Aggression and social experience: genetic analysis of visual circuit activity in the control of aggressiveness in Drosophila
© Ramin et al.; licensee BioMed Central Ltd. 2014
Received: 12 June 2014
Accepted: 24 July 2014
Published: 13 August 2014
Animal aggressiveness is controlled by genetic and environmental factors. Among environmental factors, social experience plays an important role in modulating aggression in vertebrates and invertebrates. In Drosophila, pheromonal activation of olfactory neurons contributes to social suppression of aggression. While it was reported that impairment in vision decreases the level of aggression in Drosophila, it remains unknown if visual perception also contributes to the modulation of aggression by social experience.
In this study, we investigate the role of visual perception in the control of aggression in Drosophila. We took several genetic approaches to examine the effects of blocking visual circuit activity on fly aggressive behaviors. In wild type, group housing greatly suppresses aggressiveness. Loss of vision by mutating the ninaB gene does not affect social suppression of fly aggression. Similar suppression of aggressiveness by group housing is observed in fly mutants carrying a mutation in the eya gene leading to complete loss of eye. Chronic visual loss does not affect the level of aggressiveness of single-housed flies that lack social experience prior to behavioral tests. When visual circuit activity is acutely blocked during behavioral test, however, single-housed flies display higher levels of aggressiveness than that of control flies.
Visual perception does not play a major role in social suppression of aggression in Drosophila. For single-housed individuals lacking social experience prior to behavioral tests, visual perception decreases the level of aggressiveness.
Aggression is an innate behavior that allows animals to compete for limited resources, such as food, mating partners and habitats. The level of aggressiveness is influenced by both genetic and environmental factors . Accumulated evidence supports that social experience is one of the most important environmental factors that affect aggression in humans , rats – and Drosophila.
Recent studies have shed light on molecular mechanisms underlying the control of aggression by social experience. For instance, Cyp6a20, a cytochrome P450, is identified as a common genetic target for the control of aggressiveness by social experience in Drosophila. It has also been reported that chronic activation of Or65a olfactory neurons by the volatile pheromone 11-cis-vaccenyl acetate (cVA) contributes to social suppression of aggressiveness in Drosophila,. However, it remains unknown if other sensory stimuli such as vision, also contributes to social suppression of aggression in Drosophila.
A previous study reports that mutations in the white (w) gene that regulates eye pigmentation, greatly decrease aggressiveness of single-housed flies, suggesting that vision is required for normal aggression . To determine if visual perception contributes to social suppression of aggressiveness, we investigated if the blockade of visual circuit activity affects social suppression of aggression. We also examined the effects of visual impairment on aggressiveness of single-housed flies that lack social experience prior to behavioral tests.
Loss of vision in ninaB mutants does not prevent social suppression of aggression
We then performed experiments to examine the level of aggressiveness in flies with or without social experience. Wild-type flies reared in isolation (single housing) displays a much higher level of aggressiveness compared to flies reared in group (group housing) (Figure 1C), indicating that social experience prior to aggression assays suppresses the level of aggressiveness. Similar to that of wild-type flies, group housing greatly decreased the level of aggressiveness of ninaB1 mutants (Figure 1C). This result suggests that visual perception does not contribute significantly to social suppression of fly aggressiveness.
Complete loss of eye does not prevent social suppression of aggression
Chronic visual loss does not affect aggressiveness of single-housed flies lacking social experience prior to behavioral assays
When we examined the effects of chronic visual loss on social suppression of aggression, we found that single-housed flies in which vision is impaired still showed high levels of aggressiveness (Figures 1 and 2). Such results are in marked contrast to a previous report that suggests that visual impairment greatly decreases aggressiveness of single-housed flies, based on analysis of white-eyed flies carrying mutations in the w gene . To further test the potential role of visual perception in regulating aggressiveness of single-housed flies, we performed more detailed analysis of flies with chronic visual loss.
Acute blockade of visual circuit activity increases aggressiveness of single-housed flies
We then examined if temporal blockade of visual circuit activity during the period of aggression assays affects aggressiveness of flies that were single-housed prior to behavioral assay. To test this, synaptic transmission from photoreceptor cells was temporally blocked by eye-specific expression of a temperature-sensitive form of shibire (shits) that encodes the fly homolog of dynamin. This allows the blockade of synaptic transmission in photoreceptor cells at restrictive temperature ,.
Social suppression of aggression is observed in both vertebrates and invertebrates. While pheromonal activation of olfactory neurons has been implicated in this process in Drosophila, it remains unclear if other sensory cues also contribute to the modulation of aggressiveness by social experience. In this study, we investigated the effects of manipulating visual circuit activity in the control of fly aggression. We showed that blockade of visual circuit activity does not prevent social suppression of aggression. While chronic blockade of visual circuit activity does not affect aggressiveness of single-housed flies that lack social experience prior to behavioral tests, acute blockade of visual circuit activity increases the level of aggressiveness of single-housed flies.
Our results indicate that visual perception is not a major factor that allows male flies to recognize and interact with each other for suppressing aggressiveness by social experience. Whereas pheromonal activation of certain neurons in the olfactory system has been shown to contribute significantly to social suppression of aggression ,. Recent studies showed that the gustatory system also plays a role in modulating fly aggression –. Future studies are required to determine if gustatory cues contribute to social suppression of fly aggressiveness.
Our result showing that acute blockade of visual circuit activity increases the level of aggressiveness of single-housed flies is surprising. Previous work by Heisenberg and colleagues showed that mutants defective in the w gene display much lower levels of aggressiveness . Since the w gene mediates eye pigmentation, this result suggests that visual perception promotes aggressiveness. However, since removing w gene in the brain also causes a decrease in aggression , together with that white-eyed cricket mutants display normal levels of aggressiveness , we speculate that the decrease in the level of aggressiveness of white-eyed flies may not be caused by vision impairment.
While acute blockade of visual circuit activity increases the level of aggressiveness of isolated flies, chronic blockade of visual circuit activity does not affect fly aggressiveness. One possible explanation is that chronic blockade of visual circuit activity increases the sensitivity of fly response to other sensory cues, which may compensate for loss of visual perception in decreasing aggressiveness. Future studies are required to address these possibilities.
Visual circuit activity does not contribute significantly to social suppression of aggression in Drosophila. For individuals reared in isolation and thus lack social experience prior to behavioral tests, however, visual perception helps decrease the level of aggressiveness.
Materials and methods
Stocks and rearing condition
ninaB1 and eya2 mutants were obtained from Bloomington stock center. ninaB rescue experiments were performed by generating GMR-GAL4/+; ninaB1, UAS-ninaB/ninaB1, UAS-ninaB flies. UAS-Shits were provided by Dr. Greg Suh (NYU). GMR-GAL4;UAS-Shits flies were generated by crossing male UAS-Shits with female GMR-GAL4 flies. Canton-S flies were used as wild-type controls. Flies were reared at 25°C with 50-60% humidity and 12 hour light–dark cycle. Newly emerged males from pupal cases were single-housed in a 2 ml microfuge tube containing 1 ml of fly food for 6 days prior to aggression and locomotion assays. For experiments testing social influence of fly aggressiveness, flies were grouped in vials (10 flies per vial) and reared for 6 days prior to aggression assay. For vision tests, flies were group housed (7–12 flies) per vial and reared for 5–7 days prior to vision tests.
To examine fly vision, we used standard vials with 1.2 cm radius and 9.5 cm height. One vial was completely covered with dark duct tape except for the tip where flies were aspirated, and was indicated as dark zone. Another uncovered transparent vial was indicated as light zone. The two vials were attached together, separated by a paper cardboard, and horizontally placed under a light source (Figure 1A). For each experiment, ~7-12 flies were gently aspirated into either dark or light zone. Flies were allowed to get accustomed to new environment for 5 min. The cardboard was then removed gently in a way that did not agitate the flies. Flies were let freely move between light and dark zone for 10 min. The number of flies in light zone and dark zone was counted. Vision index is defined as: (number of light-zone flies – number of dark-zone flies)/total number of flies.
Aggression assay was performed by placing a pair of male flies in a circular fighting chamber (7 mm radius and 3.5 mm height), which has a central pad (4 mm radius) covered with food, and outer space filled with agar to reduce the dehydration of food. Behavioral tests were carried out at 22°C. For experiments involving acute blockade of visual circuit activity, aggression tests were performed at 22°C (permissive temperature) or 32°C (restrictive temperature). Two male flies of the same genotype were gently aspirated to the fighting chamber. After 5 minutes for flies to get accustomed to the environment, their behaviors were recorded with a high definition (HD) camera under fluorescent lamp for 10 minutes. The total number of aggressive events (i.e. lunges, wing threats, tussles, boxing, and holding) per 10-min period was used to indicate the level of aggressiveness.
Movement of two flies in a small round chamber was videotaped and analyzed by CADABRA software . Two flies were gently aspirated into a chamber similar to the fighting chamber used for aggression assay. Movement of flies was recorded for 10 min.
Data were expressed as mean ± SEM and processed by commercially available GraphPad Prism® 5.0. Mann Whitney test, or Kruskal-Walis ANOVA test followed by Dunn’s multiple comparison test were used in statistical analysis. p-value less than 0.05 (p < 0.05) is considered as significant.
We thank members of the Rao laboratory for comments and discussions; the Bloomington Stock Center for mutant lines; Dr. Greg Suh for UAS-shits. This work was supported by an operating grant (Rgpin 341431–10) from Natural Science and Engineer Research Council of Canada, and a China-Canada Joint Health Research Initiative Grant (FRN109611) awarded to Yong Rao.
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