Nutritional Value-Dependent and Nutritional Value-Independent Effects on Drosophila melanogaster Larval Behavior

Gustatory stimuli allow an organism not only to orient in its environment toward energy-rich food sources to maintain nutrition but also to avoid unpleasant or even poisonous substrates. For both mammals and insects, sugars—perceived as "sweet”—potentially predict nutritional benefit. Interestingly, even Drosophila adult flies are attracted to most high-potency sweeteners preferred by humans. However, the gustatory information of a sugar may be misleading as some sugars, although perceived as "sweet,” cannot be metabolized. Accordingly, in adult Drosophila, a postingestive system that additionally evaluates the nutritional benefit of an ingested sugar has been shown to exist. By using a set of seven different sugars, which either offer (fructose, sucrose, glucose, maltodextrin, and sorbitol) or lack (xylose and arabinose) nutritional benefit, we show that Drosophila, at the larval stage, can perceive and evaluate sugars based on both nutrition-dependent and -independent qualities. In detail, we find that larval survival and feeding mainly depend on the nutritional value of a particular sugar. In contrast, larval choice behavior and learning are regulated in a more complex way by nutrition value-dependent and nutrition value-independent information. The simplicity of the larval neuronal circuits and their accessibility to genetic manipulation may ultimately allow one to identify the neuronal and molecular basis of the larval sugar perception systems described here behaviorall

The serotonergic central nervous system of the Drosophila larva: anatomy and behavioral function.

The Drosophila larva has turned into a particularly simple model system for studying the neuronal basis of innate behaviors and higher brain functions. Neuronal networks involved in olfaction, gustation, vision and learning and memory have been described during the last decade, often up to the single-cell level. Thus, most of these sensory networks are substantially defined, from the sensory level up to third-order neurons. This is especially true for the olfactory system of the larva. Given the wealth of genetic tools in Drosophila it is now possible to address the question how modulatory systems interfere with sensory systems and affect learning and memory. Here we focus on the serotonergic system that was shown to be involved in mammalian and insect sensory perception as well as learning and memory. Larval studies suggested that the serotonergic system is involved in the modulation of olfaction, feeding, vision and heart rate regulation. In a dual anatomical and behavioral approach we describe the basic anatomy of the larval serotonergic system, down to the single-cell level. In parallel, by expressing apoptosis-inducing genes during embryonic and larval development, we ablate most of the serotonergic neurons within the larval central nervous system. When testing these animals for naïve odor, sugar, salt and light perception, no profound phenotype was detectable; even appetitive and aversive learning was normal. Our results provide the first comprehensive description of the neuronal network of the larval serotonergic system. Moreover, they suggest that serotonin per se is not necessary for any of the behaviors tested. However, our data do not exclude that this system may modulate or fine-tune a wide set of behaviors, similar to its reported function in other insect species or in mammals. Based on our observations and the availability of a wide variety of genetic tools, this issue can now be addressed

Nutritional value–Dependent and nutritional value–Independent effects on Drosophila melanogaster larval behavior

Gustatory stimuli allow an organism not only to orient in its environment toward energy-rich food sources to maintain nutrition but also to avoid unpleasant or even poisonous substrates. For both mammals and insects, sugars—perceived as “sweet”—potentially predict nutritional benefit. Interestingly, even Drosophila adult flies are attracted to most high-potency sweeteners preferred by humans. However, the gustatory information of a sugar may be misleading as some sugars, although perceived as “sweet,” cannot be metabolized. Accordingly, in adult Drosophila, a postingestive system that additionally evaluates the nutritional benefit of an ingested sugar has been shown to exist. By using a set of seven different sugars, which either offer (fructose, sucrose, glucose, maltodextrin, and sorbitol) or lack (xylose and arabinose) nutritional benefit, we show that Drosophila, at the larval stage, can perceive and evaluate sugars based on both nutrition-dependent and -independent qualities. In detail, we find that larval survival and feeding mainly depend on the nutritional value of a particular sugar. In contrast, larval choice behavior and learning are regulated in a more complex way by nutrition value–dependent and nutrition value–independent information. The simplicity of the larval neuronal circuits and their accessibility to genetic manipulation may ultimately allow one to identify the neuronal and molecular basis of the larval sugar perception systems described here behaviorally

Serotonergic Neurons of the CNS are not Necessary for Olfactory Chemotaxis towards Amylacetate and Benzaldehyde.

<p>Third instar larvae with almost completely ablated serotonergic neurons were tested for naïve amylacetate (AM) (A, B) and benzaldehyde (BA) (C, D) preferences. TRH-GAL4/UAS-<i>hid,rpr</i> larvae showed preference for AM (p<0.01 compared to zero) (A) and for BA (p<0.01 compared to zero) (C). Compared to the controls UAS-<i>hid,rpr</i>/+ and TRH-GAL4/+, TRH-GAL4/UAS-<i>hid,rpr</i> did not perform significantly different either in AM or in BA preference tests (p>0.05). Similar results were found by testing TPH-GAL4/UAS-<i>hid,rpr</i> larvae. They preferred AM (p<0.01) (B) as well as BA (p<0.001) (D) and showed in both assays no significant difference to any control line (p>0.05). Under each boxplot of the figure for each genotype the sample size is shown; n = 15−20. Asterisks above each boxplot indicate, if the data is significantly different from zero. *<0.05; **<0.01; ***<0.001.</p

Expression Pattern of the Driver Line TRH-GAL4 in the Larval CNS.

<p>Triple staining of TRH-GAL4/UAS-mCD8::GFP third instar larvae in the first column shows cell membrane-bound CD8 labeling (green) combined with 5HT-immunoactivity (red) and anti-FasII/anti-ChAT staining for visualizing the neuropil (blue). The second (CD8), third (5HT) and fourth columns illustrate the three channels separately. The first row (A–A’’’) shows the whole CNS. The other rows represent higher magnifications of the brain in frontal view (B–B’’: posterior; C–C’’: anterior) and the ventral nerve cord (VNC) (D–D’’). A high co-localization of CD8- and 5HT-positive cells is found in the posterior hemisphere clusters SP1, SP2 and LP1 (B–B’’) as well as in the anterior clusters IP and SE0-3 (C–C’’). Nearly all cells of the VNC clusters T1-3 and A1–A8/A9 (D–D’’) show anti-CD8 and anti-5HT double staining. In addition some non-serotonergic CD8-expressing cells were detected (asterisks). Scale bars: 50 µm.</p

Morphology of the SP2 Cells.

<p>SP2-1 and SP2-2 type 5HT cells shown in single-cell flp-out clones via anti-CD8 (green), anti-5HT (red) and anti-FasII/anti-ChAT (blue) staining (A and E). The three channels are presented individually in panels B–D and G–H. In A and B three cells are labeled by the flp-out technique. Besides the SP2-1 cell (arrow), weak expression was detectable in an additional cell body (arrowhead) and a third cell of the LP cluster (asterisk). The SP2-2 cell was only weakly labeled and therefore likely misses a comprehensive visualization of its entire morphology. Scale bars: 25 µm.</p

Anatomy of the Serotonergic System in the Larval CNS Based on anti-5HT Staining.

<p>5HT positive cells (green) of Canton-S wild type larvae are shown in combination with anti-FasciclinII (FasII)/anti-Cholineacetyltransferase (ChAT) neuropil markers (magenta) (A and D–G). (A) The CNS of the third instar larva comprises 19 different 5HT-positive bisymmetrical clusters of one to three cells each. (B–G) In the brain hemispheres, five serotonergic clusters, SP1, SP2, LP1, SE0 and IP, were detected (in B and C only the anti-5HT channel is shown). (D) 5HT cells innervate the antennal lobe (AL; right arrow) and the suboesophageal ganglion (SOG; left arrowhead). (E) The mushroom body lobes (MB; arrow) and the (E) MB calyx (arrow) show only very week – if any - innervation. (F) By contrast, the larval optic neuropil (LON; arrow) is innervated by serotonergic arborizations. (B) and (C) show a frontal view of the anterior or posterior half of the brain, respectively. In (D–G) lateral is always to the right and medial to the left. Scale bars: A–C: 50 µm; D–G: 25 µm.</p

Morphology of the IP Cells.

<p>IP1-1, IP1-2 and IP1-3 type 5HT cells shown in single-cell or two-cell flp-out clones via anti-CD8 (green), anti-5HT (red) and anti-FasII/anti- ChAT (blue) staining (A, E and I). The three channels are presented individually in panels B–D, G–H and J–L. The IP1-1 cell (B, arrow) is visualized in a double flp-out clone that shows an additional weakly labeled cell body in the right hemisphere (arrowhead). The IP1-2 cell (F) is also visualized in a double flp-out clone together with the SP1-1 cell (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047518#pone-0047518-g006" target="_blank">Figure 6</a>). The arrow marks the cell body of the IP1-2 cell that innervates the ipsi- and contralateral hemispheres by crossing the midline more dorsal (arrow) compared to the SP1-1 cell that crosses the midline next to the pharynx. The expression in the SOG belongs to third cell of a different type that does not innervate the brain hemispheres. Scale bars 25 µm.</p

The Serotonergic Neurons of the CNS are not Necessary for Appetitive and Aversive Olfactory Learning.

<p>For testing appetitive olfactory learning, we utilized a two-group, reciprocal training design consisting of two half trials that give rise to a final performance index. Third instar larvae lacking serotonergic neurons preferred an odor that was paired with 2-M fructose (A, B). Using a single odor, non-reciprocal standard assay for aversive odor-shock learning third instar larvae lacking serotonergic neurons avoided the odor paired with pulses of electric shock (C, D). In both learning experiments, TRH-GAL4/UAS-<i>hid,rpr</i> larvae achieved relatively high performance scores (A) (p<0.01) (C) (p<0.001). Similar results were obtained for TPH-GAL4/UAS-<i>hid,rpr</i> larvae, which showed significant sugar learning (p<0.01) and electric shock learning (p<0.01). In none of the learning assays significant differences between experimental and control larvae were found. Under each boxplot of the figure for each genotype the sample size is shown; n = 10−16. Asterisks above each boxplot indicate, if the data is significantly different from zero. **<0.01; ***<0.001.</p