Methods
Fly stocks and
culture
tßhnM18
#(Monastirioti et al., 1996#; FBal0061578), oamb #(Han et al., 1998#;
OctαR, oamb286 FBti0038368, oamb584 FBti0038361), honoka #(Kutsukake
et al., 2000#; Oct-TyrR, FBal0104701), hsp-tßh #(Schwaerzel et al.,
2003#; FBal0152162) and w+;;UAS-tßh #(Monastirioti, 2003#;
FBti0038601) were obtained from Henrike Scholz, Cologne; Hiromu
Tanimoto, Martinsried; Andreas Thum, Konstanz; and Amita Seghal,
Chevy Chase. Mutants that deleted large stretches of genomic DNA
coding for the Octß2R (CG6989; FBgn0038063) were generated by
remobilization of FRT-containing P-elements, i.e. f05679 and f07155
excised 17,539 bp giving rise to deletion mutant Octß2R∆3.22;
f00021and f01537 excised 33,489 bp resulting in Octß2R∆4.3.
Deletions were confirmed by genomic PCR. TyrRf05682 (CG7431f05682,
FBal0184987), TyrRII∆29 (CG16766, FBgn0038541) and TyrRII-TyrR∆124
were kindly provided prior to publication by Edward Blumenthal,
Milwaukee (Zhang and Blumenthal, in preparation). Receptor mutants
and their respective control lines were outcrossed for at least six
generations into CS background.
Flies were kept on
standard cornmeal/molasses-food in a 12/12 h light/dark cycle
(light on at 8:00 hrs) at 60% relative humidity and 25°C except
for hsp-tßh which were raised at 18°C without humidity control and
except for flies used in electrophysiological experiments (see
Electrophysiological recording).
Starvation procedure
Newly hatched to one
day old flies were collected and transferred to fresh food vials. The
following day, between 16:00 and 19:00 hrs, 20 to 30 flies of
mixed gender were transferred into starvation vials (68 ml,
Greiner bio-one, Frickenhausen, Germany) by a fly aspirator. The
starvation vial contained a cotton pad moistened with 2.5 to 3 ml
of Evian® water. If not otherwise indicated, starvation was
performed at 25°C and 60% relative humidity and lasted for 20 h.
Survival experiments
Newly hatched to one
day old flies were collected and transferred to fresh food vials. The
following day, flies were briefly CO2-anesthetized and sorted by
gender and genotype. At 17:00 hrs, around 20 female flies were
transferred into a starvation vial (see Starvation procedure). Dead
flies were counted every 3 h and not removed. Daily counting
sessions were repeated from 9:00 to 18:00 hrs, until all flies
were found dead.
For the survival
rescue attempt, fly vials with eggs were stored in an incubator
without humidity control and heated up every 4 h to 37°C for
30 min until hatching. Temperature in between heat shocks ranged
from 25 to 30°C. Hatched flies were collected and transferred to
fresh food vials and kept in the incubator with continuing heat
shocks every 4 h.
Sugar response test
Newly hatched to one
day old flies were collected and transferred to fresh food vials. The
following day, they were starved as described (see Starvation
procedure). Four hours before the end of the starvation period,
female flies (if not stated otherwise) were briefly immobilized by
cold-anesthesia. Their head and thorax were glued to a
triangle-shaped copper hook (0.05 mm in diameter) using a UV
sensitive glue (3M ESPE, Sinfony Indirect Lab Composite,
Minneapolis, USA). Animals were then kept individually in small
chambers (14 mm in diameter x 28 mm in height, custom-made)
with ad libitum access to water until the test. hsp-tßh flies were
glued before receiving the heat shock.
Tests were performed
between 12:00 and 16:00 hrs. Using forceps, we transferred flies
by their hook and fixed them to a magnetic clamp, which was then
attached to a rack. This treatment established free movement of the
flies’ tarsi and proboscis and was a modification from a previously
described PER assay #(Scheiner et al., 2004)# in order to prevent
unnecessary stress and pressure on the abdomen of the flies. A group
of six to eight flies was tested in parallel. A filter paper soaked
with sucrose solution was presented for 5 s to all six tarsi but
not the proboscis. Seven different concentrations (0, 0.1%, 0.3%,
0.6%, 1%, 3%, and 30%) were presented in series with an
inter-stimulus interval of 80 s. The proboscis extension
response was recorded. Finally, the proboscis was stimulated by 30%
sucrose solution. Flies not responding to this last stimulation or
responding to the first stimulation (water only) were discarded from
the analysis.
For the first sugar
response rescue attempt (Fig. 1A), flies were starved at 18°C and
put into an incubator without humidity control and heated up to 37°C
for 30 to 45 min. After the heat shock, flies were kept in a
25°C incubator with humidity control for 3 h until testing. For
the second rescue attempt (Fig. 1B), the first heat shock was given
with beginning of starvation every 23 h for 45 min until
one day before testing. Temperature in between heat shocks was 18°C.
PER assay validation
As a positive
control for the locomotion-independent sugar response assay, we
tested the sugar response of wild type flies starved for 14 h
versus 21 h #(Colomb et al., 2009)#. There was a statistically
significant increase in sugar response with the longer starvation
regime (p = 0.0409, Wilcoxon rank sum test, n = 48;
#Damrau et al., 2014)#. We thus conclude that the assay is sensitive
enough for detecting slight differences in sugar responsiveness.
Carbohydrate
measurement
Newly hatched to one
day old flies were collected and transferred to fresh food vials. The
following day at 17:00 hrs, 20 flies of mixed gender were either
transferred into starvation vials (see Starvation procedure) or kept
in the food vials. After 20 h, approximately 40 female flies per
group were cold-anesthetized, pierced through the thorax by the tip
of a dissecting needle (0.5 mm in diameter), and collected on ice
within a sieve composed of two tubes. The hemolymph was centrifuged
out of the fly into the bottom tube at 4°C. 0.5 µl of the
extracted hemolymph was transferred by a capillary (0.5 µl,
Hirschmann Laborgeraete, Eberstadt, Germany) into 19.5 µl PBS
(see #Damrau et al., 2013)#. Trehalose and glucose content in the
hemolymph was measured according to the protocols provided by the
manufacturer (Sigma Aldrich, Seelze, Germany). 10 µl of the
hemolymph-PBS mixture were added to 30 μl citric acid buffer
(135 mM, pH 5.7 at 37°C) and 10 μl of a trehalase
enzyme solution (Sigma Aldrich, 3% in citric acid buffer). After
incubation overnight at 37°C, 50 μl of Tris buffer were added.
80 µl of the resulting solution were added to 156.8 μl
Glucose oxidase and 3.2 μl o-Dianisidine (Glucose Assay Kit,
Sigma Aldrich) and incubated for 30 min at 37°C. Finally,
160 μl of 33% sulfuric acid were added. Absorbance at 540nm was
measured for the resulting solution using a nanoDrop® (nanoDrop
Technologies, Wilmington, USA) spectrometer.
Electrophysiological
recording
Flies were raised on
cornmeal-yeast-glucose-agar medium under a 12/12 h light/dark
cycle (lights on at 06:00 hrs) at 25°C. Newly hatched to one day old
flies were collected and transferred into a vial containing Kimwipe
paper soaked with 100 mM glucose for one to two days as
previously described #(Zhang et al., 2010)#. Starved flies were kept
in a vial containing Kimwipe paper soaked with Evian® water for 20 h
before testing.
Electrophysiological
recordings from l-type labellar chemosensilla were done by the
tip-recording method, as previously described #(Hodgson et al., 1955;
Hiroi et al., 2002)#. Briefly, the proboscis was fixed at the base of
the labellum. A glass capillary filled with Drosophila Ringer
solution served as an indifferent electrode. 100 mM sucrose
solution for stimulation contained 1 mM KCl as electrolyte. The
recorded signals were digitized and analyzed using the custom
software dbWave #(Marion-Poll, 1995, 1996)#. Action potentials were
detected by a visually-adjusted threshold set across the digitally
filtered signal. The total number of spikes within 1 second was
counted.
Statistics
Figures and
statistical analyses were performed in R (
http://r-project.org). If
not stated otherwise, data are illustrated as boxplots representing
the median (line), the 25% and 75% quartiles (boxes), the data within
1.5 times the interquartile range (whiskers), and data outside that
range (outliers, depicted as points).
The sugar response
score was calculated as the sum of all positive responses over the
seven sucrose presentations and therefore ranges from 0 to 7 (Total
PER).
For survival
measurement, the mean proportion of animals still alive was
calculated over time. The LD50 for each tested group, i.e. the time
point at which 50% of the flies were dead, was estimated using
MASS-package in R.
Hemolymph
carbohydrate content was read from a calibration curve showing the
absorbance of standard glucose/trehalose solutions that were treated
identically to hemolymph. Change in sugar content was calculated as
(intensitystarved - intensityfed) / (intensitystarved + intensityfed).
The significance
level of statistical tests was set to 0.05. Depending on data
distribution, we used parametric (Welch Two-Sample t-Test or two-way
ANOVA followed by TukeyHSD post hoc test) or non-parametric (Wilcoxon
rank sum test or paired Wilcoxon rank sum test with Bonferroni
correction) tests as depicted in the figure legends.