Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance
The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 functions in transport of JHE such that over- or under-expression of DmP29 results in increased or decreased JH degradation at specific sites respectively. Overexpression of DmP29 during the first or second instar was lethal, while overexpression during the third instar resulted in eclosion of small adults. Overexpression of DmP29 in newly eclosed flies reduced ovarian development and fecundity in addition to reducing the abundance of aggregation pheromone (cis-vaccenyl acetate) in males and courtship pheromone (cis,cis-7,11-heptacosadiene) in females. Both sexes also had lower levels of 23 and 25 carbon monoenes. Females exhibited reduced receptivity to mating, and males exhibited male-male courtship behavior, with both sexes being hyperactive: Male flies covered 2.7 times the distance of control flies at 2.9 times the maximum velocity. Application of the JH analog methoprene reversed impaired ovarian development, supporting a role for reduced JH in production of this phenotype. Rather than increasing lifespan as expected from a JH deficiency, overexpression of DmP29 reduced the life span of adult flies which may result from the hyperactivity of these flies. Underexpression of DmP29 resulted in reduced longevity, increased fecundity and reduced titers of pupal JHE. An alternative hypothesis, that mitochondrial dysfunction rather than JHE results in the JH-mediated phenotypes, is discussed.
Overexpression of Drosophila juvenile hormone esterase binding
protein results in anti-JH eﬀects and reduced pheromone abundance
, Xiuli Li, Jarrad R. Prasifka, Russell Jurenka, Bryony C. Bonning
Department of Entomology and Program in Genetics, 418 Science II, Iowa State University, Ames, IA 50011-3222, USA
Received 28 September 2007; revised 10 December 2007; accepted 4 January 2008
Available online 12 January 2008
The titer of juvenile hormone (JH), which has wide ranging physiological eﬀects in insects, is regulated in part by JH esterase
(JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent
anti-JH eﬀects. We hypothesize that DmP29 functions in transport of JHE such that over- or under-expression of DmP29 results in
increased or decreased JH degradation at speciﬁc sites respectively. Overexpression of DmP29 during the ﬁrst or second instar was
lethal, while overexpression during the third instar resulted in eclosion of small adults. Overexpression of DmP29 in newly eclosed
ﬂies reduced ovarian development and fecundity in addition to reducing the abundance of aggregation pheromone ( cis-vaccenyl ace-
tate) in males and courtship pheromone (cis,cis-7,11-heptacosadiene) in females. Both sexes also had lower levels of 23 and 25 car-
bon monoenes. Females exhibited reduced receptivity to mating, and males exhibited male–male courtship behavior, with both sexes
being hyperactive: Male ﬂies covered 2.7 times the distance of control ﬂies at 2.9 times the maximum velocity. Application of the JH
analog methoprene reversed impaired ovarian development, supporting a role for reduced JH in production of this phenotype.
Rather than increasing lifespan as expected from a JH deﬁciency, overexpression of DmP29 reduced the life span of adult ﬂies which
may result from the hyperactivity of these ﬂies. Underexpression of DmP29 resulted in reduced longevity, increased fecundity and
reduced titers of pupal JHE. An alternative hypothesis, that mitochondrial dysfunction rather than JHE results in the JH-mediated
phenotypes, is discussed.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Juvenile hormone esterase binding protein; Juvenile hormone esterase; Juvenile hormone; Pheromone production; Longevity; Hyperactivity;
The central and pleiotropic role of the sequiterpenoid
juvenile hormone (JH) in modulating ecdysteroid action
is fundamental to understanding many developm ental pro -
cesses in insects, and has provided a target for successful
development of technologies for insect pest management
(Minakuchi and Riddiford, 2006). In addition to the well
established roles of JH in maintenance of ‘‘status quo” in
larvae and the stimulation of vitellogenesis in adults (Bow-
nes, 1982; Riddiford, 1994; Riddiford et al., 2003; Truman
and Riddiford, 2007; Williams, 1961; Wyatt and Davey,
1996), low titers of JH are also associated with diapause
and increased lifespan (Denlinger and Tanaka, 1989; Tatar
et al., 2001; Tatar and Yin, 2001). In Drosophila melanogas-
ter, JH is known to be involved in pre-adult development
and metamorphosis (Riddiford and Ashburner, 1991), ini-
tiation and continuation of vitellogenin uptake, oocyte
development and ovarian maturation (Handler and Post-
lethwait, 1977; Ringo et al., 2005). JH also functions in
female receptivity to mating (Manning, 1967; Ringo
et al., 2005) and stimulates synthesis of male accessory
gland proteins (Herndon et al., 1997). In addition, JH
0016-6480/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
Corresponding author. Fax: +1 515 294 5957.
E-mail address: email@example.com (B.C. Bonning).
Present address: Massachusetts Eye and Ear Inﬁrmary, 243 Charles
Street, Boston, MA 02114, USA.
Available online at www.sciencedirect.com
General and Comparative Endocrinology 156 (2008) 164–172
and ecdysone have been implicated in the developmental
regulation of hydrocarbon and pheromone production
(Jallon and Wicker-Thomas, 2003).
Juvenile hormone esterase (JHE: EC 184.108.40.206) is critical
for the appropriate regulation of JH (Hammock, 1985).
For example, in the tobacco hornworm, Manduca sexta,
inhibition of JHE with a triﬂuoroketone inhibitor resulted
in giant larvae (Abdel-Aal and Hammock, 1986), mimick-
ing the eﬀects of application of a JH analog (Hatakoshi
et al., 1988). Although there are both extracellular (hemo-
lymph) and intracellular pools of JHE (Mitsui et al., 1979;
Vince and Gilbert, 1977; Wroblewski et al., 1990), hemo-
lymph JHE is considered to play the primary role in regu-
lating the titer of JH. In lepidopteran hemolymph,
decreased JH titers are positively correlated with increased
JHE abundance (Hammock, 1985). The function of intra-
cellular JHE has not been determined.
To better understand the regulation and diﬀerential tar-
geting of JHE to intracellular and extracellular locations,
we isolated a D. melanogaster JHE binding protein that is
primarily located in the mitochondria (DmP29: Drosophila
mitochondrial protein 29), and determined that DmP29
binds D. melanogaster JHE (Liu et al., 2007a,b). To deter-
mine whether DmP29 indeed interacts with JHE in vivo ,
and to elucidate the role of DmP29 in relation to JHE,
we examined the phenotypes resulting from DmP29 misex-
pression. Based on a series of apparent JH-mediated phys-
iological changes, we show here that the titer of DmP29
appears to be inversely correlated with JH action. Our data
are consistent with a role for JHE in mediation of the anti-
JH eﬀects resulting from DmP29 overexpression. Our data
also support a role for JH in pheromone production in
2.1. Drosophila strains
EP835 and EP840 were obtained from the Szeged Drosophila Stock
Centre (Rorth, 1996). In EP835 and EP840 the EP element is inserted
35 and 37 bp upstream of the DmP29 ORF (CG3776), respectively.
Expression of DmP29 is reduced in EP835. GAL4-HSP70/CyO (Bloom-
ington Drosophila Stock Center, stock 2077) expresses GAL4 in all tissues
under control of the Hsp70 promoter. All ﬂies were maintained at 25 °C
with a 12:12 L:D cycle in vials containing Drosophila food.
2.2. Misexpression of DmP29
Virgin females and males were collected within 6 h of eclosion. GAL4-
HSP70/CyO virgin females were mated to EP835 males. Five pairs of ﬂies
were maintained in food vials. F1 progeny were heat shocked at diﬀerent
stages for overexpression of DmP29. Two genotypes EP835/GAL4-HSP70
(straight wing) and EP835/CyO (curly wing) were generated from the
EP835 and GAL4-HSP70/CyO cross.
For heat shock in adults, ﬂies were sorted by genotype and gender
within 6 h of eclosion, and then heat shocked at 37 °C for 1 h in a water
bath. Flies were then frozen at diﬀerent times after heat shock. To test
for DmP29 overexpression, proteins were extracted, quantiﬁed by Brad-
ford assay and separated by SDS–PAGE (12% gel). After transfer to
Hybond-P membrane (Amersham Biosciences, Piscataway, NJ) proteins
were detected by using puriﬁed DmP29 antibody (Liu et al., 2007a). To
test whether the 2 EP lines underexpressed DmP29, proteins from
EP835, EP840 and Oregon R were extracted, separated by SDS–PAGE
and examined by Western blot.
2.3. Juvenile hormone esterase activity assay
JHE activity was tested using staged ﬂies with underexpressed DmP29.
Prepupae and pupae (11 h after pupariation) of EP835, EP840 and Oregon
R were frozen at 80 °C. Individual staged pupae were ground in eppen-
dorf tubes with 60 ll PBS on ice and centrifuged at 8000g at 4 °C brieﬂy to
remove debris. JHE activity was measured in triplicate by a partition assay
H-JHIII as substrate (Hammock and Sparks, 1977).
2.4. Fertility assays
Newly eclosed adults from the cross of EP835 and GAL4-HSP70/CyO
were sorted according to gender and genotype and heat shocked at 37C
for 1 h. Flies were maintained for 1 day in food vials to allow for
DmP29 expression and then crossed. Female ﬂies with overexpressed
DmP29 (EP/Gal4$) were crossed with male ﬂies with overexpressed
DmP29 (EP/Gal4#) or with control males (EP/CyO#). Control females
(EP/CyO$) were crossed with male ﬂies with overexpressed DmP29 (EP/
Gal4#) or with control males (EP/CyO#). Four pairs of ﬂies were crossed
in vials or apple juice plates with yeast. Progeny adults were counted for
the crosses in vials. Eggs laid per day were counted for the crosses in apple
juice plates for 80 pairs of ﬂies. To determine whether overexpression of
DmP29 aﬀected the fecundity of older females, newly eclosed ﬂies were
sorted and maintained separately in vials for 7 days. The ﬂies were then
heat shocked at 37 °C for 1 h and maintained at 25 °C for an additional
7 days. The ﬂies were crossed and eggs laid per day counted as above with
60 pairs of ﬂies. To determine whether ovary development was aﬀected by
overexpression of DmP29 and whether ovary development could be recov-
ered by application of the JH analog, methoprene, 1 lg of methoprene in
0.2 ll acetone was applied to the abdomen of anesthetized ﬂies 1 day after
heat shock (control, acetone only). Ovaries were dissected under a light
microscope. Heat shocked EP/CyO ﬂies were used as controls.
To determine whether ovary development was aﬀected by underexpres-
sion of DmP29, EP835 and control (Oregon R) ﬂies were also heat
shocked at 37 °C for 1 h and maintained at 25 °C for 1 day before being
crossed. EP835 females were crossed with EP835 or Oregon R males. Ore-
gon R females were crossed with Oregon R or EP835 males. Four pairs of
ﬂies were crossed in apple juice plates with yeast. One hundred and twenty
pairs of ﬂies were used and eggs laid were counted daily for the ﬁrst 3 days.
2.5. Overexpression of DmP29 in larval stages
Five pairs of GAL4-HSP70/CyO virgin females and EP835 males
were crossed in vials with Drosophila food. Adult ﬂies were removed 4
days later. Flies were reared at 25 °C with a 12:12 L:D cycle. The vials with
eggs and larvae were heat shocked at 37 °C for 1 h immediately after the
adult ﬂies were removed. Progeny adults were counted daily. This experi-
ment was repeated 3 times.
2.6. Mating behavior and pheromone assay
Newly eclosed EP/Gal4 were heat shocked and incubated at 25 °C for
3 or 5 days. Mating was observed for 1 h when 5 pairs of ﬂies were put into
a tube with ﬂy food. Crosses were as for the fertility assays.
For pheromone assays, newly eclosed Oregon R, EP/Gal4, EP/EP and
EP/CyO were heat shocked at 37 °C for 1 h and maintained at 25 °C.
Cuticular lipids were extracted 1 day after heat shock. Five ﬂies were
immersed in 100 ll hexane for 10 min after which the hexane was dried
and subjected to GC/MS. Analyses were conducted by capillary GC-MS
using a Hewlett–Packard 5890 GC equipped with a DB-5 column
(30 m 0.25 mm). The GC was interfaced with a Hewlett–Packard 5972
Z. Liu et al. / General and Comparative Endocrinology 156 (2008) 164–172 165
Mass Selective Detector operated in scan mode. Separations were con-
ducted in splitless mode with temperature programming at 80 °C for
1 min, then 10 °C /min to 320 °C(Choi et al., 2005).
2.7. Longevity study of DmP29 misexpression mutants
EP/Gal4 and EP835 were used to study whether misexpression of
DmP29 aﬀected life span with EP/CyO and Oregon R as controls. Males
and females were examined separately for this experiment. One hundred ﬂies
were kept in 32 oz transparent plastic food containers with adapter tubes
with Drosophila food at 25 °C with a 12:12 L:D cycle (Spencer et al.,
2003). Fresh food vials were provided and ﬂies were scored for survival every
2–3 days. The starting population for each genotype and gender was 300.
Flies were heat shocked at 37 °C for 1 h daily. Food vials were replaced with
vials that contained ﬁlter paper soaked with 1% sucrose solution during heat
shock. Two replicates of the longevity experiments were conducted.
2.8. Quantiﬁcation of locomotor activity by video-tracking
Newly eclosed male ﬂies (EP835/GAL4-HSP70 and Oregon R) were
heat shocked at 37 °C for 1 h, maintained at 25 °C for 24 h and then trans-
ferred to 35 mm Petri dishes with standard Drosophila food. The ﬂies were
heat shocked at 37 °C again for 2 h. The locomotor behavior was moni-
tored using an automated video-tracking system. Six ﬂies (3 Oregon R,
3 EP835/Gal4) housed individually were monitored simultaneously with
8 replicates (total of 48 ﬂies).
Dishes containing ﬂies were placed onto a sheet of white acrylic glass
illuminated from below by infrared LED arrays (Tracksys LTD, Notting-
ham, United Kingdom) and from above by ﬂuorescent lights. The paths of
ﬂies, called tracks, were collected using EthoVision software (Noldus,
2002), which captured the location of the center of gravity for each ﬂy
as time-series coordinates (x, y). EthoVision calculated total distance
moved, maximum velocity, and percentage of time moving over 2 h. To
eliminate apparent movement caused by small diﬀerences in x, y coordi-
nates (captured each 0.2 s), displacement of at least 0.028 cm was required
before considering a ﬂy to have actually moved. Other ﬁlters previously
used for Drosophila (Martin, 2004) were modiﬁed to estimate the percent-
age of time moving (i.e., periods where initial velocity exceeded 3.0 mm/s
and was maintained above 2.0 mm/s). Log-transformation (total distance
moved, maximum velocity) and arcsine square root transformations (per-
centage of time moving) were used before analyses of variance were
2.9. Statistical analysis
Analysis of variance was run using JMP 6 (SAS-Institute, 1990) and
SAS to test for diﬀerences among treatments. F-test was used to test sig-
niﬁcance among treatments. All pairwise comparisons were made using
Tukey’s HSD method. For the lifespan assay, pairwise comparisons
among genotypes for age-speciﬁc survival were conducted with Kaplan–
Meier survival analyses (log-rank test and Wilcoxon test) using PROC
LIFETEST in SAS with signiﬁcance corrected for multiple tests (Bonfer-
3.1. Misexpression of DmP29
The EP lines EP835 and EP840 have an EP element
inserted upstream of the start site of the dmp29 ORF. Wes-
tern blot was conducted to address whether insertion of the
EP element in these lines aﬀected expression of DmP29.
The predicted size of mature DmP29 following cleavage
of the 4.4 kD N-terminal targeting sequence is 25.9 kD
(Liu et al., 2007a). Expression of DmP29 by EP835 was sig-
niﬁcantly reduced relative to both Oregon R and EP840
(Fig. 1A). EP835 was used for examination of the eﬀects
of underexpression of DmP29.
Two genotypes EP835/GAL4-HSP70 and EP835/CyO
were generated from the EP835 and GAL4-HSP70/CyO
cross and DmP29 expression examined by Western blot.
Heat shock for overexpression of DmP29 in EP835/Gal4
ﬂies resulted in appearance of a 30 kD protein with peak
expression at 24 h after heat shock, which was present until
48 h after heat shock (Fig. 1B). This 30kD protein is the
size of unprocessed DmP29, which retains the mitochon-
drial leader sequence. In wild type ﬂies, import into the
mitochondia and coincident removal of the mitochondrial
leader sequence appears to be eﬃcient such that the
30 kD protein is not detected by Western blot. The 35 kD
protein on the blot was previous ly shown to be tropomyo-
sin (Liu et al., 2007b). Levels of this protein were not
aﬀected by heat shock.
3.2. JHE activity
JHE activity in ﬂies underexpressing DmP29 (EP835)
was signiﬁcantly lower than that of control ﬂies (EP840
and Oregon R). JHE activity was analyzed in individual
prepupae, and in pupae 11 h after pupariation, at peak
JHE titer (Campbell et al., 1992 ). For prepupae, there
was a signiﬁcant diﬀerence in JHE activity for the three
genotypes Oregon R, EP835 and EP840 (ANOVA,
df = 26, F = 3.98, P < 0.05). The JHE activity in prepupae
was signiﬁcantly lower in the EP835 line than in Oregon R,
and JHE activity in Oregon R was not signiﬁcantly diﬀer-
+ 835 840 kDa
1 2 5 9 24 EP/CyO +
EP/Gal4 (hr after HS)
Fig. 1. Misexpression of DmP29 in D. melanogaster. (A) Western blot of
proteins from Oregon R (+), EP835 and EP840 with puriﬁed DmP29
antiserum showing underexpression of the 25 kDa DmP29 in EP835. (B)
Western blot of proteins from the oﬀspring of EP835 and GAL4-HSP70/
CyO. Newly eclosed adults were heat shocked at 37C for 1 h and
maintained in tubes with food at 25C. Flies were collected at diﬀerent
times after heat shock (an hour after HS). EP/CyO was also heat shocked
and maintained for 24 h. Twenty micrograms of protein were loaded per
lane for both gels.
166 Z. Liu et al. / General and Comparative Endocrinology 156 (2008) 164–172
ent from that of EP840 (data not shown). There was also a
signiﬁcant diﬀerence in JHE activity for the three geno-
types at the pupal stage (ANOVA, df = 14, F = 8.74,
P < 0.005) with JHE activity of EP835 signiﬁcantly lower
than that of Oregon R (Fig. 2).
3.3. Impact of Dm29 misexpression on fecundity
To address whether overexpression of DmP29 in females
reduced fecundity, 1 day old male and fema le ﬂies that
overexpressed DmP29 were mated, and females allowed
to lay eggs for 3 days. Overexpression of DmP29 in newly
eclosed female ﬂies resulted in a reduced number of adult
progeny ﬂies even when mated with normal males. Over ex-
pression of DmP29 in males did not aﬀect the number of
adult progeny (data not shown). To determine whether
female ﬂies laid fewer eggs, apple juice plates were used
to collect eggs, which were counted daily. Again, overex-
pression of DmP29 in females that were mated with either
normal or DmP29-overexpressing males resulted in signiﬁ-
cantly fewer eggs than those laid by control females.
DmP29 overexpression in the males did not aﬀect fecundity
(Fig. 3A). Fecundity was not aﬀected when ﬂies were heat
shocked 7 days after eclosion (data not shown). Hypo-
expression of DmP29 in females resulted in signiﬁcantly
more eggs than those laid by control females (Oregon R).
Hypoexpression of DmP29 in the males did not aﬀect
fecundity (Fig. 3B). Dissection of ovaries from four day
old females showed that females with overexpressed
DmP29 had fewer eggs than control fema les. Application
of the JH analog methoprene recovered egg development
3.4. DmP29 overexpression reduced survival
At 25 °C with a photop eriod at 12-h light/12-h dark
cycle, Drosophila embryonic and larval stages were com-
pleted in 24 and 96 h, respectively. Four days after mating
of the parent ﬂies, larvae were heat shocked. At this time
1st to 3rd instar larvae of both EP/Gal4 and EP/CyO were
present in the vial. Both EP/Gal4 and EP/CyO ﬂies began
to eclose at the same time, but EP/Gal4 ﬂies only eclosed
over a period of 2 days, with EP/Gal4 adults smaller than
those of EP/CyO (Fig. 4). Based on the timing of eclosion,
these ﬂies resulted from heat shock of third instar larvae. In
the absence of heat shock, EP/Gal4 ﬂies had a similar body
size to EP/CyO ﬂies (Fig. 4). After 2 days, no more ﬂies
with overexpressed DmP29 (EP/Gal4) eclosed, while con-
trol ﬂies eclosed normally (EP/CyO), indicating that heat
shock of ﬁrst and second instar EP/Gal4 was lethal. In
the control vial without heat shock, both EP/Gal4 and
Oregon R EP840 EP835
(pmol JH hydrolyzed/min/fly)
Fig. 2. Underexpression of DmP29 results in reduced JHE activity in
D. melanogaster pupae. JHE activity was determined for pupae of Oregon
R, EP840 and EP835 11 h after puparium formation (APF), which
corresponds to the peak JHE activity (Campbell et al., 1992). Columns
with diﬀerent letters are signiﬁcantly diﬀerent (p < 0.05).
P29 x P29
Cross (newly eclosed fly)
Mean eggs laid/fly/day
P29 x P29
P29 x P29
Cross (newly eclosed fly)
Mean eggs laid/fly/day
P29 x P29
P29 xP29 P29 x P29 P29 xP29
Fig. 3. Impact of DmP29 misexpression on egg production. (A) DmP29
overexpression resulted in a signiﬁcant reduction in the numbers of eggs
laid by newly eclosed females, while (B) DmP29 underexpression resulted
in a signiﬁcant increase in the numbers of eggs laid by newly eclosed
females. (C) Overexpression of DmP29 resulted in reduced ovary size
which could be restored by application of the JH analog methoprene.
Ovaries from ﬂies with normal (EP/CyO) or overexpressed DmP29 (EP/
Gal4), treated with methoprene or acetone. Newly eclosed ﬂies were heat
shocked at 37 °C for 1 h and methoprene (1 l g) or acetone (control)
applied to the abdomen of anesthetized ﬂies 1 day after heat shock.
Ovaries were dissected 11 days after treatment.
Z. Liu et al. / General and Comparative Endocrinology 156 (2008) 164–172 167
EP/CyO ﬂies eclosed over a period of 5 days and the pop-
ulation size from the third to the ﬁfth day was twice that of
the heat shocked vial. Hence, overexpr ession of DmP29
(EP/Gal4) during the ﬁrst and second instar was lethal,
while overexpression of DmP29 during the third instar
resulted in eclosion of small adults.
3.5. Mating behavior
To examine the impact of DmP29 overexpression on
mating behavior, 5 virgin females and males were intro-
duced into a food vial and mating behavior observed.
Males with overexpressed DmP29 (EP/Gal4) had a high
frequency of mating behavior and showed male–male
courtship behavior (tapping, singing and attempting)
(Amrein, 2004). EP/Gal4 ﬂies courted similar numbers of
male and female EP/Gal4. Male ﬂies occasionally formed
chains of three males with one female, with each male
courting the ﬂy in front and being courted by the ﬂy
behind. We also noted that EP/Gal4 males were tapping,
singing and attempting and formed lines at the beginning
of the longevity study with 100 male ﬂies per 32oz plastic
container. No such behavior was observed for Oregon R,
EP/CyO or EP835/EP835 ﬂies. EP/Gal4 males had a lower
frequency of mating behavior with EP/CyO females than
with EP/Gal4 females.
3.6. Pheromone and hydrocarbon abundance
Cuticular lipids were extracted from the ﬂy cuticle and
GC-MS used to identify pheromones. Overexpression of
DmP29 in males resulted in decreased production of the
aggregation pheromone, cis-vaccenyl acetate (Z11-
18:OAc) compared to control ﬂies (EP/CyO). Overexpres-
sion of DmP29 in females (EP/Gal4) resulted in decreased
production of co urtship pheromone cis,cis-7,11-heptacos-
adiene (7,11–27:Hc) and cis,cis-7,11-nonacosadiene com-
pared to control ﬂies (EP/CyO) (Fig. 5). Lower levels of
23C and 25C monoenes were also observed (Fig. 5).
EP835/Gal4 which overexpressed DmP29 and EP835/
EP835 which underexpressed DmP29 were used to address
whether misexpression of DmP29 aﬀected life span, with
EP835/CyO and Oregon R as controls. The lifespan of
male ﬂies with under- and overexpressed DmP29 was
reduced by 38.8 and 42.6% respectively when compared
with Oregon R ﬂies. The lifespan of female ﬂies with under-
and overexpressed DmP29 was reduced by 31.6 and 35%
respectively when compared to Oregon R ﬂies. Among
males and females, relative to Oregon R and EP835/CyO,
the age-speciﬁc survival of EP835/EP835 and EP835/
Gal4 was reduced in both log-rank and Wilcoxon tests
(P < 0.001); survival of EP835/EP835 and EP835/Gal4 dif-
Fig. 4. Overexpression of DmP29 during the third instar results in small
adult ﬂies. Overexpression of DmP29 (EP/Gal4 with heat shock, HS+)
during the third larval instar resulted in an approximately 50% reduction
in the size of adult ﬂies compared to EP835/CyO (EP/CyO), or EP835/
Gal4 (EP/Gal4) in the absence of heat shock (HS).
18 19 20 21 22 23 24 25
Retention time, min
Fig. 5. Overexpression of DmP29 reduced pheromone and hydrocarbon
abundance. Partial chromatograms of cuticular lipids isolated from ﬂy
cuticles showing a decrease in cis,cis-7,11-heptacosadiene (7,11–27:Hc)
and cis,cis-7,11-nonacosadiene (7,11–29:Hc) in female ﬂies and cis-
vaccenyl acetate (Z11–18:OAc) in male ﬂies following overexpression of
DmP29. Lower levels of 23 and 25 carbon monoenes were found in both
sexes. EP/CyO was analysed as a control.
168 Z. Liu et al. / General and Comparative Endocrinology 156 (2008) 164–172
fered using the log-rank test (male: P < 0.001; female:
P = 0.027: Fig. 6A). The mean ages were compared using
Tukey–Kramer HSD (alpha = 0.05; JMP 6; SAS Institute).
The mean ages of EP835/EP835 and EP835/Gal4 were sig-
niﬁcantly lower than those of Oregon R and EP835/CyO
for both males and females. The mean ages of EP835/
EP835 and EP835/Gal4 did not diﬀer signiﬁcantly
Based on observations of hyperactivity associ ated with
overexpression of DmP 29 in adult ﬂies, we compared the
locomotor behavior of EP835/Gal4 and Oregon R ﬂies.
The total distances moved by the EP835/Gal4 ﬂies during
the 2 h recording period were signiﬁcantly greater than
by wild type Oregon R ﬂies (P = 0.0098: Table 1). The
maximum velocity of the EP835/Gal4 ﬂies was also signif-
icantly faster than that of wild type ﬂies (P = 0.0002: Table
1). The percentage of time ﬂies were in motion was signif-
icantly higher for EP835/Gal4 than for wild type ﬂies
(P = 0.0004). Given that solitary ﬂies exhibit reduced activ-
ity (Martin, 2004 ), these data likely underestima te the
extent of hyperactivity exhibited by EP835/Gal4 ﬂies when
4.1. Overexpression of DmP29 resulted in phenotypes
associated with decreased JH
JH is essential for larval development. Disruption of JH,
particularly during early instars, tends to be fatal (Minaku-
chi and Riddiford, 2006). Overexpression of DmP29 in the
ﬁrst and second larval instar was lethal, which is consistent
with reduced JH action. Overexpression of DmP29 during
the third instar resulted in reduced size of adult ﬂies, which
is consistent with premature pupation associated with early
elimination of JH. Overexpression of DmP29 in newly
eclosed but not in older females resulted in reduced fecun-
dity (Fig. 3), which is consistent with the role of JH early in
ovarian maturation (Handler and Postlethwait, 1977;
Ringo et al., 2005). Females with overexpressed DmP29
were less receptive, similar to wild type ﬂies treated with
the anti-JH agent precocene I ( Manning, 1967; Ringo
et al., 2005). We also found that males and females with
overexpressed DmP29 had less aggregation and courtship
pheromone, respectively (Fig. 5). While studies in other
insects suggest that JH is involved in pheromone biosyn-
thesis (Cusson and McNeil, 1989; Ignell et al., 2001; Till-
man et al., 1998), a direct role for JH in Drosophila
pheromone biosynthesis has not been demonstrated.
% of survival
% of survival
Oregon R EP835/Cyo EP835/EP835 EP835/Gal4
Oregon R EP835/Cyo EP835/EP835 EP835/Gal4
Fig. 6. DmP29 misexpression reduced life span. (A) The survival of ﬂies
with low expression of DmP29 (EP835/EP835) and overexpression of
DmP29 (EP835/Gal4) was compared to that of wild-type Oregon R and
control ﬂies (EP835/CyO). Females and males were monitored separately
and results expressed as percentage of surviving ﬂies on each day. (B) The
mean ages of the genotypes shown in A. Bars with diﬀerent letters are
signiﬁcantly diﬀerent from each other (p < 0.05). Both over- and under-
expression of DmP29 resulted in a signiﬁcant reduction in longevity
compared to Oregon R and EP835/CyO ﬂies.
Overexpression of DmP29 increased ﬂy locomotor activity
Genotype Distance (cm) ± SE
Maximum velocity (cm/s) ± SE Time moving (%) ± SE
EP835/GAL4 78.27 ± 18.85 A 1.79 ± 0.41 A 6.97 ± 1.52 A
Oregon R 28.91 ± 9.91 B 0.61 ± 0.10 B 1.82 ± 0.68 B
Activity of individual male ﬂies was monitored over a period of 2 h using EthoVision.
Diﬀerent letters within the same column indicate signiﬁcant diﬀerences.
Z. Liu et al. / General and Comparative Endocrinology 156 (2008) 164–172 169
Instead, JH appears to regulate the developmental switch
in hydrocarbon production that occurs between young
and mature adult ﬂies (Herndon et al., 1997).
EP/Gal4 males had a lower frequency of mating behav-
ior with EP/CyO females than with EP/Gal4 females. This
behavior may result from the fact that EP/Gal4 females
were less receptive than EP/CyO females and escaped from
the males. Alternatively, EP/Gal4 females were more
active, and hence more attractive to EP/Gal4 males on
the basis that males court moving females more vigorously
than immobile females (Tompkins et al., 1982).
Overexpression of DmP29 resulted in hyperactivity and
reduced longevity of adult ﬂies. Hyperactivity and reduced
longevity are also characteristic of the response of D. mel-
anogaster to starvation (Lee an d Park, 2004). Remarkably,
apart from responses induced in D. melanogaster by drugs
and insecticides, starvation is the only other condition for
which a phenotype of hyperactivity has been observed.
Starvation of Drosophila also results in a drop in the titer
of JH. Starved ﬂies display prolonged hyperactivity prior
to death, which is likely related to ﬁnding food (Lee and
Park, 2004). However, persistent hyperactivity hastens
starvation-induced death through rapid consumption of
energy. During periods of starvation there is a trade-oﬀ
between the allocation of resources for survival and egg
production (Harshman and Zera, 2006; Wayne et al.,
2006). Nutr itional shortage leads to decreased egg produc-
tion which can be reversed by topical application of JH or
by feeding (Terashima et al., 2005).
4.2. Underexpression of DmP29 resulted in phenotypes
associated with increased JH
Underexpression of DmP29 resulted in signiﬁcantly
increased fecundi ty and a shorter life span, which is consis-
tent with the increased action or titer of JH. The titer of
JHE in prepupae and pupae was signiﬁcantly lower when
compared to titers in Oregon R, which may account for
the apparent JH-mediated physiological changes in these
ﬂies. Given that the JHE titers were only reduced by 30
and 36% for prepupae and pupae, respectively, a reduction
in JHE activity at a particular stage, or in a particular loca-
tion may account for the observed phenotypes. Underex-
pression of DmP29 had no eﬀect on the pheromone
abundance (data not shown).
All of the phenotypes described above indicate that the
titer of DmP29 is negatively correlated with JH action.
Although reduced titers of JHE were associated with ﬂies
in which DmP29 was underexpressed, we did not detect
increased titers of JHE when DmP29 was overexpressed
(data not shown). We hypothesize that DmP29 plays a role
in JHE transport, with underexpression of DmP29 resulting
in impaired transport of JHE and increased action of JH at
speciﬁc target sites. The alteration of JH action may result
from diﬀerential regulation of JH degradation at speciﬁc
sites, rather than alteration of the overall titer of JH. This
is similar to the mode of action of the putative JH binding
protein Takeout which is expressed in response to starva-
tion in structures related to feeding (Tu et al., 2005).
4.3. Overexpression of DmP29 resulted in decreased life span
Despite the apparent anti-JH impact of DmP29 overex-
pression, ﬂies with overexpressed DmP29 diﬀered from ﬂies
with reduced JH synthesis. Reducing insulin-like peptides
(InR mutant) increased the life-span of Drosophila by
decreasing JH synthesis and the extension of lifespan could
be reversed by applying methoprene (Tatar et al., 2003; Tu
et al., 2005 ). The theory that reproduction and lifespan are
two trade-oﬀ phenotypes is not the case in ﬂies with over-
expressed DmP29, which had lower fecundity and shorter
lifespan. The hyperactivity of these ﬂies, which may result
from pheromone-mediated disruption of mating processes,
may account for exhaustion of resourc es and consequent
reduced longevi ty.
4.4. Overexpression of DmP29 reduced pheromone and
Overexpression of DmP29 resulted in reduced abun-
dance of aggregation pheromone in males and courtship
pheromone in females (Fig. 5). A reduction of 23 and 25
carbon monoenes was also observed. The absence of
Z11–18:OAc and 7–23:Hc could contribute to the observed
male-male courtship behavior in the EP/Gal4 males as
these compounds play an inhibitory role in male-male
courtship interactions. The reduction of unsaturated
hydrocarbons and Z11–18:OAc could be due to lowered
expression levels of desat1, the ﬁrst desaturase that func-
tions in the biosynthetic prod uction of monenes and dienes
(Herndon et al., 1997).
4.5. Function of DmP29
Drosophila melanogaster JHE is diﬀerentially targeted
according to developmental stage, with relatively low
amounts located in the mitochondria: D. melanogaster
JHE in pupae is primarily soluble, while in adults JHE is
equally distributed between the microsomal and soluble
fractions with a lesser portion in the mitochondria (Camp-
bell et al., 1992). The low level of JHE activity in larvae is
also distributed between the mitochondrial, microsomal
and soluble fractions. Interestingly, JH epoxide hydrolase,
which is the predominant JH hydrolytic enzyme in larvae
and functi ons to an equal degree with JHE in the adult,
is primarily located in the mitochondria (Campbell et al.,
1992). Cricklet, which may also play a role in JH regula-
tion, also has a mitochondrial targeting sequence (Li u
et al., 2007b; Shirras and Bownes, 1989). DmP29 which
is signiﬁcantly more abundant than JHE, is primarily mito-
chondrial and based on the presence of a Tim44 domain,
may be membrane-associated (
Liu et al., 2007a). Because
of the relative abundance of these two proteins, it is likely
170 Z. Liu et al. / General and Comparative Endocrinology 156 (2008) 164–172
that DmP29 has other functions in addition to its interac-
tion with JHE.
The data presented herein, suggest that DmP29 may
function in the appropriate targeting of JHE to speciﬁc
sites of JH action. The link between reduced JH and star-
vation provides a possible alte rnative hypothesis for the
function of DmP29, that DmP29 overexpression causes
mitochondrial dysfunction resulting in a perceived nutri-
tional sho rtage, which in turn elicits a starvation response.
A perceived nutritional shortage, which could result from
depletion of energy substrates by overactive mitochondria,
or the inability of mitochondria to use available resources,
would result in a drop in JH and suppression of JH-medi-
ated phenotypes. Decreased egg production resulting from
nutritional shortage can be reversed by topical application
of JH (Terashima et al., 2005). Conversely, on underex-
pression of DmP29, the perception of food abundance
may promote JH synthesis and JH-mediated phenotypes.
Further analyses will be required to determine whether
the link between DmP29 and JH is mediated by mitochon-
drial activity or by JHE.
We thank Drs. Jack Girton and Clark Coﬀman for help-
ful advice. This journal paper of the Iowa Agriculture and
Home Economics Experiment Station, Ames, Iowa, Pro-
ject No. 6657, is based upon work supported by the Na-
tional Science Foundation under Grant No. 0090874 and
by Hatch Act and State of Iowa funds.
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