Figure 1.
Illustration of fluorescence microscopy and electrical stimulation setups.
The filter and mirror set in green font is used to observe Fluo-8, Rhodamine 123, and DiBAC4(3). The filter and mirror set in red font is used to observe Cell Tracker and Rhodamine B. Inset, dorsal view of the beetle fixed upside down on dissection plate using clay, rubber bands (in red), and insect pins. Two PFA-coated thin silver wire electrodes (one working electrode and one counter electrode) were inserted into the ends of the muscle that are marked by 2 yellow dots. The insertion depth is approximately 2 mm, as measured from the outer cuticle. The red circle indicates the observation window that was used to observe the flexion muscle, which located inside the femur of the beetle leg.
Figure 2.
Difference in fluorescence intensity of insect flexion leg muscles of the control beetle and the beetle after oral dosing with chemical indicators.
(A) Fluo-8; (B) Rhodamine 123; (C) DiBAC4(3); (D) Rhodamine B; and (E) Cell Tracker. The Fluo-8, Rhodamine 123, and DiBAC4(3) dosed beetle leg was observed under 460–480 nm excitation light and fluorescence emitted was collected within 495–540 nm. The Rhodamine B and Cell Tracker dosed beetle leg was observed under 535–555 nm excitation light and fluorescence emitted was collected within 570–625 nm. Fluorescence intensity was measured at the 2 regions of interest (ROIs) shown in S1A Fig. The images obtained were digitized by ImageJ software, and the averaged intensity is shown in each bar graph. The graphs in the right column show the fluorescence intensities of each beetle leg dosed with different chemical indicators (center) compared with the control (left) beetle leg. The control beetles were fed with the home-made jelly (no chemical indicator added) for 2 days prior to observation. The error bars represent the standard deviation (S.D.) (N = 5 beetles, n = 30 beetle legs for control, DiBAC4(3), Rhodamine B, and Rhodamine 123; N = 9 beetles, n = 30 beetle legs for Fluo-8 and Cell Tracker). Each data set was compared with control leg data set by student’s t-test (Fluo-8, p = 1.42×10-4; Rhodamine 123, p = 4.65×10-5; DiBAC4(3), p = 6.26×10-9; Rhodamine B, p = 1.15×10-9 and Cell Tracker, p = 7.10×10-3). The color scale is given on the bottom left corner of the image. The increase in fluorescence intensity for the chemical indicator-dosed beetle compared with the control beetle indicates that the oral dosing method successfully administers and delivers various chemical indicators in order to label the beetle leg muscle.
Figure 3.
Ca2+ dynamics and muscle displacement of the beetle leg muscle under electrical stimulation with multiple pulse trains (100 Hz, 10% duty cycle, 2 V) for 3 s.
Pseudocolor time series images of beetle leg muscle dosed with (A) Fluo-8 (60 µM) and (C) Cell Tracker (60 µM) indicators. ROIs are indicated by yellow region (as also shown in S1B Fig.). The color scale is given on the right side of each image (A) or (C) respectively. Fluorescence intensity dynamics of (B) Fluo-8 and (D) Cell Tracker under electrical stimulation, digitized with ImageJ software from the ROI shown in (A) and (C) respectively. The stimulus timing is indicated by grey shading. The Fluo-8 pseudocolor images illustrate the fluorescence intensity dynamics that correspond to the Ca2+ dynamics inside the leg muscle: it increased at the start of electrical stimulation, was maintained during the application of the stimulus, and finally slowly decreased after the stimulus stopped. The Cell Tracker pseudocolor images display that the muscle displacement also causes intensity change during electrical stimulation, which slightly affects the Fluo-8 measurement.
Figure 4.
Effect of various electrical stimulation frequencies on Ca2+ dynamics and muscle displacement in beetle leg muscle.
Images of beetle leg muscle that was dosed with (A) Fluo-8 (60 µM) and (C) Cell Tracker (60 µM), with the yellow selection indicating the ROI that was used for analysis (as also shown in S1B Fig.). The color scale is given at the bottom left corner of the image. Representative time courses showing the fluorescence intensity dynamics of (B) Fluo-8, and (D) Cell Tracker under various electrical stimulations of multiple pulse trains (50 Hz, 10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz; 10% duty cycle; 2 V) observed from the ROI that are displayed in (A) and (C) respectively. All electrical stimulations were applied for 3 seconds periods with a 27 seconds resting period in between stimulations. The stimulus timing is indicated by grey shading. (E) Relative change in fluorescence intensity ((ΔF/F0)×100%) for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulation frequencies (50 Hz, 10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz; 10% duty cycle; 2 V). The small numbers next to each plot indicate the order of stimulation; i.e., first from 50 Hz followed by varying frequency pulses (10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz). The Fluo-8 intensity plot shows that Ca2+ dynamics are dependent on electrical stimulation frequency, whereas the Cell Tracker plot shows that the muscle displacement contributes a small amount.
Figure 5.
Relationship of Ca2+ dynamics with electrical stimulation frequency.
Relative changes in fluorescence intensity ((ΔF/F0)×100%) for leg muscle of (A) beetle orally dosed with Fluo-8 (blue) and Cell Tracker (red) and (B) control beetle measured with the filter setting used for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulations (1 Hz, 10 Hz, 50 Hz, and 100 Hz; 10% duty cycle; 2 V). Data were analyzed from the ROI adjacent to the stimulated site (S1B Fig.). The error bars represent the S.D. (N = 8 beetles, n = 24 beetle legs for (A); N = 2 beetles, n = 8 beetle legs for (B)). The small numbers next to each plot indicate the order of stimulation. Cell Tracker data set was compared with Fluo-8 data set at each stimulation frequency evaluated by student’s t-test both for dosed beetles in (A) (1st 50 Hz, p = 9.37×10-4; 10 Hz, p = 7.45×10-3; 1st 1 Hz, p = 2.30×10-1; 100 Hz, p = 4.17×10-4; 2nd 1 Hz, p = 4.49×10-2; and 2nd 50 Hz, p = 8.16×10-3) and for control beetles in (B) (1st 50 Hz, p = 4.10×10-1; 10 Hz, p = 9.30×10-1; 1st 1 Hz, p = 9.29×10-1; 100 Hz, p = 5.69×10-2; 2nd 1 Hz, p = 6.85×10-1; and 2nd 50 Hz, p = 2.67×10-2). The significant differences are displayed by an asterisk (p < 0.05). Fluo-8 intensity dynamics show that Ca2+ dynamics inside the muscle have a positive correlation with electrical stimulation frequency; i.e., higher stimulation frequency induces larger increase in [Ca2+]. On the other hand, Cell Tracker intensity dynamics show that the frequency-dependent intensity change due to muscle displacement is not apparent.