1230 E. Johnson et al. Journal of Insect Physiology 46 2000 1229–1236
Nichols, 1992. Hewes et al. 1998, tested eight of these peptides, synthesized from predicted sequences, on the
neuromuscular junction NMJ of Drosophila larval muscle. All but one were excitatory at the level of the
NMJ, and there was no evidence of synergy when the peptides were injected in combination. This led these
workers to propose a functional redundancy among the peptides encoded by this gene. They cautioned, however,
that for evolutionary conservation of these peptides to have occurred, differential effects in other tissue targets
is likely Hewes et al., 1998. We measured the response of the Drosophila pupal heart to these and other peptides
in the hope of discovering differential effects. Any dif- ferences between the response of the heart and that of
the NMJ, or differences in response among the peptides within the cardiac system, would reveal functional diver-
sity among the peptides, a critical step in a search for receptors and modes of action. Cardioinhibitory peptides
have also been isolated from Limulus polyphemus; these peptides affect the modulation of this animal’s neuro-
genic heart Gaus et al. 1993, 1994. We tested these substances as well for activity in the Drosophila heart.
2. Materials and methods
2.1. Stocks and rearing All flies tested were Canton-S and were raised on a
standard cornmeal-molasses-malt-agar medium. Propi- onic acid was added to control mold. Flies were cultured
in 250-ml bottles at 25 °
C, 12:12 LD. 2.2. Recording
Heartbeat was recorded at 25 °
C by placing an early pupa P1; Ashburner, 1989 on a glass slide set in the
light beam of an Olympus binocular compound micro- scope. At this early stage the pupa is nearly transparent,
easily passing a light beam. A drop of distilled water placed on the slide concentrated the light passing
through the animal. The heart was centered in the field of view. A phototransistor positioned at the center of
the exit pupil of one of the eyepieces served to detect movements of the organ through the pupa. Microscope
stage temperature was held constant by a Sensortek TS- 4 unit. The signal was pre-amplified with a 741C oper-
ational amplifier circuit, and then conditioned with a low-pass electronic filter World Precision Instruments,
LPF-30; 200 Hz cutoff and amplified by a Grass 79D polygraph. The data were recorded directly by a 486i
computer through a Metrabyte DAS8 analog to digital converter at 100 Hz.
2.3. Data analysis The data were analyzed with our own software. Rate
was estimated by Maximum Entropy Spectral Analysis MESA Ulrych and Bishop, 1975; Dowse and Ringo
1989, 1991. Autocorrelation analysis Chatfield, 1980; Dowse and Ringo, 1989 provided a test of significance
for the MESA peaks. Regularity of heartbeat was esti- mated from the autocorrelation function by determining
the numerical value of the second peak. As the envelope of the autocorrelation function decays as a direct func-
tion of the regularity of periodicity Chatfield, 1980, this provides a numerical measurement of the strength of the
signal see Johnson et al., 1998 for details. We term this correlation coefficient the “rhythmicity” of the signal.
Records with low rhythmicity scores had some or all of the following irregularities: intervals of arrhythmicity
i.e. widely varying intervals between beats, skipped beats, varying frequency or amplitude, and occasional
total cessation of cardiac activity. Means of rate and rhythmicity before and after treatment were compared
using t-tests.
2.4. Injection of peptides 2.4.1. Preparation of neuropeptides and injection
techniques All peptides were dissolved in Ca
2 +
-free Drosophila Ringer
solution Ashburner,
1989 because
Ca
2 +
increases heart rate. Injections were done dorsally in the caudal end of the animal near the heart, through needles
pulled from capillary glass on a Narishige PB-7 unit. The tips were adjusted to a 3-
µ m aperture with a Narishige
microforge. Needle placement was by micromanipulator. The substances were metered by a World Precision
Instruments Picopump expressing a 0.4-s pulse of N
2
. The volume dispensed at the instrument setting was esti-
mated to be 2 ±
1 nl by measuring the image on a video monitor which had been calibrated with a stage
micrometer; the droplet was assumed to be nearly spheri- cal. Once set, the injection volume did not vary percep-
tibly when multiple droplets were measured.
2.4.2. Substances injected, concentrations, and dilution factor
Substances injected are listed in Table 1 and II with their abbreviations. All peptides were injected at four
dosage concentrations: 1 mM, 100 µ
M, 10 µ
M, and 1 µ
M. An exception was DPKQDFMRFamide which, owing to its exceptional effectiveness, was tested at two
lower concentrations, 0.1 and 0.01 µ
M. Five animals were injected at each concentration, and each animal was
injected once. In Limulus, these substances had been tested at concentrations ranging from 0.1 nM to 1
µ M.
Our choice of higher dosage concentrations lies in a dif- ference in technique: In the Limulus study Gaus et al.,
1231 E. Johnson et al. Journal of Insect Physiology 46 2000 1229–1236
Table 1 Effects of native neuropeptides on Drosophila heart rate HR and regularity r
a
Peptide Rate before
Rate after DHR
r before r after
Dr Saline control
2.41 ±
0.1 2.4
± 0.1
21.0 0.55
0.52 ±
0.07 25.0
TDVDHVFLRF-NH
2
2.32 ±
0.1 1.64
± 0.04
229.2
†
0.68 ±
0.06 0.51
± 0.03
225.4 Dromyosuppresin
SAPQDFRS-NH
2
2.43 ±
0.2 2.48
± 0.06
+ 1.6
0.78 ±
0.09 0.58
± 0.2
225.1 MDSNFVIRF-NH
2
2.30 ±
0.1 2.4
± 0.09
22.7 0.69
± 0.9
0.56 ±
0.1 218.8
PDNFMRF-NH
2
2.65 ±
0.05 1.54
± 0.1
241.8
†
0.52 ±
0.2 0.44
± 0.1
216.0 SPKQDFMRF-NH
2
2.5 ±
0.06 2.41
± 0.1
23.6 0.50
± 0.1
0.46 ±
0.1 24.8
SDNFMRF-NH
2
2.54 ±
0.1 2.44
± 0.1
23.9 0.60
± 0.1
0.52 ±
0.1 212.5
TPAEDFMRF-NH
2
2.39 ±
0.1 2.22
± 0.1
27.0 0.54
± 0.09
0.47 ±
0.1 214.4
DPKQDFMRF-NH
2
2.61 ±
0.1 1.47
± 0.1
243.6
†
0.51 ±
0.08 0.33
± 0.1
235.0 DPKQDFMRF-OH
2.45 ±
0.05 2.37
± 0.05
+ 2.4
0.51 ±
0.09 0.48
± 0.1
26.6 SVQDNFMHF-NH
2
2.46 ±
0.08 2.37
± 0.05
23.6 0.62
± 0.1
0.60 ±
0.1 22.9
a
Five individuals were tested at every dosage. The results for the highest dosage are reported. Means before and after each injection were compared by t-test.
†
Indicates the change was significant at the P ,0.05 level.
1994, hearts were isolated and perfused with seawater containing the desired concentration. Based on our esti-
mates of hemolymph volume in Drosophila pupae and published values for a wide range of insects Jones,
1977, we estimate a total dilution factor of approxi- mately 200:1 for the injected agents.
2.5. Recording protocol Heartbeat was recorded for 30 s after allowing 120 s
for the animal to acclimate to 25 °
C. Within 3 min of this recording, the animal was injected. A second 30-s
recording was made 120 s after injection. This interval is sufficient to allow for even diffusion throughout the
animal Johnson et al., 1998, but we also did systematic test recordings at 0, 300, and 600 s post injection data
not shown, and the results were not significantly differ- ent from those taken from recordings done at 120 s.
These results also indicate that injection near the heart did not cause transient effects that were a result of an
initial pulse of concentrated peptide.
3. Results
