acetyldopamine (NADA), in the compound eyes and optic lobes of praying mantis (Polyspilota sp. and Tenodera sinensis) during postembryonic development. After hatching, DA and 5-HT concentrations (pmol/ mg ww) were relatively high (DA, 5.43 6 1.13; 5-HT, 5.65 6 1.0 for Polyspilota), but the NADA concentration was more than 25 times higher than those of DA and 5-HT (143.7 6 16.71 for Polyspilota). Subsequently, the concentrations decreased constantly into the middle larval instar and then rose to reach their highest peak in the last larval instar (DA) or a very high concentration in the seventh instar (5-HT and NADA). In adults, DA, 5-HT and NADA concentrations decreased again. The concentration profile for NADA was similar to that of 5-HT. The values per structure (compound eye and optic lobe complex) and per ommatidial column channel were also calculated. It is significant that changes in the amine levels during postembryonic development went hand in hand with development changes in the activity and behavior of the mantids. Copyright 1997 Elsevier Science Inc. comp biochem physiol 116A;4:379–386, 1997. KEY WORDS. dopamine, N-acetyldopamine, Polyspilota sp., postembryonic development, praying mantis, serotonin, Tenodera sinensis, visual system
INTRODUCTION There are many indications that the two biogenic amines, dopamine (DA) and 5-hydroxytryptamine (5-HT, or serotonin), play an important role as neuromodulators in the visual systems of insects (8,10,23). The morphological correlates are the large serotonergic and dopaminergic neurons that have been demonstrated using immunohistochemical methods in the optic lobe (15,26–28) and studied electrophysiologically (17). Behavioral studies have demonstrated the neuromodulatory effect of 5-HT on certain closely defined visual behavioral responses (4,9). There is an indication in worker honeybees of a relationship between neuronal DA and 5-HT concentrations and morphological and behavioral development (35). Other quantitative works have shown relatively large variations in DA and 5-HT concentrations in insect visual systems, depending on the time of day (20,21), which indicate an influence of the time of the circadian rhythm (36), the time of year, various other external factors (12) and visual experience (11,24). N-aceAddress reprint requests to: M. Germ, Yamaguchi University, Faculty of Science, Department of Biology, Physics and Informatics, Yamaguchi 753, Japan. Fax 181-839-33-5768; Tel. 181-839-33-5714; E-mail: germ@po. cc.yamaguchi-u.ac.jp. Received 23 February 1996; accepted 19 August 1996.
tyldopamine (NADA) is a DA metabolite and has an important role in sclerotization of the cuticle of insects (33), but its biochemical and physiological role in the insect visual system is still unknown. In the present study, HPLC with electrochemical detection (ECD) was used to measure DA, 5-HT and NADA concentrations in the tissue of the visual system (compound eyes and optic lobes) in praying mantids throughout postembryonic development (i.e., from hatching through adulthood). The aim was to determine whether the amine levels are age dependent and whether there are chronological parallels between changes in amine level and the changes that occur in animal behavior and locomotor activity (18,32). MATERIALS AND METHODS Animals Larvae (2L, first motile instar to 8L or 9L) and adults of the Chinese (Tenodera sinensis) and South African (Polyspilota sp.) praying mantis species were used. From hatching until the fifth instar, the mantids were kept in an illuminated incubator; older larvae and adults were kept in a breeding room. The animals were individually kept in plastic containers. Rearing containers were changed according to the stage of nymphal development. The daily cycle was 06: 00–
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FIG. 1. Mean fresh weight (wet weight, 6SD) of compound eye and optic lobe (lamina, medulla, lobula) of praying mantis
Polyspilota sp. (A) and Tenodera sinensis (B) during postembryonic development until adulthood. L, larval instar; n 5 12– 48 eyes and optic lobes. Numbers of ommatidia concerning to the postembryonic development, n 5 3–8: * 5 estimated value for seventh instar (C). Wet weight in mg/ommatidium, n 5 12–48 (D). Wet weight in mg concerning to the numbers of ommatidia (E). For linear regression see text.
18: 00 light and 18 : 00–06 : 00 dark at about 28°C and 55% relative humidity. Illumination was given by white fluorescent light tubes, and the light intensity in the incubator was about 500–800 lx and in the breeding room about 300– 600 lx. The animals were fed with fruit flies until the fifth instar and then with Tenebrio larvae and crickets and provided with water 2 to 3 times per day. Sample Preparation All animals were prepared 2 days after moult from one larval instar to the next one and 2 days after emergence to the adult stage, respectively. One hour after light-on, at 07:00, the animals were removed from the incubator, placed on ice and the compound eyes and optic lobes quickly dissected out with microscissors under a stereo microscope and then weighed on a microscale (Mettler). Depending on the larval
instar, one to eight eyes were homogenized in 300 µl of 0.3 N perchloric acid, centrifuged and stored at 270°C until analysis. Sample Analysis From each sample, 100 µl were injected directly into the Beckman HPLC system using a Beckman S07 microsampler. A C18 reversed-phase column (LI Chrospher 100 RP18; 5 µm average particle size; LI Chrocart, 125-4 Merck) and a Beckman electrochemical detector (range of 0.5 µA; 1-Hz filter; 0.5 V) were used. The mobile phase was composed of 14% methanol, consisting of 13.8 g NaH2PO4– H2O, 600 mg heptane sulfonic acid, one spatula tip EDTA (100 mg), 280 ml methanol and 1720 ml distilled water. This medium was placed in an ultrasonic bath for 10 min. The flow rate was 1.25 ml/min and passage time was 20
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FIG. 2. Mean DA concentrations in pmol per compound eye and optic lobe complex (6SD, n 5 6–12 samples), pmol/mg ww
(6SD, n 5 6–12) of praying mantis Polyspilota sp. (A and B) and Tenodera sinensis (C and D) during postembryonic development and in adulthood and in fmol/ommatidial column channel for Tenodera sinensis (6SD, n 5 6–12; E). All measurements were made 2 days after moult from one instar to the next and 2 days after emergence to the adult stage, respectively. L, larval instar, 2L, first motile larval instar. See text for significance level.
min. DA hydrochloride, NADA monohydrate and 5-HT creatine sulfate complex were used as standards. The DA, NADA and 5-HT values were related to the weight of the fresh tissue and given in pmol substance/mg tissue (wet wt). Because of the differences in size of the structures at different stages, it was necessary to relate the concentration to the wet weight of fresh tissue to produce values that could be compared directly with each other. The mean and SDs were calculated, and significant differences between the different age groups were studied with the nonparametric Wilcoxon test (P , 0.05). The concentration was also given in pmol/compound eye and optic lobe complex. Numbers of Ommatidia The numbers of ommatidia were counted from the larval corneal exuvia by means of an Olympus BH2 RFCA micro-
scope only in T. sinensis and the numbers were compared with papers for Sphrodomantis (3,5). For the seventh instars, unfortunately no exuvia was available; therefore, the numbers of ommatidia for this instar was estimated concerning to the regression line (Fig. 1C). The concentrations of biogenic amines were also calculated for one ommatidium (per ommatidial/neuropile column channel) in the case of T. sinensis. RESULTS Fresh Weight of the Visual System In the course of postembryonic development (about 2.5–3 months), the fresh weight of the compound eyes and optic lobes of Polyspilota sp. and T. sinensis increased approximately exponentially (Fig. 1, A and B, n 5 12–48 eyes and optic lobes). This means that for every moult, weight
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FIG. 3. Mean NADA concentrations in pmol per compound eye and optic lobe complex (6SD, n 5 6–12 samples), pmol/mg
ww (6SD, n 5 6–12) of praying mantis Polyspilota sp. (A and B) and Tenodera sinensis (C and D) during postembryonic development and in adulthood and in fmol/ommatidial column channel for Tenodera sinensis (6SD, n 5 6–12; E). All measurements were made 2 days after moult from one instar to the next and 2 days after emergence to the adult stage, respectively. L, larval instar; 2L, first motile larval instar. See text for significance level.
roughly doubled. In case of Tenodera, the wet weight was also calculated for one ommatidium (Fig. 1D). Numbers of Ommatidia The numbers of ommatidia for T. sinensis increased from the second instar to the adult more than 15 times, and there was a clear positive correlation (linear regression, R 5 0.99, P , 0.001; Fig. 1C). There was a positive correlation between the numbers of ommatidia and the wet weight (linear regression, R 5 0.89, P , 0.01; Fig. 1E). Dopamine The DA concentration profile per compound eye and optic lobe complex for Polyspilota sp. increased until the ninth instar; a high concentration could also be found in adult animals (Fig. 2A). A relatively large amount (pmol/mg ww) of DA was measured in the visual system in the second lar-
val instar (5.43 6 1.13). The differences in DA values were all significant (P , 0.01, n 5 6–12 samples for each point) between all instars. DA decreased steadily until the sixth instar by about 75% (Wilcoxon test, P , 0.01, n 5 6–12; 1.38 6 0.41) compared with the second instar. From the seventh instar onward, however, the values increased and reached a maximal value in the ninth instar (P , 0.001; 6.59 6 1.41). Distinctly lower DA values were subsequently measured in adult animals (Fig. 2B). For T. sinensis, the changes in values were similar. The concentration per compound eye and optic lobe complex gradually increased from the second instars to the adulthood (Fig. 2C). The DA content (pmol/mg ww) in the second instar reached a value about 4.34 6 1.46 and then it decreased exponentially until the fifth instar (by 68% of the second instar; P , 0.01, n 5 6–12; 1.37 6 0.34) but then increased again until the eighth instar (by 74% of the fifth instar; P , 0.01; 5.31 6 0.84) and adulthood (Fig. 2D). The concentration for one ommatidium (ommatidial/neuropile
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FIG. 4. Mean 5-HT concentrations in pmol per compound eye and optic lobe complex (6SD, n 5 6–12 samples), pmol/mg
ww (6SD, n 5 6–12) of praying mantis Polyspilota sp. (A and B) and Tenodera sinensis (C and D) during postembryonic development and in adulthood and in fmol/ommatidial column channel for Tenodera sinensis (6SD, n 5 6–12; E). All measurements were made 2 days after moult from one instar to the next and 2 days after emergence to the adult stage, respectively. L, larval instar; 2L, first motile larval instar. See text for significance level.
column channel) was high in the eighth instar, and in adult animals and the lowest concentration could be found in the fifth instar (Fig. 2E). N-Acetyldopamine In Polyspilota sp., very high values per compound eye and optic lobe complex for NADA could be found from the seventh instar to the adulthood; in the other instars the concentrations were relatively low (Fig. 3A). The largest value for the concentration of NADA (pmol/mg ww) was measured in the second instar (143.7 6 16.71). There was about 25 times more NADA than DA in the visual system of Polyspilota sp. in the second larval instar. In the following instars, however, the values decreased drastically to the sixth instar (20.46 6 2.46), corresponding to a decrease of 86% of the second instar (P , 0.001, n 5 6–12). In the seventh instar there was again a distinct increase in values by 83% of the sixth instar (P , 0.001; 119.2 6 20.51). From the
eighth instar onward, however, NADA concentration again decreased continuously to the adulthood (Fig. 3B). In case of concentration per compound eye and optic lobe complex, the highest level of NADA was reached in the seventh instar (T. sinensis; Fig. 3C). A relatively large value (pmol/mg ww) for NADA was measured in the second instar (107.8 6 19.51), which then decreased in the following instars until it reached a minimum in the fifth instar (25.57 6 6.52), corresponding to a decrease of 76% of the second instar (P , 0.01). The highest value occurred in the seventh instar (108 6 20.78); this means an increase by 76% of the fifth instar (P , 0.01) followed by a decrease until adulthood (Fig. 3D). In the seventh instar, the highest value for NADA per ommatidium also could be found (Fig. 3E). Serotonin In Polyspilota sp., high 5-HT values per compound eye and optic lobe could be found in the seventh and ninth instar
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as well as in adulthood (Fig. 4A). In the second larval instar, 5-HT concentration (pmol/mg ww) in the visual system is about the same as DA concentration (5-HT, 5.65 6 1). It then decreased continuously until the fifth instar (by 77% of the second instar, P , 0.01, n 5 6–12), when it reached its lowest value (1.29 6 0.35). Values increased slightly from the sixth instar; in the seventh instar the 5-HT value reached its maximum (5.81 6 1.05), which means an increase as compared with the fifth instar of 78% (P , 0.001, n 5 6–12). The concentration of 5-HT then decreased again in the ninth instar and in adults (Fig. 4B). In T. sinensis, the highest 5-HT value per compound eye and optic lobe complex was reached in the seventh instar (Fig. 4C). The 5-HT concentration (pmol/mg ww) decreased until the fifth instar (by 86% of the second instar, P , 0.001, n 5 6–12; 0.67 6 0.19). In the seventh instar there was, as in Polyspilota sp., a pronounced increase in 5HT values (by 83% of the fifth instar, P , 0.01; 4.04 6 0.61; Fig. 4D). The highest 5-HT concentration per ommatidium was reached in the seventh instar (Fig. 4D). DISCUSSION HPLC-ECD measurements showed that concentrations of DA, NADA and 5-HT in the visual system of praying mantis depend on the stage of postembryonic development of the animals. It is remarkable that measurements in the respective age groups showed relatively small interindividual variation. DA and 5-HT moreover have similar concentrations, and there are parallel trends until the middle instars. From then on to adulthood, there are also opposite effects in the two transmitter systems. Common or different roles of DA and 5-HT have been described in various systems, such as in locust salivary glands (1) or in the brain of the pond snail Lymnaea stagnalis (13). A 5-HT metabolite has been described to inhibit DA biosynthesis in the nervous system of the snail Helix pomatia (30). The generally parallel trends between 5-HT and NADA concentrations during postembryonic development are remarkable. The high concentration of NADA seems to speak against this substance being a simple breakdown product of DA (6). The morphology of the DA immunoreactive (IR) and 5HT immunoreactive neurons has been well studied, and the 5-HT-IR cells have been shown to supply all three optic neuropiles in most insect species (19,25,26). The morphological correlates of differences in 5-HT concentration during postembryonic development are currently under study. The gradual decrease in concentration for all three transmitter/metabolite might merely reflect the fact that 5HT and DA cells are already present at hatching and that some or all are widefield cells (25,26). As new ommatidia are added to the eye, these cells extend and grow into the new neuropile columns that are assembled at the growing edge of the optic neuropiles. Yet the number of their somata and primary arborizations do not change, so that total trans-
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mitter content per unit neuropile volume gradually decreases: therefore, the total concentration of transmitter is also important. On the other hand, for a direct comparison of the instars, it is necessary to find a common unit (mg ww, ommatidium). The concentration per ommatidial/neuropile column channel provide a more solid cellular basis for the comparison with immunocytochemical and immunohistochemical data. These concentration profiles (fmol/ ommatidium) are very similar to the diagrams per mg ww (pmol). Therefore, these values could be established for direct comparison between the different instars. It was interesting to see in this context that under certain laboratory conditions, there was a recognizable correlation between concentration of the three amines (DA, NADA and 5-HT) and measureable locomotor activity on the part of the animals (31,32,37). For the interpretation of a comparison of the amine concentration and the behavior, we should be very careful, because only the concentration not the release was measured. But there are some reports demonstrating a relationship between the concentrations of biogenic amines and specific behavioral activities in other insects (7,22,29,35). The relationship between aggressiveness and serotonin in crustaceas was also studied (16). Freshly emerged mantid larvae showed the greatest tendency to change location, as measured by the rate of scanning-peering jumps in an arena (32), exactly when DA and 5-HT concentrations (pmol/mg ww, fmol/ommatidium) are relatively high. This locomotor tendency then decreases distinctly toward the middle instars, when DA and 5-HT concentrations are lowest. The changes in the concentrations of biogenic amines in visual tissue can be considered in the context of the postembryonic development of visually controlled preycapturing behavior (2,18). This requires the mobilization of new neuronal and motor systems at the correct developmental time. The pre-adult instars become completely dependent on the binocular prey-capturing mechanism; earlier in their development, monocular capture is also possible (18). The seventh instar is the particular stage at which the mantids loose their visual plasticity in binocular vision, and this is the stage at which the concentrations of 5-HT and NADA are also very high. Other examples are described in which the significance of biogenic amines in the introduction of a new behavioral condition is demonstrated (12,35). An increase in 5-HT due to social stress and a related drastic increase in motor activity have been demonstrated in crickets (11). A distinct and positive relationship between stress and 5-HT has also been found, for example, in cockroaches (14) and honeybees (12) and between stress and DA in Drosophila (34). To find out more about the relationship between the release of biogenic amines and behavioral activities, further critical study using the injection of these substances directly to the neuronal structures would be deserved.
Concentrations of Biogenic Amines in Mantids’ Eyes
I thank Dr. Karl Kral (Institute for Zoology, Graz, Austria) as my supervisor, Dr. J. Donnerer (Institute for Experimental and Clinical Pharmacology, Karl-Franzens University, Graz, Austria) for the use of the HPLC equipment, Maria Theny for assistance in the measurements, Mr. R. Ehrmann for determination of Polyspilota sp. (a new species), Prof. Dr. K. Tomioka for reading the manuscript critically and Genie Lamont for translation of the text into English. This project was supported by Grant No. P09510BIO to Dr. K. Kral from the Austrian Science Foundation.
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ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00362-3
Dopamine, N-Acetyldopamine and Serotonin Concentrations in the Visual System of Praying Mantis During Postembryonic Development Monika Germ Institut fu¨r Zoologie, Karl-Franzens-Universita¨t Graz, Abteilung Neurobiologie, Universita¨tsplatz 2, A-8010 Graz, Austria ABSTRACT. High-performance liquid chromatography with electrochemical detection was used to quantify the two biogenic amines dopamine (DA) and 5-hydroxytryptamine (5-HT, serotonin) as well as a metabolite of DA, N-acetyldopamine (NADA), in the compound eyes and optic lobes of praying mantis (Polyspilota sp. and Tenodera sinensis) during postembryonic development. After hatching, DA and 5-HT concentrations (pmol/ mg ww) were relatively high (DA, 5.43 6 1.13; 5-HT, 5.65 6 1.0 for Polyspilota), but the NADA concentration was more than 25 times higher than those of DA and 5-HT (143.7 6 16.71 for Polyspilota). Subsequently, the concentrations decreased constantly into the middle larval instar and then rose to reach their highest peak in the last larval instar (DA) or a very high concentration in the seventh instar (5-HT and NADA). In adults, DA, 5-HT and NADA concentrations decreased again. The concentration profile for NADA was similar to that of 5-HT. The values per structure (compound eye and optic lobe complex) and per ommatidial column channel were also calculated. It is significant that changes in the amine levels during postembryonic development went hand in hand with development changes in the activity and behavior of the mantids. Copyright 1997 Elsevier Science Inc. comp biochem physiol 116A;4:379–386, 1997. KEY WORDS. dopamine, N-acetyldopamine, Polyspilota sp., postembryonic development, praying mantis, serotonin, Tenodera sinensis, visual system
INTRODUCTION There are many indications that the two biogenic amines, dopamine (DA) and 5-hydroxytryptamine (5-HT, or serotonin), play an important role as neuromodulators in the visual systems of insects (8,10,23). The morphological correlates are the large serotonergic and dopaminergic neurons that have been demonstrated using immunohistochemical methods in the optic lobe (15,26–28) and studied electrophysiologically (17). Behavioral studies have demonstrated the neuromodulatory effect of 5-HT on certain closely defined visual behavioral responses (4,9). There is an indication in worker honeybees of a relationship between neuronal DA and 5-HT concentrations and morphological and behavioral development (35). Other quantitative works have shown relatively large variations in DA and 5-HT concentrations in insect visual systems, depending on the time of day (20,21), which indicate an influence of the time of the circadian rhythm (36), the time of year, various other external factors (12) and visual experience (11,24). N-aceAddress reprint requests to: M. Germ, Yamaguchi University, Faculty of Science, Department of Biology, Physics and Informatics, Yamaguchi 753, Japan. Fax 181-839-33-5768; Tel. 181-839-33-5714; E-mail: germ@po. cc.yamaguchi-u.ac.jp. Received 23 February 1996; accepted 19 August 1996.
tyldopamine (NADA) is a DA metabolite and has an important role in sclerotization of the cuticle of insects (33), but its biochemical and physiological role in the insect visual system is still unknown. In the present study, HPLC with electrochemical detection (ECD) was used to measure DA, 5-HT and NADA concentrations in the tissue of the visual system (compound eyes and optic lobes) in praying mantids throughout postembryonic development (i.e., from hatching through adulthood). The aim was to determine whether the amine levels are age dependent and whether there are chronological parallels between changes in amine level and the changes that occur in animal behavior and locomotor activity (18,32). MATERIALS AND METHODS Animals Larvae (2L, first motile instar to 8L or 9L) and adults of the Chinese (Tenodera sinensis) and South African (Polyspilota sp.) praying mantis species were used. From hatching until the fifth instar, the mantids were kept in an illuminated incubator; older larvae and adults were kept in a breeding room. The animals were individually kept in plastic containers. Rearing containers were changed according to the stage of nymphal development. The daily cycle was 06: 00–
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FIG. 1. Mean fresh weight (wet weight, 6SD) of compound eye and optic lobe (lamina, medulla, lobula) of praying mantis
Polyspilota sp. (A) and Tenodera sinensis (B) during postembryonic development until adulthood. L, larval instar; n 5 12– 48 eyes and optic lobes. Numbers of ommatidia concerning to the postembryonic development, n 5 3–8: * 5 estimated value for seventh instar (C). Wet weight in mg/ommatidium, n 5 12–48 (D). Wet weight in mg concerning to the numbers of ommatidia (E). For linear regression see text.
18: 00 light and 18 : 00–06 : 00 dark at about 28°C and 55% relative humidity. Illumination was given by white fluorescent light tubes, and the light intensity in the incubator was about 500–800 lx and in the breeding room about 300– 600 lx. The animals were fed with fruit flies until the fifth instar and then with Tenebrio larvae and crickets and provided with water 2 to 3 times per day. Sample Preparation All animals were prepared 2 days after moult from one larval instar to the next one and 2 days after emergence to the adult stage, respectively. One hour after light-on, at 07:00, the animals were removed from the incubator, placed on ice and the compound eyes and optic lobes quickly dissected out with microscissors under a stereo microscope and then weighed on a microscale (Mettler). Depending on the larval
instar, one to eight eyes were homogenized in 300 µl of 0.3 N perchloric acid, centrifuged and stored at 270°C until analysis. Sample Analysis From each sample, 100 µl were injected directly into the Beckman HPLC system using a Beckman S07 microsampler. A C18 reversed-phase column (LI Chrospher 100 RP18; 5 µm average particle size; LI Chrocart, 125-4 Merck) and a Beckman electrochemical detector (range of 0.5 µA; 1-Hz filter; 0.5 V) were used. The mobile phase was composed of 14% methanol, consisting of 13.8 g NaH2PO4– H2O, 600 mg heptane sulfonic acid, one spatula tip EDTA (100 mg), 280 ml methanol and 1720 ml distilled water. This medium was placed in an ultrasonic bath for 10 min. The flow rate was 1.25 ml/min and passage time was 20
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FIG. 2. Mean DA concentrations in pmol per compound eye and optic lobe complex (6SD, n 5 6–12 samples), pmol/mg ww
(6SD, n 5 6–12) of praying mantis Polyspilota sp. (A and B) and Tenodera sinensis (C and D) during postembryonic development and in adulthood and in fmol/ommatidial column channel for Tenodera sinensis (6SD, n 5 6–12; E). All measurements were made 2 days after moult from one instar to the next and 2 days after emergence to the adult stage, respectively. L, larval instar, 2L, first motile larval instar. See text for significance level.
min. DA hydrochloride, NADA monohydrate and 5-HT creatine sulfate complex were used as standards. The DA, NADA and 5-HT values were related to the weight of the fresh tissue and given in pmol substance/mg tissue (wet wt). Because of the differences in size of the structures at different stages, it was necessary to relate the concentration to the wet weight of fresh tissue to produce values that could be compared directly with each other. The mean and SDs were calculated, and significant differences between the different age groups were studied with the nonparametric Wilcoxon test (P , 0.05). The concentration was also given in pmol/compound eye and optic lobe complex. Numbers of Ommatidia The numbers of ommatidia were counted from the larval corneal exuvia by means of an Olympus BH2 RFCA micro-
scope only in T. sinensis and the numbers were compared with papers for Sphrodomantis (3,5). For the seventh instars, unfortunately no exuvia was available; therefore, the numbers of ommatidia for this instar was estimated concerning to the regression line (Fig. 1C). The concentrations of biogenic amines were also calculated for one ommatidium (per ommatidial/neuropile column channel) in the case of T. sinensis. RESULTS Fresh Weight of the Visual System In the course of postembryonic development (about 2.5–3 months), the fresh weight of the compound eyes and optic lobes of Polyspilota sp. and T. sinensis increased approximately exponentially (Fig. 1, A and B, n 5 12–48 eyes and optic lobes). This means that for every moult, weight
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FIG. 3. Mean NADA concentrations in pmol per compound eye and optic lobe complex (6SD, n 5 6–12 samples), pmol/mg
ww (6SD, n 5 6–12) of praying mantis Polyspilota sp. (A and B) and Tenodera sinensis (C and D) during postembryonic development and in adulthood and in fmol/ommatidial column channel for Tenodera sinensis (6SD, n 5 6–12; E). All measurements were made 2 days after moult from one instar to the next and 2 days after emergence to the adult stage, respectively. L, larval instar; 2L, first motile larval instar. See text for significance level.
roughly doubled. In case of Tenodera, the wet weight was also calculated for one ommatidium (Fig. 1D). Numbers of Ommatidia The numbers of ommatidia for T. sinensis increased from the second instar to the adult more than 15 times, and there was a clear positive correlation (linear regression, R 5 0.99, P , 0.001; Fig. 1C). There was a positive correlation between the numbers of ommatidia and the wet weight (linear regression, R 5 0.89, P , 0.01; Fig. 1E). Dopamine The DA concentration profile per compound eye and optic lobe complex for Polyspilota sp. increased until the ninth instar; a high concentration could also be found in adult animals (Fig. 2A). A relatively large amount (pmol/mg ww) of DA was measured in the visual system in the second lar-
val instar (5.43 6 1.13). The differences in DA values were all significant (P , 0.01, n 5 6–12 samples for each point) between all instars. DA decreased steadily until the sixth instar by about 75% (Wilcoxon test, P , 0.01, n 5 6–12; 1.38 6 0.41) compared with the second instar. From the seventh instar onward, however, the values increased and reached a maximal value in the ninth instar (P , 0.001; 6.59 6 1.41). Distinctly lower DA values were subsequently measured in adult animals (Fig. 2B). For T. sinensis, the changes in values were similar. The concentration per compound eye and optic lobe complex gradually increased from the second instars to the adulthood (Fig. 2C). The DA content (pmol/mg ww) in the second instar reached a value about 4.34 6 1.46 and then it decreased exponentially until the fifth instar (by 68% of the second instar; P , 0.01, n 5 6–12; 1.37 6 0.34) but then increased again until the eighth instar (by 74% of the fifth instar; P , 0.01; 5.31 6 0.84) and adulthood (Fig. 2D). The concentration for one ommatidium (ommatidial/neuropile
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FIG. 4. Mean 5-HT concentrations in pmol per compound eye and optic lobe complex (6SD, n 5 6–12 samples), pmol/mg
ww (6SD, n 5 6–12) of praying mantis Polyspilota sp. (A and B) and Tenodera sinensis (C and D) during postembryonic development and in adulthood and in fmol/ommatidial column channel for Tenodera sinensis (6SD, n 5 6–12; E). All measurements were made 2 days after moult from one instar to the next and 2 days after emergence to the adult stage, respectively. L, larval instar; 2L, first motile larval instar. See text for significance level.
column channel) was high in the eighth instar, and in adult animals and the lowest concentration could be found in the fifth instar (Fig. 2E). N-Acetyldopamine In Polyspilota sp., very high values per compound eye and optic lobe complex for NADA could be found from the seventh instar to the adulthood; in the other instars the concentrations were relatively low (Fig. 3A). The largest value for the concentration of NADA (pmol/mg ww) was measured in the second instar (143.7 6 16.71). There was about 25 times more NADA than DA in the visual system of Polyspilota sp. in the second larval instar. In the following instars, however, the values decreased drastically to the sixth instar (20.46 6 2.46), corresponding to a decrease of 86% of the second instar (P , 0.001, n 5 6–12). In the seventh instar there was again a distinct increase in values by 83% of the sixth instar (P , 0.001; 119.2 6 20.51). From the
eighth instar onward, however, NADA concentration again decreased continuously to the adulthood (Fig. 3B). In case of concentration per compound eye and optic lobe complex, the highest level of NADA was reached in the seventh instar (T. sinensis; Fig. 3C). A relatively large value (pmol/mg ww) for NADA was measured in the second instar (107.8 6 19.51), which then decreased in the following instars until it reached a minimum in the fifth instar (25.57 6 6.52), corresponding to a decrease of 76% of the second instar (P , 0.01). The highest value occurred in the seventh instar (108 6 20.78); this means an increase by 76% of the fifth instar (P , 0.01) followed by a decrease until adulthood (Fig. 3D). In the seventh instar, the highest value for NADA per ommatidium also could be found (Fig. 3E). Serotonin In Polyspilota sp., high 5-HT values per compound eye and optic lobe could be found in the seventh and ninth instar
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as well as in adulthood (Fig. 4A). In the second larval instar, 5-HT concentration (pmol/mg ww) in the visual system is about the same as DA concentration (5-HT, 5.65 6 1). It then decreased continuously until the fifth instar (by 77% of the second instar, P , 0.01, n 5 6–12), when it reached its lowest value (1.29 6 0.35). Values increased slightly from the sixth instar; in the seventh instar the 5-HT value reached its maximum (5.81 6 1.05), which means an increase as compared with the fifth instar of 78% (P , 0.001, n 5 6–12). The concentration of 5-HT then decreased again in the ninth instar and in adults (Fig. 4B). In T. sinensis, the highest 5-HT value per compound eye and optic lobe complex was reached in the seventh instar (Fig. 4C). The 5-HT concentration (pmol/mg ww) decreased until the fifth instar (by 86% of the second instar, P , 0.001, n 5 6–12; 0.67 6 0.19). In the seventh instar there was, as in Polyspilota sp., a pronounced increase in 5HT values (by 83% of the fifth instar, P , 0.01; 4.04 6 0.61; Fig. 4D). The highest 5-HT concentration per ommatidium was reached in the seventh instar (Fig. 4D). DISCUSSION HPLC-ECD measurements showed that concentrations of DA, NADA and 5-HT in the visual system of praying mantis depend on the stage of postembryonic development of the animals. It is remarkable that measurements in the respective age groups showed relatively small interindividual variation. DA and 5-HT moreover have similar concentrations, and there are parallel trends until the middle instars. From then on to adulthood, there are also opposite effects in the two transmitter systems. Common or different roles of DA and 5-HT have been described in various systems, such as in locust salivary glands (1) or in the brain of the pond snail Lymnaea stagnalis (13). A 5-HT metabolite has been described to inhibit DA biosynthesis in the nervous system of the snail Helix pomatia (30). The generally parallel trends between 5-HT and NADA concentrations during postembryonic development are remarkable. The high concentration of NADA seems to speak against this substance being a simple breakdown product of DA (6). The morphology of the DA immunoreactive (IR) and 5HT immunoreactive neurons has been well studied, and the 5-HT-IR cells have been shown to supply all three optic neuropiles in most insect species (19,25,26). The morphological correlates of differences in 5-HT concentration during postembryonic development are currently under study. The gradual decrease in concentration for all three transmitter/metabolite might merely reflect the fact that 5HT and DA cells are already present at hatching and that some or all are widefield cells (25,26). As new ommatidia are added to the eye, these cells extend and grow into the new neuropile columns that are assembled at the growing edge of the optic neuropiles. Yet the number of their somata and primary arborizations do not change, so that total trans-
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mitter content per unit neuropile volume gradually decreases: therefore, the total concentration of transmitter is also important. On the other hand, for a direct comparison of the instars, it is necessary to find a common unit (mg ww, ommatidium). The concentration per ommatidial/neuropile column channel provide a more solid cellular basis for the comparison with immunocytochemical and immunohistochemical data. These concentration profiles (fmol/ ommatidium) are very similar to the diagrams per mg ww (pmol). Therefore, these values could be established for direct comparison between the different instars. It was interesting to see in this context that under certain laboratory conditions, there was a recognizable correlation between concentration of the three amines (DA, NADA and 5-HT) and measureable locomotor activity on the part of the animals (31,32,37). For the interpretation of a comparison of the amine concentration and the behavior, we should be very careful, because only the concentration not the release was measured. But there are some reports demonstrating a relationship between the concentrations of biogenic amines and specific behavioral activities in other insects (7,22,29,35). The relationship between aggressiveness and serotonin in crustaceas was also studied (16). Freshly emerged mantid larvae showed the greatest tendency to change location, as measured by the rate of scanning-peering jumps in an arena (32), exactly when DA and 5-HT concentrations (pmol/mg ww, fmol/ommatidium) are relatively high. This locomotor tendency then decreases distinctly toward the middle instars, when DA and 5-HT concentrations are lowest. The changes in the concentrations of biogenic amines in visual tissue can be considered in the context of the postembryonic development of visually controlled preycapturing behavior (2,18). This requires the mobilization of new neuronal and motor systems at the correct developmental time. The pre-adult instars become completely dependent on the binocular prey-capturing mechanism; earlier in their development, monocular capture is also possible (18). The seventh instar is the particular stage at which the mantids loose their visual plasticity in binocular vision, and this is the stage at which the concentrations of 5-HT and NADA are also very high. Other examples are described in which the significance of biogenic amines in the introduction of a new behavioral condition is demonstrated (12,35). An increase in 5-HT due to social stress and a related drastic increase in motor activity have been demonstrated in crickets (11). A distinct and positive relationship between stress and 5-HT has also been found, for example, in cockroaches (14) and honeybees (12) and between stress and DA in Drosophila (34). To find out more about the relationship between the release of biogenic amines and behavioral activities, further critical study using the injection of these substances directly to the neuronal structures would be deserved.
Concentrations of Biogenic Amines in Mantids’ Eyes
I thank Dr. Karl Kral (Institute for Zoology, Graz, Austria) as my supervisor, Dr. J. Donnerer (Institute for Experimental and Clinical Pharmacology, Karl-Franzens University, Graz, Austria) for the use of the HPLC equipment, Maria Theny for assistance in the measurements, Mr. R. Ehrmann for determination of Polyspilota sp. (a new species), Prof. Dr. K. Tomioka for reading the manuscript critically and Genie Lamont for translation of the text into English. This project was supported by Grant No. P09510BIO to Dr. K. Kral from the Austrian Science Foundation.
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