Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance

Article (PDF Available)inGeneral and Comparative Endocrinology 156(1):164-72 · April 2008with39 Reads
DOI: 10.1016/j.ygcen.2008.01.006 · Source: PubMed
Abstract
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.

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Available from: Bryony Bonning
Overexpression of Drosophila juvenile hormone esterase binding
protein results in anti-JH effects and reduced pheromone abundance
Zhiyan Liu
1
, 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
Abstract
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;
Fecundity
1. Introduction
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.
doi:10.1016/j.ygcen.2008.01.006
*
Corresponding author. Fax: +1 515 294 5957.
E-mail address: bbonning@iastate.edu (B.C. Bonning).
1
Present address: Massachusetts Eye and Ear Infirmary, 243 Charles
Street, Boston, MA 02114, USA.
www.elsevier.com/locate/ygcen
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 3.1.1.1) 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
D. melanogaster.
2. Methods
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
using
3
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
conducted.
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-
roni’s method).
3. Results
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-
EP EP
+ 835 840 kDa
45
31
20
A
B
1 2 5 9 24 EP/CyO +
EP/Gal4 (hr after HS)
35
25
35
30
25
kDa
kDa
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
(Fig. 3C).
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
0
50
100
150
200
250
Oregon R EP840 EP835
Pupae (11APF)
a
a
b
JHE activity
(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).
0
2
4
6
8
10
12
14
16
P29 x P29
Cross (newly eclosed fly)
Mean eggs laid/fly/day
aa
b
b
0
1
2
3
4
5
6
7
P29 xP29
P29 x P29
P29 x P29
Cross (newly eclosed fly)
Mean eggs laid/fly/day
a
b
a
b
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).
3.7. Lifespan
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-
EP/Gal4 EP/CyO
P29
HS
-
+
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
Male
EP/CyO
Male
EP/Gal4
Female
EP/CyO
Female
EP/Gal4
Retention time, min
7,11-27:Hc
7,11-29:Hc
Z11-18:OAc
23C
unsat
25C
unsat
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
(Fig. 6B).
3.8. Hyperactivity
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
housed together.
4. Discussion
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.
0
0.2
0.4
0.6
0.8
1
02040 60
% of survival
Female
0
0.2
0.4
0.6
0.8
1
0204060
Age (days)
% of survival
0
10
20
30
40
50
Oregon R EP835/Cyo EP835/EP835 EP835/Gal4
Male
Ages (days)
a
b
c
c
Male
Oregon R
EP835/EP835
EP835/Gal4
EP835/CyO
0
10
20
30
40
Oregon R EP835/Cyo EP835/EP835 EP835/Gal4
Female
Ages (days)
a
c
c
b
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.
Table 1
Overexpression of DmP29 increased fly locomotor activity
Genotype Distance (cm) ± SE
a
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.
a
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
hydrocarbon abundance
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.
Acknowledgments
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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    • "Our screen also identified three sterol-related genes. Previously, juvenile hormone was shown to be involved in CHC regulation [12,[52][53][54] . Our results found that ecdysteroid hormones are important not only for maintaining levels of CHCs in adults but are needed, as well, for the viability of oenocytes. "
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