Nociceptive neurons protect Drosophila larvae from parasitoid wasps

Abstract

Background: Natural selection has resulted in a complex and fascinating repertoire of innate behaviors that are produced by insects. One puzzling example occurs in fruit fly larvae that have been subjected to a noxious mechanical or thermal sensory input. In response, the larvae "roll" with a motor pattern that is completely distinct from the style of locomotion that is used for foraging.

Results: We have precisely mapped the sensory neurons that are used by the Drosophila larvae to detect nociceptive stimuli. By using complementary optogenetic activation and targeted silencing of sensory neurons, we have demonstrated that a single class of neuron (class IV multidendritic neuron) is sufficient and necessary for triggering the unusual rolling behavior. In addition, we find that larvae have an innately encoded preference in the directionality of rolling. Surprisingly, the initial direction of rolling locomotion is toward the side of the body that has been stimulated. We propose that directional rolling might provide a selective advantage in escape from parasitoid wasps that are ubiquitously present in the natural environment of Drosophila. Consistent with this hypothesis, we have documented that larvae can escape the attack of Leptopilina boulardi parasitoid wasps by rolling, occasionally flipping the attacker onto its back.

Conclusions: The class IV multidendritic neurons of Drosophila larvae are nociceptive. The nociception behavior of Drosophila melanagaster larvae includes an innately encoded directional preference. Nociception behavior is elicited by the ecologically relevant sensory stimulus of parasitoid wasp attack.

Figure 1. GAL4 drivers which target distinct subsets of multidendritic neurons

(A-D) Confocal microscope images of third instar larval multidendritic neurons (dorsal cluster labeled with UAS-MCD8-GFP). (A₁-D₁) Immunostaining with the pan-neuronal marker anti-HRP-FITC (green). (A₂-D₂) Co-immunostaining with anti-GFP (magenta). (A₃-D₃) Merge. (A₄-D₄) Schematic diagrams of a dorsal cluster of MD neurons labeled by class and name for each GAL4 driver. (A) Expression pattern of md-GAL4 (Class I-IV). (B) Expression pattern of c161-GAL4 (Class I-II). (C) Expression pattern of 1003.3-GAL4 (Class II-III). (D) Expression pattern of ppk1.9-GAL4 (Class IV). Roman numerals represent multidendritic neuron class. Asterisk indicates the dmdI neuron. Scale bar is 10 μm.

Figure 2. Class IV multidendritic neurons are necessary for nocifensive behavior

(A-F) The distribution of latencies of thermal nocifensive behavior in third instar larvae that were lightly touched with a 47°C probe. (A) Nocifensive response of the UAS-TnTE/+ larvae without a GAL4 driver (n=112). (B) Blocking the synaptic output of all four classes of multidendritic neurons completely blocked thermal nocifensive responses (n=27). (C) Blocking the output of class I and II md neurons using the C161-GAL4 driver slightly increases the latency of thermal nociception behavior (n=195). (D) Blocking the output of class II and III md neurons using the 1003.3-GAL4 driver did not affect thermal nociception behavior (n=63). (E) Silencing of class IV multidendritic neurons dramatically impaired thermal nociception behavior (n=139). (F) Effects of blocking different subsets of md neurons upon the frequency of mechanical nociception behavior (UAS-TnTE/+ n=179, c161-GAL4 n=79, 1003.3-GAL4 n=61, ppk-GAL4 n=65). In all panels, error bars indicate standard error of mean.

Figure 3. Optogenetic activation of Class IV multidendritic neurons is sufficient to elicit the nocifensive response

(A_1–3) Expression of Channelrhodpsin2-YFP under control of ppk-GAL4. (A₁) pan-neuronal marker anti-HRP (green). (A₂) anti-GFP (magenta). (A₃) Merge of a₁ and a₂. (B) Expression levels of UAS-ChR2-YFP lines measured by pixel intensity of confocal images and normalized to staining intensity of line C. (C) Optogenetic activation of various subsets of multidendritic neurons triggers the “accordion” phenotype at high frequency. (D) Optogenetic activation of Class IV multidendritic neurons elicited nocifensive rolling behavior at high frequency. Rolling behavior with activation of classes I-IV was also elicited (15%). (E) ChR2-YFP expression levels are correlated with efficiency of nocifensive behavior. Sample sizes for c and d (Class I neuron driver 2–21-GAL4: atr+ n=54, atr- n=43), (Class I-IV neuron driver md-GAL4: atr+ n=103, atr- n=92 ), (Classes I & II driver c161-GAL4: atr+ n=117, atr- n=21), (Class II & III driver 1003.3-GAL4: atr+ n=46, atr- n= 52), (Class IV ppk-GAL4 atr+ n=181, atr- n=112). Sample sizes for e (Chop2 n=173, Line 1 n=20, Line 2 n=84, Line AB n=181, Line C n=80). UAS-Chop2 is an untagged Channelrhodopsin-2 line from the Fiala laboratory[34] with an insertion on the third chromosome. Error is standard error of the mean.

Figure 4. Optogenetic activation of Class IV md neurons triggers nocifensive response

(A-C) Still images of third instar larvae and optogenetically activated behaviors extracted from videotaped responses. (A,C) ppk-GAL4 UAS ChR2-YFP larvae that been fed atr- yeast (A) or atr+ yeast and illuminated with blue light. (B) c161-GAL4 UAS ChR2-YFP larva fed ATR+ and illuminated with blue light. (A) A larva expressing ChR2YFP in Class IV md neurons showed little response to the blue light when fed yeast paste lacking all-trans retinal. (B) A larva expressing ChR2YFP in Class I and II md neurons simultaneously contracts muscles of every segment to produce the accordion like behavior (straight white arrows denote reduction in length of larva from contractions). (C) A larva fed atr+ yeast and also expressing ChR2YFP in the Class IV md neurons produced a rolling motor response (curved arrow) that was indistinguishable from nocifensive rolling behavior. Note the net lateral direction of movement (straight white arrows). The time point of the sequence is shown in the left with illumination occurring at time zero.

Figure 5. Paradoxical directionality of rolling behavior

(A.) Directionality of larval rolling is biased. Top: Larvae had a strong tendency to roll towards the heat stimulus. When stimulated on the right (n=114), they predominantly rolled to the right. When stimulated on the left (n=102) they had a strong tendency to roll to the left. Roll direction was determined according to the first complete (ie. 360°) roll. Bottom: schematic representation of roll direction. (B) Hypothetical effects of rolling away from a wasp attack causes increased penetration. (C) Hypothetical effects of rolling towards the side of the body where attacked. In B and C the cross section of the larva is depicted as a circle and the ovipositor of the wasp attacking from above is depicted as a curved line.

Figure 6

Rolling behavior allows Drosophila larvae to escape from parasitoid wasp attack. Leptopilina boulardi female and an early third instar Drosophila melanogaster larva. (a) Wasp ovipositor penetrates larval cuticle and epidermis; (b) Nocifensive rolling behavior is triggered. The long threadlike ovipositor of the wasp is clearly visible (arrowhead). (c) Nocifensive rolling results in the ovipositor being wrapped around the larva. (d-f) Rolling of larva flips parasitoid wasp onto her side; (note that the image of the wasp in panel e is blurred due to its rapid movement during the exposure). (g) larva moves quickly away from parasitoid wasp; (h) larva is freed from ovipositor. Note: Images have been digitally adjusted to increase the contrast of the larva relative to the background (so that it could be easily seen). The original data can be seen in Supplementary Video 4.

Figure 7

Model for sensory control of alternate patterns of larval locomotion. The Class IV neurons are both necessary and sufficient to trigger rolling escape behavior. Other Classes of md-neurons modulate peristaltic locomotion through a distinct Central Pattern Generator. When inappropriately activated via Channelrhodopsin the accordion phenotype is manifested.