Dynamics of locomotor activity in the wild-type Canton-S males previously housed individually for 3 days from the emergence
Locomotor activity in the fruit fly depends on many environmental factors, including temperature, humidity, atmospheric pressure, nutritional conditions, etc. Not all factors may be satisfactorily controlled and equalized between different experiments. Figure 1 demonstrates the 5-h dynamics of locomotor activity in 4 different experiments with the wild-type Canton-S males. The common trend may be seen for majority of repetitions. It is a gradual decrease in AI from the beginning to the end of registration. However, in each of the successive 30-min intervals significant differences between some repetitions may be found. This supports the requirement of synchronizing the recording of locomotor activity in the control and experiment.
Modification of locomotor activity level in the wild-type Canton-S males after their housing in a unisex group for 3 days from the emergence (in comparison to control males housed individually)
Earlier we showed that previous housing Drosophila males in unisex groups reduced their level of locomotor activity in comparison to naïve males having no social experience (Panova et al. 2013). This behavioral modification was long-lasting: the reduced activity index retained up to 5 days after isolating males from a group. However, the extent of this reduction may vary from one experiment to another (Fig. 2). The naïve controls in Fig. 2 are the same samples of males kept individually as in Fig. 1. The variability in the extent of manifestation of this behavioral modification appears to be related mainly to the uncontrollable experimental conditions such as atmospheric pressure or flies’ state of health. Nevertheless, the significant reduction of locomotor activity is always noted in males previously housed in a group in comparison to naïve control. It is noteworthy that the maximal difference between control and experimental males may be observed either at the beginning, in the middle or at the end of 5-h registration period.
Modification of locomotor activity level in various mutants after housing males in a group for 3 days from the emergence (in comparison to control males housed individually)
Since the mechanisms of changes in locomotor activity resulted from housing in a group situation are unknown, we have tested the locomotor activity modifications in the mutants with impaired learning or memory, circadian rhythms, some biochemical pathways and sensory systems (Table 1).
Table 1
Activity index in the wild-type and mutant males previously housed either individually or in a group situation (means with standard errors calculated for the whole 5-h period, N = 20–60).
Mutant lines | Previous housing | P (two-sided randomization test) |
individually | in groups of 20 |
Wild-type Canton-S (four repetitions) |
1 | 69.7 ± 4.8 | 41.0 ± 9.7 | 0.0196 |
2 | 77.5 ± 1.9 | 45.5 ± 2.6 | 0.0000 |
3 | 61.4 ± 4.5 | 17.0 ± 2.2 | 0.0001 |
4 | 65.5 ± 1.2 | 43.8 ± 3.0 | 0.0000 |
Genes affecting learning or memory |
dunce1 | 41.8 ± 4.3 | 21.3 ± 5.1 | 0.0082 |
rutabaga1 | 34.4 ± 2.5 | 16.7 ± 1.6 | 0.0002 |
rutabaga2080 | 52.4 ± 2.7 | 47.1 ± 3.0 | 0.2053 |
amnesiacХ8 | 32.3 ± 3.4 | 24.9 ± 4.3 | 0.1997 |
nemyP153 | 73.4 ± 3.7 | 46.1 ± 8.7 | 0.0118 |
Ent2P124 | 50.0 ± 5.4 | 33.2 ± 5.0 | 0.0457–0.0502 |
Genes affecting biochemical pathways |
vermillion | 70.5 ± 2.5 | 23.7 ± 5.4 | 0.0000 |
cinnabar | 66.7 ± 4.2 | 7.7 ± 3.5 | 0.0000 |
cardinal | 84.5 ± 0.7 | 52.4 ± 3.0 | 0.0000 |
ebony | 50.0 ± 3.0 | 46.3 ± 2.7 | 0.3842 |
TβhnM18 | 76.6 ± 1.4 | 71.5 ± 1.8 | 0.0377 |
Genes affecting circadian rhythms |
period | 42.9 ± 6.3 | 14.3 ± 5.8 | 0.0049 |
ClockJrk | 60.3 ± 5.1 | 12.0 ± 5.8 | 0.0001 |
takeout | 53.4 ± 5.9 | 6.0 ± 4.1 | 0.0000 |
Genes affecting sensory systems |
white | 75.7 ± 4.5 | 51.8 ± 9.9 | 0.0438 |
nompA | 40.7 ± 3.8 | 17.1 ± 1.7 | 0.0000 |
Orco1 | 31.6 ± 3.8 | 33.4 ± 3.4 | 0.7283 |
Orco2 | 39.1 ± 1.9 | 36.0 ± 3.6 | 0.4589 |
It should be noted that a relatively low basic level of locomotor activity (e.g., in rutabaga, dunce or nompA) does not affect the ability of flies for its further reduction due to acquisition of social experience. In some cases we have observed the reduction of locomotor activity to the near zero level (e.g., in cinnabar or takeout).
Examination of learning/memory mutants showed the following (Table 1). The learning and short-term memory defects in the mutants dunce1, rutabaga1 (Davis 1996) and nemyP153 (Kamyshev et al. 2002b) did not prevent the reduction of locomotor activity resulted from housing males in a group. The fact points to the conclusion that this behavioral modification is not based on learning/memory mechanisms. At the same time, previous housing in a group did not lead to a severe reduction of locomotor activity in the two other learning mutants: amnesiacХ8 and Ent2P124 (Table 1). However, if to consider the time course of locomotor activity, one can note a slight but statistically significant reduction at certain time points in the mutant amnesiacХ8 and a well pronounced reduction, but only in the first half of 5-h period, in the mutant Ent2P124 (Fig. 3). Thus, the behavior of these two mutants does not contradict the above conclusion.
The kynurenine pathway is the main route of tryptophan metabolism both in the invertebtate and vertebrate animals. Kynurenines participate in regulation of functional state of the central nervous system, cognitive processes (learning and memory) and the mechanisms of stress development (Zhuravlev et al. 2020). All three mutations tested – vermilion (accumulation of tryptophan and lack of all kynurenines), cinnabar (accumulation of kynurenine and lack of 3-hydroxykynurenine and its derivatives) and cardinal (accumulation of 3-hydroxykynurenine and lack of xanthommatin) – did not affect the ability of males to reduce locomotor activity after their housing in a group situation (Table 1).
Quite a different result was obtained when testing the mutants with impaired synthesis of biogenic amines. Dopamine plays an important role in the mechanisms of learning and stress response (Neckameyer 1998), as well as in regulation of the basal level of locomotion (Ueno et al. 2012). In our study of the mutant ebony (a high level of dopamine), the control males housed individually demonstrated the intermediate level of locomotor activity among all mutants tested. In contrast to other biochemical mutants tested, the previous housing of the ebony males for 3 days in a group did not lead to a reduction of their locomotor activity (Table 1, Fig. 4). Deficient in octopamine mutant TβhnM18 (Monastirioti et al. 1996) demonstrate a high level of locomotor activity both in naïve males without social experience and in males previously housed in a group situation. However close values of their locomotor activity differ significantly (Table 1, Fig. 4). Thus, it seems likely that dopaminergic system plays a principal role in plastic changes of locomotor activity while the octopaminergic system may modulate them (Hoyer et al. 2008).
We have tested three circadian rhythm mutants: periodKG00546, ClockJrk (Dushay et al. 1990), takeoutc00632. No effect of these mutations on the ability of males to modify their locomotor activity under influence of social experience was found (Table 1).
Next, we have tested the dependence of males’ ability to reduce locomotor activity after housing in a group situation on their ability to perceive visual and olfactory stimuli (Table 1). Visual defect in the mutant white did not prevent the reduction of locomotor activity. Oppositely, loss of olfaction in the mutants Orco1 (olfactory receptor coreceptor) and Orco2 made impossible plastic changes resulted from previous social experience. The gene Orco encodes a co-receptor to a variety of olfactory receptors and is required for perception of almost all odors. It plays a role in olfactory recognition of potential sexual partners (Tan et al. 2013). Olfaction is necessary for ability of flies to form nonrandom social interaction networks (Schneider et al. 2012). Using environmental and genetic manipulations, Bentzur et al. (2021) showed the importance of visual and olfactory cues for social interactions in groups of flies. Thus, olfaction seems to be the leading sensory modality in modification of locomotor activity by social experience.
At last, we tried to test the importance of mechanoreception for the plastic changes considered. Loss of function of the gene nompA (no mechanoreceptor potential A) causes sensory defects including deafness, touch-insensitivity, and lack of coordination due to loss of proprioception (http://flybase.org/reports/FBgn0016047.html). The insertional mutant nompAMI07959 demonstrated well pronounced response to housing males in a group situation (Table 1) implying that mechanoreception does not participate in reducing the locomotor activity resulted from group housing.
Screening the collection of PdL insertions for inability of males to modify the level of locomotor activity after their previous housing in a group for 3 days (in comparison to the control perpetual individuals)
To broaden the list of candidate genes participating in the plastic changes of Drosophila behavior resulted from social experience we have tested a part of the previously created collection of PdL insertions. PdL (Ponce de Leon) is a kind of non-autonomous P element (Landis et al. 2001). The collection consists of lines, each carrying a random insertion of PdL transposon into second or third chromosome. Here we tested 103 lines, in which the insertion was localized and a presumptive gene affected was identified. As usually, the locomotor activity of males housed for 3 days from the moment of their emergence either individually or in groups of 20 males was compared. Each line was tested several times, and only those lines showing no effect of previous housing in a group in at least 3 successive independent repetitions were selected as carrying a mutation in the candidate gene. The levels of locomotor activity of the four lines selected are presented in Table 2 (joined repetitions), and characteristics of PdL insertions in these lines are shown in Table 3.
Table 2
Activity index in the selected PdL lines showing no effect of previous housing in a group situation (means with standard errors calculated for the whole 5-h period, N = 60–100).
PdL line | Previous housing | P (two-sided randomization test) |
individually | in groups of 20 |
309-s2 | 35.6 ± 3.7 | 34.0 ± 2.7 | 0.7468 |
2561 | 33.3 ± 2.6 | 32.3 ± 2.0 | 0.7832 |
3041-t3 | 47.3 ± 2.8 | 51.4 ± 1.0 | 0.1930 |
3356a | 28.3 ± 2.7 | 22.6 ± 2.5 | 0.1404 |
Table 3
Description of genes – candidates for regulation of locomotor activity after social experience (http://flybase.org/)
PdL line | Breakpoints | PdL insertion site relative to gene(s) | Molecular function Biological process | Human orthologs |
309-s2 | 2R:16856423 | 203 bp after 5-end of Dek (noncoding region) | Encodes DNA binding protein involved in DNA repair and heterochromatin integrity | DEK |
2561 | 3R:15973359 | 61 bp after 5-end of Hel89B (noncoding region) | Encodes a protein that belongs to the SNF2 family of chromatin regulators. | BTAF1 |
3041-t3 | 2R:24903903 | 32 bp after 5-end of RpL41 (noncoding region) 1st intron of NaCP60E | Ribosomal protein L41 predicted to be a structural constituent of ribosome. Na channel protein 60E (NaCP60E) encodes a voltage-gated calcium-selective cation channel that likely modulates the stability of neural circuits, particularly under environmental stresses. Plays a role in processing of olfactory information during the olfactory avoidance response. | - several human genes |
3356a | 3R:25050241 | 625 bp after 5-end of CG11791 (noncoding region) | Unknown | |
This study reveals the five candidate genes playing role in behavioral modifications resulted from social experience. In four cases the PdL transposon inserted into non-coding region at the beginning of the gene (genes Dek, Hel89B, RpL41, and CG11791), and in one case the insertion took place into the first intron of the NaCP60E gene.
The genes Dek and Hel89B encode proteins related to formation of the epigenetic code (histone modification, chromatin remodeling and genome stability). Thus, one may expect an essential role of the epigenetic factors in the behavioral changes resulted from social experience.
Because the product of gene NaCP60E seems to modulate stability of neural circuits (Table 3), its role in the social experience-mediated modification of neural circuit controlling locomotor behavior is very probable. Also, the data about its participation in processing of olfactory information (Table 3) are in accordance with our above conclusion that olfaction is the leading sensory modality in modification of locomotor activity by social experience.
Probable role of genes RpL41 and CG11791 is yet unclear.
A forward screen for «genes regulating sensitivity to isolation» was recently performed by Eddison (2021). In fact, he also compared behavior of males previously housed for 5 days either individually (isolation) or in groups of 20. Three P insertional mutants that had abnormal levels of aggression, locomotor activity and ethanol resistance when tested in isolated males were selected and the candidate genes identified. However, none of these mutants affects all three behaviors in the same direction, suggesting the relationship between each gene and these behaviors is complex. In one of the mutants, sex pistol, P-element was inserted in hts, an actin regulator of synaptic stability (Eddison 2021).