Chem. Senses 26: 223-230,
2001
© Oxford University Press 2001
SYMPOSIUM: AChemS XXII Symposium |
Foraging Behaviour in Drosophila Larvae: Mushroom Body Ablation
1 Department of Biological Sciences, University of Nevada, Las Vegas, NV 89154, USA and 2 Department of Zoology, University of Toronto, Mississauga, Ontario, Canada L5L 1C6 3 Present address: National Institute of Mental Health, Laboratory of Molecular Biology, Bethesda, MD 20892, USA
Correspondence should be sent to: Marla B. Sokolowski, Department of Zoology, University of Toronto, 3359 Mississauga Rd, Mississauga, Ontario, Canada L5L 1C6. e-mail: msokolow{at}credit.erin.utoronto.ca
Abstract
Drosophila larvae and adults exhibit a naturally occurring genetically based behavioural polymorphism in locomotor activity while foraging. Larvae of the rover morph exhibit longer foraging trails than sitters and forage between food patches, while sitters have shorter foraging trails and forage within patches. This behaviour is influenced by levels of cGMP-dependent protein kinase (PKG) encoded by the foraging (for) gene. Rover larvae have higher expression levels and higher PKG activities than do sitters. Here we discuss the importance of the for gene for studies of the mechanistic and evolutionary significance of individual differences in behaviour. We also show how structure–function analysis can be used to investigate a role for mushroom bodies in larval behaviour both in the presence and in the absence of food. Hydroxyurea fed to newly hatched larvae prevents the development of all post-embryonically derived mushroom body (MB) neuropil. This method was used to ablate MBs in rover and sitter genetic variants of foraging to test whether these structures mediate expression of the foraging behavioural polymorphism. We found that locomotor activity levels during foraging of both the rover and sitter larval morphs were not significantly influenced by MB ablation. Alternative hypotheses that may explain how variation in foraging behaviour is generated are discussed.
Introduction
The single gene mutant approach developed by Benzer (Benzer, 1967
) has been used by Drosophila behaviour geneticists to identify genes involved in a variety of phenotypes, including courtship (Hall, 1994), rhythms (Hall, 1998), learning (Dubnau and Tully 1998) and olfaction (described in companion papers). More recently natural variants have been employed to identify and isolate genes involved in normal individual differences in behaviour [reviewed by Sokolowski (Sokolowski, 1998)]. Here we discuss research on foraging, a naturally polymorphic gene, and present some new data on rover and sitter adult responses to yeast and on mushroom body ablation and larval behaviour.
Drosophila larval foraging behaviour
In Drosophila melanogaster, foraging behaviour occurs as a naturally occurring behavioural dimorphism. In 1980, M.B. Sokolowski first noticed that foraging third instar D. melanogaster larvae taken directly from the wild can be separated into two distinct groups based on the locomotory component of their foraging behaviour. She called larvae that travel a long distance while foraging in the presence of food (a yeast and water paste) ‘rovers’ and those that moved significantly less on the same substrate ‘sitters’ (Figure 1A). Rover/sitter differences are only expressed in the presence of food; their path lengths do not differ in non-nutritive (agar) environments (Pereira and Sokolowski, 1993). When more than one food patch is present in the environment rover larvae have a much higher probability of leaving the food. Rovers move from patch to patch whereas sitters move to the nearest patch and remain feeding on it (Figure 1B).
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The adult fly
These naturally derived strains of rover and sitter animals also differ in two food-based adult behaviours. The first is post-feeding locomotion. In the assay designed by Nagle and Bell (Nagle and Bell, 1987), a single fly is placed on a drop of sucrose solution in the centre of a circular arena and allowed to feed. The distance the fly travels from the drop of sugar during the first 30 s post-feeding is scored. In this assay rover adults travel in a relatively straight line away from the drop of sucrose after feeding, while sitter adults circle close to it (Pereira and Sokolowski, 1993). These different responses to the same assay conditions are interpreted as the adults making qualitatively different decisions about the quality of food (Tortorici and Bell, 1988; Bell, 1990; Pereira and Sokolowski, 1993). Remaining close to the drop of sucrose (called ‘intensive search behaviour’) indicates that the animal evaluates the food source highly, while moving in a straight line away from the drop (‘ranging’) is indicative of search behaviour for an alternative food source (Tortorici and Bell, 1988; Bell, 1990). The apparent difference in the quality rating given to the same sucrose solution by the rover and sitter naturally occurring foraging morphs may reflect a difference in food search strategies. If so, this difference in food quality assessment may provide insight into the environment in which each behavioural morph would thrive. It was predicted that rovers would be more successful than sitters in locating food in a patchy environment, while sitters would be better able to exploit a more homogeneous food environment than their rover counterparts by utilizing less energy in search behaviour. Indeed, Sokolowski et al. showed that both rover and sitter alleles are selected for under different environmental conditions (Sokolowski et al., 1997). When raised at high densities for many generations, the frequency of the rover allele (forR) increases, while under low-density conditions the sitter allele (fors) is preferentially selected. Examination of the foraging behaviour in D. melanogaster has identified one way in which natural selection can act on individual differences in behaviour.
The second adult behaviour studied shows that rovers and sitters differ in their ability to locate food (yeast) using both the T-maze (Tully and Quinn, 1985) and the olfactory trap assays (Woodard et al., 1989). The T-maze is constructed such that flies placed at a choice point are exposed to odorants from both arms of the assay. A given concentration of yeast is placed in an odour cup and air is pumped over the cup so that yeast odour fills one arm of the maze. Water is placed in a second odour cup so that humidified air fills the other arm of the maze. Flies are scored for their attraction to yeast by moving into the appropriate arm of the maze. The olfactory trap assay is similar to the T-maze in that flies must again move to the yeast source, this time placed in a small trap. One significant difference between the T-maze and the olfactory trap assay is that the T-maze test is performed over several minutes, while the olfactory trap assay is conducted over several days. Under both test conditions, sitter adults locate the yeast at a significantly higher rate than do rover adults [olfactory trap (Shaver et al., 1998); T-maze (Figure 2)]. These results can be interpreted in a number of ways. For example, (i) rover adults may have a reduced olfactory acuity to yeast compared with their sitter counterparts—a difference not observed in the larval life stage (Shaver et al., 1998); or (ii) sitter adults which exhibit higher levels of intensive search may be generally more ‘motivated’ to search, resulting in a more rapid movement towards yeast odours than their rover counterparts. Like larvae, rover and sitter adults do not differ in their locomotion in the absence of food (Pereira and Sokolowski, 1993). Neither the rover nor the sitter morph is a sick animal as determined from general fitness assays that include development rate, longevity, fecundity, muscle movement and pupation height (Sokolowski et al., 1984; Graf and Sokolowski, 1989; Sokolowski, 1980; Sokolowski and Hansell, 1992). The fact that rovers and sitters are equally healthy, combined with the realization that both morphs are found in appreciable quantities in nature (30% sitters, 70% rovers in Ontario orchards), allows each to be considered wild type.
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Genetic analysis of foraging behaviour
Extensive genetic analysis localized the single major gene responsible for foraging behaviour (foraging; for) to the left arm of chomosome 2 (Sokolowski, 1980; de Belle and Sokolowski, 1987, 1989; de Belle et al., 1989). In order to further map and ultimately clone for, de Belle et al. (de Belle et al., 1989, 1993) employed ethyl methanosulphonate (EMS) and gamma radiation to generate sitters derived from rover mutants. Each of the resulting mutant lines was also marked with recessive lethality. de Belle et al. (de Belle et al., 1989) developed this technique, termed lethal tagging, to enable genes responsible for continuously varying traits to be mapped. Continuously varying traits are characterized by a mean behavioural score and some measure of variance. Larval foraging behaviour is a good example of a continuously varying trait. While foraging third instar larvae may be genetically identical, these larvae will rarely move exactly the same distance in the assay. As a result, populations of animals of a given genotype must be tested and their means compared statistically. Continuously varying traits are also more susceptible to subtle changes in both the external and genetic environments than discontinuous traits (e.g. lethality), and therefore test conditions must remain rigorously constant between and within strains to be compared. Lethal tagging allowed de Belle et al. (de Belle et al., 1989, 1993) to indirectly localize the foraging gene by mapping a discontinuously varying trait (lethality) which was closely linked to the foraging (for) gene.
Cloning of the foraging gene
The paper by Osborne et al. (Osborne et al., 1997) was the first to identify a molecular basis for a naturally occurring behavioural polymorphism in any organism. dg2, one of the two cGMP-dependent protein kinases (PKG) found in D. melanogaster (Kalderon and Rubin, 1989), was found to be synonymous with for (Osborne et al., 1997). The importance of this work was highlighted in a review of this paper by Pennisi (Pennisi, 1997). The evidence that for is synonymous with dg2 was:
- The level of transcription of dg2 was reduced in sitter animals compared with their rover counterparts. This result was also reflected in the level of dg2 protein determined by Western blots, and the level of PKG kinase activity measured in the rover and sitter behavioural morphs.
- Disruption of dg2 by insertional mutagenesis reduced the level of dg2 expression and changed larval foraging behaviour from rover to sitter.
- Transgenic rescue, regarded by many as the most convincing evidence that a single gene is responsible for a specific phenotype, was used to demonstrate that an increase in the abundance of dg2 was responsible for rover behaviour. The increase in the level of PKG in sitter behaving lines, through the addition of a transgenic dg2–T2 transcript under the control of the heat shock promoter, changed larval foraging behaviour from sitter to rover. A correlated increase in PKG activity in the larval CNS was also found.
It was not surprising to find that PKG, a member of the cGMP signal transduction pathway, is involved in foraging behaviour. Signal transduction occurs when a cell translates an external cue, such as an odour, photon of light or hormone in the blood stream, to an internal signal that then elicits a cellular response. The case of learning and memory in Drosophila is arguably the most successful examination to date of the role that components of a signal transduction pathway play in the generation of behaviour. By examining a series of mutants in the cAMP signal transduction system for their effect on learning and memory {including cAMP phosphodiesterase [PDE; dunce; dnc (Nighorn et al., 1991)]; particulate adenylyl cyclase [AC; rutabaga; rut (Levin et al., 1992)]; the catalytic domain of cAMP-dependent protein kinase [PKA; DCO (Skoulakis et al., 1993)]; cAMP response element binding protein [CREB (Frank and Greenberg, 1994)]}, the cAMP signal transduction system has eloquently been implicated in the generation of learning and memory. Also, the elements of the cAMP signal transduction system identified as being important in learning and memory are found in enhanced levels in the adult mushroom bodies (Skoulakis et al., 1993), structures independently implicated in olfactory learning by structure–function analysis (Heisenberg et al., 1985).
In the past five years many aspects of the cGMP signal transduction pathway have come to light [reviewed recently (Wang and Robinson, 1997; Lohmann et al., 1997; Ruth, 1999)]. As can be seen in Figure 3, PKG is not the only compound to mediate a cellular response to an increased level of cGMP. Although the cGMP signal transduction pathway is involved in regulating systems throughout the body (such as vision, olfaction, taste), this does not a priori implicate PKG in these areas. Osborne et al. (Osborne et al., 1997) demonstrated a function for PKG in the modulation of foraging behaviour in D. melanogaster. [See Sokolowski and Riedl for a review of PKG and foraging behaviour (Sokolowski and Riedl, 1999).]
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There are two important questions to be asked. The first is how do rovers and sitters differ in their for DNA sequences? The higher levels of PKG expression in the rover compared with the sitter morph suggests that they likely differ in their regulatory regions. The T2 transcript of for/dg2 was used to successfully rescue sitter larval behaviour to that of rover (Osborne et al., 1997
). The sequences of the T2 transcript from natural rover and sitter morphs did not differ, suggesting that rovers and sitter do not differ in the exons encoding T2 (M.B. Sokolowski, unpublished data). Investigation of rover and sitter sequence variation in the regulatory regions of for is currently under investigation. The second question is one concerning RNA and protein expression. Specifically, when during development and where in the organisms must for be expressed to produce rover as compared with sitter behaviour? This question requires analyses of patterns of gene expression in combination with targeted gene expression. We are interested in determining whether transcript-specific functions can be applied to the natural rover and sitter behavioural variants and to the behavioural and developmental phenotypes exhibited by for mutants. This work is also underway. A complementary approach is structure–function analysis. This addresses the requirement of certain structures (e.g. mushroom bodies) for the performance of behaviour. The remainder of this paper addresses the question of whether the mushroom bodies are involved in the generation of foraging-specific locomotion. Structure–function mapping
The paired mushroom bodies (MBs) in the insect brain are most closely associated with sensory input (Strausfeld, 1976; Schürmann, 1987; Strausfeld et al., 1998). In Drosophila adults, MBs are third-order chemosensory neuropils with prominent links to the antennal lobes (ALs) via several antenno-cerebral fiber tracts (Stocker et al., 1990, 1994; Heisenberg, 1994). Although direct connections from the visual system to the MBs are described in several insect groups (Strausfeld et al., 1998), only a single neuron providing this link has been reported in Drosophila (Technau, 1983). Surgical interference, chemical ablation and genetic studies have suggested that MBs either influence or are required for a variety of different behavioural functions [reviewed by several groups (Erber et al., 1987; Heisenberg, 1998; de Belle and Kanzaki, 1999)]. In Drosophila, MBs are necessary for normal olfactory learning and memory (Heisenberg et al., 1985; de Belle and Heisenberg, 1994, 1996) [reviewed by Heisenberg (Heisenberg, 1989, 1994)]; they are implicated in aspects of courtship behaviour (Ferveur et al., 1995; O’Dell et al., 1995; Neckameyer, 1998; Joiner and Griffith, 2000); and they have modifying effects on both walking activity (Martin et al., 1998) (R. Strauss and J.S. de Belle, unpublished observations) and visual learning (Liu et al., 1999). Genes mediating odour learning and memory are also reported to have preferential expression in the MBs (Crittenden et al., 1998), and MB-targeted transgene expression studies have further linked signalling pathways underlying memory formation to these structures (Connolly et al., 1996; Zars et al., 2000).
Drosophila MBs consist mainly of 
2500 intrinsic neurons known as Kenyon cells (KCs) arranged in parallel arrays (Technau and Heisenberg, 1982) [reviewed by Heisenberg (Heisenberg, 1989, 1994)]. KCs are derived from four neuroblasts in each hemisphere (Ito and Hotta, 1992; Prokop and Technau, 1994) that begin proliferating in stage 13 embryos (Tettamanti et al., 1997) and continue uninterrupted until adult eclosion (Truman and Bate, 1988; Ito and Hotta, 1992). Concentric layers of KC arrays in the MB pedunculus project into different lobular structures that are added sequentially. First to appear is the 
lobe in the embryo, followed by the 
'/ß' lobes in the larva and finally the /ß lobes in the pupa (Yang et al., 1995; Ito et al., 1997; Armstrong et al., 1998; Lee et al., 1999). During metamorphosis, a large proportion of KC projections in the '/ß' lobes are retracted and then regrown, while neurogenesis contributes new KCs to the /ß lobes (Technau and Heisenberg, 1982; Armstrong et al., 1998; Lee et al., 1999). Although the meaning of this drastic reorganization of the CNS is unknown, it suggests that KCs perform functions specific to each stage of development, perhaps reflecting the contrasting sensory environments in which larvae and adults live.
Several genes are known to influence foraging behaviour in Drosophila (Sokolowski and Riedl, 1999). These include foraging [for (de Belle et al., 1989, 1993; Osborne et al., 1997; Sokolowski, 1998)], Chaser [Csr (Pereira et al., 1995)], no-bridge and ellipsoid body open [nob and ebo (Varnam et al., 1996)] and probably Shaker [Sh (Renger et al., 1999)]. The well-characterized for gene is naturally polymorphic (Sokolowski et al., 1997), with the rover allele dominant over the sitter allele [forR and fors (de Belle and Sokolowski, 1987; de Belle et al., 1989b, 1993)].
Although neural mechanisms underlying differences in foraging behaviour are poorly understood, they likely involve variation in either sensory perception and/or higher brain-determined ‘evaluation’ responses to the food environment and/or some distribution of these functions throughout the organism [i.e. sensory, central brain, motor, gut (Sokolowski and Riedl, 1999)]. Possible neuroanatomical foci for processes involved in foraging behaviour differences have been described previously. First, mutations in two genes (nob and ebo) that are required for proper development of the central complex (CC) were shown to suppress rover path length in forR larvae (Varnam et al., 1996). Secondly, a mutant for allele tagged with a P[Gal4]-enhancer trap insertion also identified elevated levels of gene activity in the MBs (Osborne et al., 1997) [J. Ewer, personal communication; Fly Trap (K. Kaiser), http://panic.molgen.gla.ac.uk/flytrap/html/enhancer/index.html].
Based on these observations and others suggesting a MB role in the regulation of motor behaviour (above), hydroxyurea (HU) ablation experiments were performed. HU ablation of MB neuroblasts in brains of newly hatched larvae prevents the birth of all KCs from this time onward (Ito and Hotta, 1992; Prokop and Technau, 1994). Only the embryonically derived neurons in the lobe persist through to later stages of development (Ito et al., 1997; Armstrong et al., 1998; Lee et al., 1999). An additional fifth neuroblast with a similar temporal pattern of proliferation gives rise to AL local and projection interneurons that are also ablated in HU-treated animals (de Belle and Heisenberg, 1994; Stocker et al., 1997). This convenient method of neuroanatomical interference is administered orally and can be targeted to defined windows of development. It has been especially useful for inferring structure–function relationships in the brains of flies (see above) and throughout the nervous systems of other insects (Sweeney et al., 2000).
We found that MB ablation with HU did not significantly affect general locomotor activity (Figure 4A) or locomotor activity levels expressed by rover and sitter larvae during foraging (Figure 4B). These data (i) show that HU does not have obvious general negative effects on larvae; and (ii) allow us to reject the hypothesis that MBs are involved in larval locomotion and foraging behaviours. However, it is important to note that ablation does not block proliferation of KCs projecting to the lobe born prior to larval hatching (Armstrong et al., 1998). Consequently, we cannot completely rule out a MB role in foraging behaviour, since our HU-ablated larvae would have had intact lobes. These structures are not detected in paraffin sections with fluorescence microscopy but can be observed in whole mounted brains with the aid of transgenic cell markers and a confocal microscope [compare figure 1 of de Belle and Heisenberg (de Belle and Heisenberg, 1994) with figure 3 of Armstrong et al. (Armstrong et al., 1998)]. It would be interesting to investigate whether variation in foraging behaviour is dependent on the MB lobe by testing transgenic larvae that have toxin gene expression driven by a -lobe-specific Gal4 enhancer [reviewed by Sweeney et al. (Sweeney et al., 2000)].
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n Among alternative regions of the brain that may be considered as potential mediators of foraging-related differences in behaviour, the CC and CC precursor cells are most notable. CC involvement in the regulation of adult motor behaviour is well documented (Strauss et al., 1992
; Strauss and Heisenberg, 1993). In addition, CC mutant-analyses suggest that the CC precursor also has a role in motor activity related to feeding (Varnam et al., 1996). Some mutants [central complex deranged (ccd), central body defect (cbd), central brain deranged (ceb) and central complex (cex)] had general defects in larval locomotion (on a non-nutritive agar surface). More interestingly, two of the mutants (ebo and nob) displayed foraging-specific reductions in locomotor activity (on a nutritive yeast surface). General locomotor activity of ebo and nob in the absence of food was normal. These CC mutations suppressed rover path length in forR larvae but only in the presence of food [nob; forR and ebo2; forR had sitter phenotypes (Varnam et al., 1996)]. Feeding-related motor behaviour in this previous study was not examined in adults because CC mutants have general walking behaviour deficits that would have confounded data interpretations (Strauss et al., 1992; Strauss and Heisenberg, 1993).
Despite our findings that MBs appear not to influence larval foraging, a parallel study of feeding-related behaviour in adults would be beneficial. Several recent reports describe MB influences on motor behaviour in Drosophila adults. For example, MB-less males actively court immature males longer (Neckemeyer, 1998); they sustain higher levels of walking activity over extended periods of time [13 h (Martin et al., 1998)]; when entrained by a 12:12 LD light regime they are subsequently more active under constant dark conditions; and they have slightly longer circadian rhythms than their unablated brothers (C. Helfrich-Förster and J.S. de Belle, unpublished results). In all of these examples there is no general loss or reduction of behaviour after MB ablation, as is observed with some of the CC mutants (Strauss and Heisenberg, 1993). On the contrary, activity increases in the absence of MB neuropil. Neuroanatomically, these observations are somewhat puzzling because Drosophila MBs do not appear to have intimate connections with motor centres or descending neurons in the brain (Ito et al., 1998). Nonetheless, if MBs do have an influence on feeding-related motor activity in adults but not in larvae, it would be reasonable to suspect /ß lobe KC fibers to be involved as they are born during the pupal stage of development (Armstrong et al., 1997; Lee et al., 1999). We anticipate that spatial and temporal control of transgene expression will be a critical tool for the assignment of foraging-specific (and other) functions to brain regions in Drosophila.
Acknowledgments
Thanks to C. and M. Riedl for help with the figures and to E. Benchimol for the unpublished data. Research was funded by a Research grant from the Medical Research Council (MRC) of Canada to M.B.S.
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Accepted October 31, 2000
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