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Biogenic amines in Drosophila melanogaster selected for differences in larval feeding behavior
BEHAVIORAL BIOLOGY 15, 213-217 (1975), Abstract No. 5134
BRIEF REPORT Biogenic Amines in Drosophila melanogaster Selected for Differences in Larval Feeding Behavior I
D. F. SEWELL
Department of Psychology, University of Hull D. M. HUNT
Department of Plant Biology and Microbiology, Queen Mary College, London and B. BURNET
Department of Genetics, University of SheffieM Levels of biogerdc amines were assayed in lines of Drosophila melanogaster selected for differences in their rate of larval feeding activity. Noradrenaline levels were low in one of the fast feeding lines (FA), but not in a second fast feeding line (FB). Dopamine levels were high in one of the slow feeding lines (SA) and low in the fast feeding line (FB). There were no significant differences in serotonin levels between the selected lines. The biochemical findings differ from those reported in previous studies on locomotor activity in adult flies. The results are consistent with the view that similar behavioral phenotypes may arise from different genetic and biochemical bases.
Studies on the relationship between behavioral characters and their genetic control systems focus our attention on the nature of the physiological and biochemical pathways linking the genome with the behavioral phenotype. Biochemical studies related to behavioral differences have largely been carried out with rodents and there are correspondingly few such studies with insects. Tunnicliff et al. (1969) investigated strains of Drosophila melanogaster previously selected by Connolly (1966) for different levels of locomotor
1This work was carried out during tenure by Dr. D. F. Sewell of a postgraduate studentship from the Medical Research Council. 213 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
214
SEWELL, HUNT AND BURNET
activity, and found that noradrenaline levels were high in the active line and low in the inactive line. Dopamine levels were high in the inactive line and low in the active line, but there were no significant differences in serotonin between the lines. They suggested that the primary target of selection for changes in locomotor activity had been a change in the frequency of alleles controlling the relative balance between the biogenic amines. Additional support for this suggestion was given by Connolly et al. (1971) who reported an increase in dopamine levels associated with a decrease in locomotor activity in flies treated with 7-hydroxybutyrate. Sewell et al. (1975) studied locomotor and feeding activity in the larvae of D. melanogaster. The larvae spend much of their period of development feeding on the substrate, and the feeding action consists of successive extensions and retractions of the cephalopharyngeal sclerites accompanied by pumping action of the pharynx. Sewell et al. applied directional selection to yield populations with fast, or slow, feeding rate, and it was found that larvae of lines with slow feeding rate showed a correlated reduction in locomotor activity. Here we report the results of an investigation into the biogenic amine levels in these selected lines. Experimental material. Fertilized eggs were harvested from adult females of each of the selected populations over an oviposition period of 1 hr in order to ensure tight synchronization of development rates. The larvae were reared at 25°C on agar gel filled watch glasses liberally coated with fresh bakers' yeast. The rate of larval feeding activity was determined by transferring individual larvae with a sable hair brush to a drop of 2% fresh aqueous bakers' yeast suspension. Each larva was allowed a settling down period of 1.5 min and then a count was made of the number of cephalopharyngeal contractions per minute of continuous feeding. Detailed description of the feeding behavior is given by Sewell et al. (1975). Larvae aged 72 hr after hatching from the egg were rapidly washed, surface dried, and plunged into liquid nitrogen. A m i n e estimates. The concentrations of dopamine, noradrenaline, and serotonin were determined in individual 0.l-g samples of synchronized larvae by the fluorimetric method of Ansell and Beeson (1968) with the following modifications. The larvae were initially homogenized in 25 vol of acid-butanol, and the procedure for the formation of the trihydroxyindole derivative of noradrenaline followed that of Anton and Sayre (1962). The distribution of feeding rates for 72-hr-old larvae are illustrated in Fig. 1. The mean feeding rate measured as the number of cephalopharyngeal retractions per minute of continuous feeding was 204.4 + 0.97 (FA) and 207.3 + 1.2 (FB), respectively, in the fast feeding lines. The means for the slow lines were 94.8 + 0.88 (SA) and 96.8 + 0.70 (SB), respectively. Larvae of the unselected control population had a mean of 138.0 +0.71 cpm. The selected populations are discrete and nonoverlapping in their rates of feeding activity.
BIOGENIC AMINES IN DROSOPtllLA
215
3O
20
A
/
FB
10 0 6o
/0
,~0
,~;
.G
,~G
,,0
200
2;°
FEEDING RATE counts/min.
Fig. L Distributions of scores for samples of t00 larvae drawn from each of the experimental populations selected for slow (SA and SB), or fast (FA or FB), feeding activity and in the unselected intrageneration control population. Values on the ordinate denote numbers of individuals.
TABLE 1 Levels of Biogenic Amines in Lines Selected for Larval Feeding Activitya Line
Serotonin
Noradrenaline
Dopamine
Fast FA Control CA Slow SA
1.33 -* 0.07 1.44 +-0.11 1.44 _+0.06
0.068 -+0.002* 0.084 +-0.007 0.100 -+0.0t0
0.99 ! 0.10 1.01 -+0.09 t.77 +_0.18"*
Fast FB Control CB Slow SB
1.10 +-0.04 1.20 ~+0.06 1.17 -+0.05
0.094 +-0.008 0.092 -+0.0I 1 0.083 -+0.008
0.89 +~0.08* 1.34 ! 0.16 1.33 ~ 0.14
aMeasurements are expressed as the mean +_SE in mg/g body weight. * and ** denote differences from the control which are significant at the 5 and t% levels, respectively.
The levels of serotonin, dopamine, and noradrenaline in the selected and control populations are shown in Table 1 for pooled homogenates of whole 72-hr-old larvae. The two independent series of selected lines were processed separately, but each group of three lines within a series was sampled, and the material processed, simultaneously for each determination. There were eight to ten replications. The appropriate comparison therefore is always with the intrageneration unselected control group. In neither series was there a significant difference between the selected lines and their control in the level of serotonin. Noradrenaline levels were significantly lower than control ( P < 0.05) in the fast line FA, but not in the fast line FB. However the fast feeding larvae o f line FB show a significant reduction in dopamine compared with the control (P < 0.05). Larvae of the slow line SA have a significantly increased level of dopamine (P < 0.01), b u t no comparable increase was found in the slow line SB.
216
SEWELL, HUNT AND BURNET
Dopamine is an intermediate in the synthesis of N-acetyldopamine which is important in the process of hardening and darkening of the insect cuticle (Sekeris and Karlson, 1966). Considerable titers of this amine are present in the adult insect immediately following eclosion from the pupa which are probably associated with this function (Hodgetts and Konopka, 1973). As Pitman (1971) has pointed out, care must be taken, on tiffs account, before attributing a transmitter role to dopamine on purely biochemical grounds. Since multiple homogenates of whole larvae were used in the present study it is an obvious possibility that the dopamine titers for which data are shown in Table 1 include a component concerned with the cuficular pathway. It is worth emphasizing that the larval populations were tightly synchronized with respect to their physiological age, and that the point in development at which the homogenates were made (72 hr after hatching from the egg) is in the middle of the third larval instar, well before activation of the enzymes of the tyrosinase complex (Mitchell, 1966; Mitchell et al., 1967). Nevertheless, there is no way at present of excluding the possibility that the observed differences relate to a pool of dopamine concerned with N-acetyldopamine synthesis rather than with a possible role in neural transmission. The results for the larval populations reveal an association between reduced feeding activity and elevated dopamine concentration comparable to that found by Tunnicliff et al. (1969) and Connolly et al. (1971). There were significantly increased dopamine levels in the slow line SA but no comparable increase could be demonstrated in the larvae of the SB line. However, the larvae of the fast line FB showed significant reduction in dopamine. The fact that pairs of selected lines, which are similar in their phenotypic expression with respect to feeding rate, are not concordant with respect to changes in dopamine indicates that where significant changes in the level of this catecholamine have occurred they are unlikely to be a secondary consequence of the change in behavior. The results are consistent with the expectation that similar behavioral phenotypes may arise from different genetic and biochemical bases. REFERENCES Ansell, G. B., and Beeson, M. F. (1968). A rapid and sensitive procedure for the combined assay of noradrenaline, dopamine, and serotonin in a single brain sample. Anal, Biochem. 23, 196-206. Anton, A. H., and Sayre, D. F. (1962). A study of the factors affecting the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Ther. 138~ 360-375. Connolly, K. (1966). Locomotor activity in Drosophila. II. Selection for active and inactive strains. Anirn. Behav. 14, 444-449. Connolly, K., Tunnicliff, G., and Rick, J. T. (1971). The effects of ~/-hydroxybutyric acid on spontaneous locomotor activity and dopamine level in a selected strain of Drosophila melanogaster. Comp. Biochem. PhysioL 40B, 321-326.
BIOGENIC AMINES IN DROSOPHILA
217
Hodgetts, R. B., and Konopka, R, J. (1973). Tyrosine and catecholamine metabolism in wild-type Drosophila melanogaster and a mutant, ebony. J. Insect Physiol. 19, 1211-1220. Mitchell, H. K. (1966). Phenol oxidases and Drosophila development. J. Insect Physiol. 12, 755-765. Mitchell, H. K., Weber, U. M., and Schaar, G. (1967). Phenol oxidase characteristics in mutants of Drosophila melanogaster. Genetics 57, 357-368. Pitman, R. M. (1971). Transmitter substances in insects: A review. Comp. Gen. Pharmacol. 2, 347-371. Sewell, D., Burnet, B., and Connolly, K. (1975). Genetic analysis of larval feeding behaviour in Drosophila melanogaster. Genet. Res. Camb. 24, 163-173. Sekeris, C. E., and Karlson, P. (1966). Biosynthesis of catecholamines in insects. Pharmacol. Rev. 18, 89-94. Tunnicliff, G., Rick, J. T., and Cormolly, K. (1969). Locomotor activity in Drosophila - V. A comparative biochemical study of selectively bred populations. Comp. Bioehe~ PhysioL 29, 1239-1245.
BRIEF REPORT Biogenic Amines in Drosophila melanogaster Selected for Differences in Larval Feeding Behavior I
D. F. SEWELL
Department of Psychology, University of Hull D. M. HUNT
Department of Plant Biology and Microbiology, Queen Mary College, London and B. BURNET
Department of Genetics, University of SheffieM Levels of biogerdc amines were assayed in lines of Drosophila melanogaster selected for differences in their rate of larval feeding activity. Noradrenaline levels were low in one of the fast feeding lines (FA), but not in a second fast feeding line (FB). Dopamine levels were high in one of the slow feeding lines (SA) and low in the fast feeding line (FB). There were no significant differences in serotonin levels between the selected lines. The biochemical findings differ from those reported in previous studies on locomotor activity in adult flies. The results are consistent with the view that similar behavioral phenotypes may arise from different genetic and biochemical bases.
Studies on the relationship between behavioral characters and their genetic control systems focus our attention on the nature of the physiological and biochemical pathways linking the genome with the behavioral phenotype. Biochemical studies related to behavioral differences have largely been carried out with rodents and there are correspondingly few such studies with insects. Tunnicliff et al. (1969) investigated strains of Drosophila melanogaster previously selected by Connolly (1966) for different levels of locomotor
1This work was carried out during tenure by Dr. D. F. Sewell of a postgraduate studentship from the Medical Research Council. 213 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
214
SEWELL, HUNT AND BURNET
activity, and found that noradrenaline levels were high in the active line and low in the inactive line. Dopamine levels were high in the inactive line and low in the active line, but there were no significant differences in serotonin between the lines. They suggested that the primary target of selection for changes in locomotor activity had been a change in the frequency of alleles controlling the relative balance between the biogenic amines. Additional support for this suggestion was given by Connolly et al. (1971) who reported an increase in dopamine levels associated with a decrease in locomotor activity in flies treated with 7-hydroxybutyrate. Sewell et al. (1975) studied locomotor and feeding activity in the larvae of D. melanogaster. The larvae spend much of their period of development feeding on the substrate, and the feeding action consists of successive extensions and retractions of the cephalopharyngeal sclerites accompanied by pumping action of the pharynx. Sewell et al. applied directional selection to yield populations with fast, or slow, feeding rate, and it was found that larvae of lines with slow feeding rate showed a correlated reduction in locomotor activity. Here we report the results of an investigation into the biogenic amine levels in these selected lines. Experimental material. Fertilized eggs were harvested from adult females of each of the selected populations over an oviposition period of 1 hr in order to ensure tight synchronization of development rates. The larvae were reared at 25°C on agar gel filled watch glasses liberally coated with fresh bakers' yeast. The rate of larval feeding activity was determined by transferring individual larvae with a sable hair brush to a drop of 2% fresh aqueous bakers' yeast suspension. Each larva was allowed a settling down period of 1.5 min and then a count was made of the number of cephalopharyngeal contractions per minute of continuous feeding. Detailed description of the feeding behavior is given by Sewell et al. (1975). Larvae aged 72 hr after hatching from the egg were rapidly washed, surface dried, and plunged into liquid nitrogen. A m i n e estimates. The concentrations of dopamine, noradrenaline, and serotonin were determined in individual 0.l-g samples of synchronized larvae by the fluorimetric method of Ansell and Beeson (1968) with the following modifications. The larvae were initially homogenized in 25 vol of acid-butanol, and the procedure for the formation of the trihydroxyindole derivative of noradrenaline followed that of Anton and Sayre (1962). The distribution of feeding rates for 72-hr-old larvae are illustrated in Fig. 1. The mean feeding rate measured as the number of cephalopharyngeal retractions per minute of continuous feeding was 204.4 + 0.97 (FA) and 207.3 + 1.2 (FB), respectively, in the fast feeding lines. The means for the slow lines were 94.8 + 0.88 (SA) and 96.8 + 0.70 (SB), respectively. Larvae of the unselected control population had a mean of 138.0 +0.71 cpm. The selected populations are discrete and nonoverlapping in their rates of feeding activity.
BIOGENIC AMINES IN DROSOPtllLA
215
3O
20
A
/
FB
10 0 6o
/0
,~0
,~;
.G
,~G
,,0
200
2;°
FEEDING RATE counts/min.
Fig. L Distributions of scores for samples of t00 larvae drawn from each of the experimental populations selected for slow (SA and SB), or fast (FA or FB), feeding activity and in the unselected intrageneration control population. Values on the ordinate denote numbers of individuals.
TABLE 1 Levels of Biogenic Amines in Lines Selected for Larval Feeding Activitya Line
Serotonin
Noradrenaline
Dopamine
Fast FA Control CA Slow SA
1.33 -* 0.07 1.44 +-0.11 1.44 _+0.06
0.068 -+0.002* 0.084 +-0.007 0.100 -+0.0t0
0.99 ! 0.10 1.01 -+0.09 t.77 +_0.18"*
Fast FB Control CB Slow SB
1.10 +-0.04 1.20 ~+0.06 1.17 -+0.05
0.094 +-0.008 0.092 -+0.0I 1 0.083 -+0.008
0.89 +~0.08* 1.34 ! 0.16 1.33 ~ 0.14
aMeasurements are expressed as the mean +_SE in mg/g body weight. * and ** denote differences from the control which are significant at the 5 and t% levels, respectively.
The levels of serotonin, dopamine, and noradrenaline in the selected and control populations are shown in Table 1 for pooled homogenates of whole 72-hr-old larvae. The two independent series of selected lines were processed separately, but each group of three lines within a series was sampled, and the material processed, simultaneously for each determination. There were eight to ten replications. The appropriate comparison therefore is always with the intrageneration unselected control group. In neither series was there a significant difference between the selected lines and their control in the level of serotonin. Noradrenaline levels were significantly lower than control ( P < 0.05) in the fast line FA, but not in the fast line FB. However the fast feeding larvae o f line FB show a significant reduction in dopamine compared with the control (P < 0.05). Larvae of the slow line SA have a significantly increased level of dopamine (P < 0.01), b u t no comparable increase was found in the slow line SB.
216
SEWELL, HUNT AND BURNET
Dopamine is an intermediate in the synthesis of N-acetyldopamine which is important in the process of hardening and darkening of the insect cuticle (Sekeris and Karlson, 1966). Considerable titers of this amine are present in the adult insect immediately following eclosion from the pupa which are probably associated with this function (Hodgetts and Konopka, 1973). As Pitman (1971) has pointed out, care must be taken, on tiffs account, before attributing a transmitter role to dopamine on purely biochemical grounds. Since multiple homogenates of whole larvae were used in the present study it is an obvious possibility that the dopamine titers for which data are shown in Table 1 include a component concerned with the cuficular pathway. It is worth emphasizing that the larval populations were tightly synchronized with respect to their physiological age, and that the point in development at which the homogenates were made (72 hr after hatching from the egg) is in the middle of the third larval instar, well before activation of the enzymes of the tyrosinase complex (Mitchell, 1966; Mitchell et al., 1967). Nevertheless, there is no way at present of excluding the possibility that the observed differences relate to a pool of dopamine concerned with N-acetyldopamine synthesis rather than with a possible role in neural transmission. The results for the larval populations reveal an association between reduced feeding activity and elevated dopamine concentration comparable to that found by Tunnicliff et al. (1969) and Connolly et al. (1971). There were significantly increased dopamine levels in the slow line SA but no comparable increase could be demonstrated in the larvae of the SB line. However, the larvae of the fast line FB showed significant reduction in dopamine. The fact that pairs of selected lines, which are similar in their phenotypic expression with respect to feeding rate, are not concordant with respect to changes in dopamine indicates that where significant changes in the level of this catecholamine have occurred they are unlikely to be a secondary consequence of the change in behavior. The results are consistent with the expectation that similar behavioral phenotypes may arise from different genetic and biochemical bases. REFERENCES Ansell, G. B., and Beeson, M. F. (1968). A rapid and sensitive procedure for the combined assay of noradrenaline, dopamine, and serotonin in a single brain sample. Anal, Biochem. 23, 196-206. Anton, A. H., and Sayre, D. F. (1962). A study of the factors affecting the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Ther. 138~ 360-375. Connolly, K. (1966). Locomotor activity in Drosophila. II. Selection for active and inactive strains. Anirn. Behav. 14, 444-449. Connolly, K., Tunnicliff, G., and Rick, J. T. (1971). The effects of ~/-hydroxybutyric acid on spontaneous locomotor activity and dopamine level in a selected strain of Drosophila melanogaster. Comp. Biochem. PhysioL 40B, 321-326.
BIOGENIC AMINES IN DROSOPHILA
217
Hodgetts, R. B., and Konopka, R, J. (1973). Tyrosine and catecholamine metabolism in wild-type Drosophila melanogaster and a mutant, ebony. J. Insect Physiol. 19, 1211-1220. Mitchell, H. K. (1966). Phenol oxidases and Drosophila development. J. Insect Physiol. 12, 755-765. Mitchell, H. K., Weber, U. M., and Schaar, G. (1967). Phenol oxidase characteristics in mutants of Drosophila melanogaster. Genetics 57, 357-368. Pitman, R. M. (1971). Transmitter substances in insects: A review. Comp. Gen. Pharmacol. 2, 347-371. Sewell, D., Burnet, B., and Connolly, K. (1975). Genetic analysis of larval feeding behaviour in Drosophila melanogaster. Genet. Res. Camb. 24, 163-173. Sekeris, C. E., and Karlson, P. (1966). Biosynthesis of catecholamines in insects. Pharmacol. Rev. 18, 89-94. Tunnicliff, G., Rick, J. T., and Cormolly, K. (1969). Locomotor activity in Drosophila - V. A comparative biochemical study of selectively bred populations. Comp. Bioehe~ PhysioL 29, 1239-1245.