For the stressor ‘cold’ the flies were kept on 18°C for 48h on ATR.

stressor: cold (18°C for 48h)

POS_CTRL: control group

Gr28bd+TrpA1(cold): experimental group

For the stressor ‘crowding’ ca. 60-65 flies were kept in one vial for 48h on ATR.

The effect of crowding was created by the volume of air available to the flies in the vial. This was altered via change in the height of the cotton plug in the vial. The control group had the cotton plug at the usual height of ca. 60mm above the food in the vial and the experimental group only 20mm.

stressor: crowding (maintained in smaller volume for 48h on ATR)

POS_CTRL: control group

Gr28bd+TrpA1(Crow): experimental group

Giant fiber Gal4 driver (BDSC 79602), green channel shows giant fiber neurons, magenta channel shows background staining with nc82:

Giant fiber neurons on Codex BANC v626 dataset:

b1 mn driver (SS40980, also targets iii1 mn, iii3 mn):

Janelia image for SS40980:

b3 mn driver (SS48311, also targets hg1 MN, XBI002, WBI009):

Janelia image for SS48311:

b3 mn driver (SS98650):

Janelia image for SS98650:

The experimental group was kept in single vials on a ATR+Sucrose mixture for 48h.

stressor: social isolation

Pos_CTRL: control group

Gr28bd+TrpA1(Iso): experimental group

A: dicer2; SNPFR-RNAi × TH-Gal4
B: w1118 × TH-Gal4
C: w1118 × dicer2; SNPFR-RNAi

Larvae were placed in the center of an agar plate with plain agar on one side and salt-containing agar (1.5 M NaCl) on the other. After 3 minutes, larval distribution was quantified using a preference index under red light. All three groups showed significant salt avoidance and no significant differences were observed between the groups.