Dear Editor,
Protein is a basic building block of all living organisms and regulates practically
every biological process. It is essential for strict heterotrophs like fruit flies
to search for desirable protein-rich food sources and consume adequate dietary protein.
Previous reports have shown that prolonged protein deprivation induces a robust feeding
preference towards protein-rich food (such as yeast extract) and enhances protein
consumption
1,2
. Conversely, consumption of protein-rich food rapidly suppresses further protein
intake via a fat body-derived circulating hormone named FIT
3
. These mechanisms altogether help to maintain protein homeostasis in fruit flies.
Notably, the regulation of protein hunger and protein consumption is sex dimorphic
4
. Female flies are generally more sensitive to protein deprivation than males. Mating
experience further enhances the requirement of protein consumption in females, likely
due to a stronger requirement of protein supply in egg production.
Food seeking is a critical yet poorly understood behavioral process in food intake.
It remains unclear whether protein deprivation also regulates food seeking in any
animal species. We have previously established a quantitative assay to study food-seeking
behavior in fruit flies, which was indirectly measured by their frequency to cross
the midline of tubes in the Drosophila Activity Monitor System (DAMS, Trikinetics)
5,6
. By using this assay, we have shown that a small group of octopaminergic neurons
in the fly brain induces food-seeking activity in starved flies
6
. In this study, we aimed to investigate whether protein deprivation modulates the
search and occupation of protein-rich food sources and its underlying neural mechanism.
We first examined whether protein deprivation increased the locomotor activity of
flies (Fig. 1a). Mated female flies raised in the presence of 5% sucrose plus 2% yeast
extract (“S + YE”) were transferred to tiny polycarbonate tubes (Day 0) and their
activities were monitored for 4 consecutive days (Days 1–4). As shown in Fig. 1b,
c, immediately after the transfer (Night 0), flies assayed in the presence of 5% sucrose
alone (“S”, light orange) exhibited comparable midline-crossing activity to those
housed in the presence of both sucrose and yeast extract (“S + YE”, dark orange).
However, flies housed on sucrose alone showed a significant increase in flies’ midline-crossing
activity compared to those housed on S + YE (Fig. 1c, d) starting from Day 1, and
such effect gradually became more salient from Day 1 to Day 4 (Supplementary Fig. S1).
Notably, mated female flies survived well in both food sources, suggesting that protein
deprivation does not harm the general health of these flies (Supplementary Fig. S2).
Fig. 1
Protein deprivation induces protein-seeking behavior in mated female flies via octopamine
signaling.
a Schematic illustration of the DAMS-based locomotion assay. Briefly, virgin female
flies were collected shortly after eclosion and raised in the presence of sucrose
and yeast extract (S + YE) for 3–5 days. Afterwards, female flies were either mated
with males for 1 day or kept as virgins. These mated or virgin females were then transferred
individually to polycarbonate tubes (5 mm (D) × 65 mm (L)) and assayed in DAMS. b
Midline-crossing activity in 30-min bins of wild-type Canton-S-mated female flies
assayed in the presence of 5% sucrose alone (“S”, light orange) or 5% sucrose plus
2% yeast extract (“S + YE”, dark orange) (n = 49–53). Yellow bars represent light-on
period of 12 h in this and all other figures. c Average daily midline-crossing activity
of flies assayed in b (n = 49–53). d Average daily midline-crossing activity of flies
assayed in b from Day 1 to Day 4 (n = 49–53). e–g Average daily midline-crossing activity
of flies assayed on different protein-rich food types (n = 38–41, 58–69, 24–28, respectively).
h, i Midline-crossing activity in 30-min bins of wild-type Canton-S male flies (h)
and virgin female flies (i) assayed in the presence of 5% sucrose alone (“S”, light
orange) or 5% sucrose plus 2% yeast extract (“S + YE”, dark orange) (n = 44–53). j
Schematic illustration of the video recording-based locomotion assay. Briefly, individual
flies were introduced into a behavioral chamber in the presence of a small food patch
located in the center, and their positions and behaviors were recorded and analyzed
by a custom computer program. k Spatial distribution of protein-deprived Canton-S
flies assayed in the presence of sucrose (left) or sucrose plus yeast extract (right)
(the heat maps showed the average duration for flies to stay in each pixel; n = 13
(left) and 17 (right); for SEM see Supplementary Fig. S5). Color temperature represents
average time spent on each pixel for the duration of the assay (11 h). l Total walking
distance of protein-deprived flies assayed in i (n = 13 and 17). m On-food and off-food
walking speed of protein-deprived flies assayed in i (n = 13 and 17). n A summary
of the behavioral analysis. Briefly, protein deprivation enhances flies’ protein-seeking
behavior, by increasing their tendency to approach protein-rich food, to reduce their
movement during their visits to protein-rich food, and to reduce their willingness
to leave protein-rich food. o–v Average daily midline-crossing activity of flies assayed
in S and S + YE food (o, n = 27–42; p, n = 26–32; q, n
= 28–39; r, n = 25–33; s, n = 44–55; t, n = 22–32; u, n = 21–39; v, n = 32–41). Mianserin
(MI) (1 mg/mL) was mixed in food in t. w A working model. Protein deprivation induces
protein-seeking behavior in mated female flies via octopamine signaling. Female flies’
mated experience is crucial for this behavioral response via SP-SPR signaling. All
error bars represent SEM. Student’s t test and one-way ANOVA were applied to statistical
analysis. NS, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For the
raw activity data from Fig. 1, see Supplementary Data S2. Materials and Methods were
described in Supplementary Data S1
We next asked whether protein deprivation caused a permanent or reversible effect
on locomotion. Mated female flies housed in the presence of sucrose alone exhibited
robust increase in locomotion, but their activity rapidly reduced and became comparable
to those protein-supplied flies shortly after being transferred to yeast-containing
food (Supplementary Fig. S3a). Conversely, protein-supplied flies rapidly increased
their activity after being transferred to sucrose food (Supplementary Fig. S3b). Collectively,
these data support the notion that protein deprivation promotes the locomotor activity
of mated females in a reversible manner.
One alternative hypothesis is that some specific structural and sensory properties
of yeast extract suppressed locomotion that was not related to flies’ internal nutrient
status. To examine this possibility, we tested three additional types of protein-rich
food, bovine serum albumin, tryptone, and brewer’s yeast in the locomotion assays
and found that all of them generated similar effect on flies’ locomotion (Fig. 1e–g).
Therefore, the locomotion-promoting effect was likely due to the lack of protein intake,
rather than some structural and sensory properties of a specific protein-rich food.
Another alternative explanation is that the calorie value of S + YE food was higher
than that of sucrose because of the addition of 2% YE. To examine this possibility,
we also tested calorie-matched food (3% sucrose + 2% YE vs. 5% sucrose), and the flies
on sucrose still showed increased locomotion than those on calorie-matched S + YE
food (Supplementary Fig. S4).
Previous studies have shown that male and virgin female flies showed a much weaker
requirement for dietary yeast than mated females
4
. We thus asked whether males and virgin females exhibited an increase in their locomotor
activity upon protein deprivation. The absence of yeast supply did not alter the midline-crossing
activity of males or virgin females (Fig. 1h, i). Therefore, these results suggest
that protein deprivation induces hyperactivity specifically in mated females.
We then asked whether hyperactivity upon protein deprivation facilitated the localization
and occupation of protein-rich food, therefore resembling a protein-seeking behavior.
To this end, we developed a computer program that tracked and analyzed the location
and moving trajectories of individual flies in a behavioral chamber with food sources
(Fig. 1j). By using this system, we found that protein-deprived flies tended to accumulate
more stably on food sources containing yeast extract (“Food: S + YE”) than those without
yeast extract (“Food: S”) (Fig. 1k and Supplementary Fig. S6), suggesting that protein-deprived
flies have a stronger tendency to seek for and accumulate on protein-rich food.
We next sought to examine the detailed behavioral kinetics of protein-deprived flies
in the presence and absence of protein-rich food. In the presence of yeast-containing
food sources, protein-deprived flies exhibited significantly shorter walking distance
and lower walking speed both on food and away from food (Fig. 1l, m). In addition,
protein-deprived flies in the presence of yeast-containing food exhibited significantly
increased total duration exploiting the food patch and significant longer stay on
food for each food visits than on sucrose food (Supplementary Fig. S6a, b). Meanwhile,
they also exhibited considerably (yet insignificant) more food visits to yeast-containing
food (Supplementary Fig. S6c).
We next examined protein-supplied flies in this video-tracking assay (Supplementary
Fig. S7). In contrast, these flies exhibited no behavioral differences in the presence
and absence of protein-rich food. Unlike protein-deprived flies, protein-supplied
flies showed comparable walking distance and walking speed in both conditions (Supplementary
Fig. S8a, b), and their total duration to stay on food, average duration for each
visit to food, and the total number of food visits were all unchanged in the presence
and absence of protein-rich food (Supplementary Fig. S8c–e).
Taken together, these results indicate that upon protein deprivation, flies exhibit
an increased willingness to visit protein-rich food and decreases their tendency to
move on food and to leave food (Fig. 1n). Therefore, protein deprivation-induced hyperactivity
likely helps the localization and occupation of protein-rich food, resembling a protein-seeking
behavior. Notably, these results are consistent with a recent study showing that protein
deprivation enhances flies’ exploitation on protein-rich food while suppressing their
general exploratory activity
7
.
We then sought to investigate how mating experience in females triggered this protein-seeking
behavior. During a copulation event, sperms and a collection of seminal fluid proteins
are transferred from males to females. We found that females mated with sperm-less
piwi
–/–
mutant males still showed protein-seeking behavior upon protein deprivation (Fig. 1o),
suggesting that sperm transfer during copulation is not the triggering factor
8
.
It has been shown that a specific seminal fluid protein transferred during copulation,
sex peptide (SP), mediates various physiological and behavioral effects in mated females,
including persistent egg laying, reduced sexual receptivity, aggression, increased
food consumption, and enhanced preference towards protein-rich food. The numerous
functions of SP is mediated by a single receptor, named SP receptor (SPR), extensively
expressed in the reproductive organ and the nervous system of females
9
. We therefore asked whether SP-SPR signaling was involved in promoting food-seeking
behavior in protein-deprived flies.
Ectopic expression of SP in the fat body of virgin female flies (yp1-SP flies) was
sufficient to recapitulate protein-seeking behavior that was observed only in mated
females (Fig. 1p). While the wild-type virgin females exhibited no increase in protein-seeking
behavior, yp1-SP virgin females showed significant increase in locomotion upon protein
deprivation. Notably, yp1-SP flies also exhibited a significant reduction in locomotion
on S + YE food (Fig. 1p). Given that overexpression of SP in virgin females promoted
egg laying
10
, and that flies tended to stop moving during oviposition
11
, it is possible that SP overexpression indeed suppresses locomotion via enhanced
oviposition. Conversely, mated females lacking a functional SPR did not exhibit increase
in locomotion after protein deprivation (Fig. 1q). Therefore, SP-SPR signaling is
both necessary and sufficient for protein seeking induced by protein deprivation in
mated females.
Our previous studies have shown that octopamine, the insect analog of mammalian norepinephrine,
regulates starvation-induced food seeking in fruit flies
5,6
. We thus asked whether a different type of food-seeking behavior, protein seeking,
also required octopamine signaling. Indeed, mated females carrying a null allele of
tyramine β-hydroxylase (TβH
M18
), a key enzyme for the biosynthesis of octopamine, were unresponsive to protein deprivation
(Fig. 1r). Neuronal knockdown of TβH expression also blocked the induction of protein
seeking by protein deprivation (Fig. 1s). Therefore, octopamine signaling is likely
involved in the induction of protein-seeking behavior in mated females. It is worth
noting that TβH
M18
mutant flies exhibited a modest yet significant reduction in locomotion compared to
the wild-type controls (Fig. 1r), which is consistent with the role of octopamine
signaling in regulating general locomotion
12,13
. Consistently, feeding mated females with an octopamine receptor antagonist, mianserin
14
, blocked the induction of protein-seeking behavior and suppressed baseline locomotion
(Fig. 1t).
We also examined the activity of octopaminergic neurons in regulating protein-seeking
behavior. We found that neuronal silencing of octopaminergic neurons by ectopically
expressing Kir2.1, an inward-rectifying potassium channel, eliminated protein-seeking
behavior in mated females (Fig. 1u). Conversely, artificial activation of these neurons
by ectopically expressing a bacterial sodium channel NaChBac enhanced flies’ locomotor
activity on S + YE food (Fig. 1v). These results are consistent with a role of octopamine
signaling in regulating protein-seeking behavior.
In addition, it has been reported that an octopamine receptor named Octβ2R was required
for increased locomotion upon food deprivation in larval flies
10
. We thus tested whether the same receptor was also involved in the regulation of
protein-seeking behavior in adult flies. As shown in Supplementary Fig. S9, protein-seeking
behavior was completely abolished by neuronal knockdown of Octβ2R, further supporting
the involvement of octopamine signaling in protein-seeking behavior. Therefore, octopamine
signaling may be required for different food-seeking behaviors induced by both starvation
and protein deprivation. These results are also in line with a role of octopamine
signaling in regulating wakefulness and arousal state in fruit flies.
In summary, we showed in this study that in mated female flies, prolonged protein
deprivation-induced hyperactivity in a reversible manner, which facilitated the localization
and occupation of desirable protein-rich food sources and hence adequate protein consumption
(Fig. 1w). Therefore, our results collectively characterized a protein-seeking behavior
that contributed to organismal protein homeostasis. Mechanistically, during copulation,
a male seminal fluid protein, SP, was transferred from male to female, and triggered
this behavioral response via its cognate receptor SPR. Interestingly, octopamine signaling
is required for protein-seeking behavior upon protein deprivation, as well as starvation-induced
food-seeking behavior. Whether the same group of octopaminergic neurons mediates food
seeking under both circumstances, or two different subsets of octopaminergic neurons
are involved, would be of significant interest for future studies.
Like in fruit flies, adequate and balanced intake of dietary protein is also critical
for the survival, reproduction, and well-being of mammals including human. The detection
of protein deprivation, and the subsequent seeking and consumption of desirable protein-rich
diets, likely also shared conserved biological mechanisms between fruit flies and
mammals. Therefore, our work has also paved a way to characterize this important yet
poorly understood behavior in more complex animal species and to investigate its underlying
neural mechanism.
Electronic supplementary material
Supplementary Data S1
Supplementary Data S2