Juvenile hormone agonists affect the occurrence of male Daphnia

Article abstract of DOI:10.1016/S0045-6535(03)00761-6

The water flea Daphnia magna reproduces primarily by cyclic parthenogenesis. Environmental stimuli that signal a change to adverse conditions induce the organisms to switch from parthenogenesis to gamogenetic reproduction. During the gamogenetic period, they produce male daphnids and dormant resting eggs, which can survive prolonged periods of environmental adversity. However, little is known about the mechanisms associated with the switch from parthenogenesis to gamogenetic reproduction. We investigated the effects of several juvenoids on sex determination in Daphnia. Females less than 24 h old were exposed to various concentrations of the test substance and were observed for 21 days. It was found that they can trigger the appearance of male daphnids: the percentage of males in the population increases to a level greater than what occurs under ordinary environmental conditions. We found that methylfarnesoate, juvenile hormone III, methoprene, and the phenoxyphenoxy derivatives pyriproxyfen and fenoxycarb (both insecticides) reduced the production of offspring and produced sex ratios dominated by male daphnids. Pyriproxyfen and fenoxycarb showed striking effects at low concentrations. Exposure to either of these chemicals at a concentration of 330 ngl ^-1 caused adult females to produce almost all male neonates. Methylfarnesoate, juvenile hormone III, and methoprene showed an effect in inducing male production at higher concentrations (3.7×10³, 3.3×10⁵, and 1.3 ×10⁵ ngl^-1, respectively). Our findings suggest that juvenile hormone agonists, including some insecticides, affect the chemical signaling responsible for inducing the production of male offspring.

Full text of DOI:10.1016/S0045-6535(03)00761-6

Chemosphere 53 (2003) 827–833
www.elsevier.com/locate/chemosphere
Juvenile hormone agonists affect the occurrence
of male Daphnia
a,b,
a
b,c
Norihisa Tatarazako
Masatoshi Morita , Taisen Iguchi
*, Shigeto Oda , Hajime Watanabe
,
a
b,c
a
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba City, Ibaraki 305-8506, Japan
b
CREST, JST (Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation),
3-13-11 Shibuya, Shibuya-ku, Tokyo 150-0002, Japan
c
Center for Integrative Bioscience, Okazaki National Research Institutes, Higashiyama 5-1, Myodaiji, Okazaki 444-8585, Japan
Received 17 March 2003; received in revised form 26 June 2003; accepted 15 July 2003
Abstract
The water flea Daphnia magna reproduces primarily by cyclic parthenogenesis. Environmental stimuli that signal a
change to adverse conditions induce the organisms to switch from parthenogenesis to gamogenetic reproduction.
During the gamogenetic period, they produce male daphnids and dormant resting eggs, which can survive prolonged
periods of environmental adversity. However, little is known about the mechanisms associated with the switch from
parthenogenesis to gamogenetic reproduction. We investigated the effects of several juvenoids on sex determination in
Daphnia. Females less than 24 h old were exposed to various concentrations of the test substance and were observed for
21 days. It was found that they can trigger the appearance of male daphnids: the percentage of males in the population
increases to a level greater than what occurs under ordinary environmental conditions. We found that methylfarne-
soate, juvenile hormone III, methoprene, and the phenoxyphenoxy derivatives pyriproxyfen and fenoxycarb (both
insecticides) reduced the production of offspring and produced sex ratios dominated by male daphnids. Pyriproxyfen
and fenoxycarb showed striking effects at low concentrations. Exposure to either of these chemicals at a concentration
of 330 ng lꢀ1 caused adult females to produce almost all male neonates. Methylfarnesoate, juvenile hormone III, and
methoprene showed an effect in inducing male production at higher concentrations (3.7 · 103, 3.3 · 105, and 1.3 · 105
ng lꢀ1, respectively). Our findings suggest that juvenile hormone agonists, including some insecticides, affect the
chemical signaling responsible for inducing the production of male offspring.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Keywords: Daphnia magna; Fenoxycarb; Methoprene; Methylfarnesoate; Pyriproxyfen; Sex determination
1. Introduction
invertebrate predators. It also plays a major role in the
control of water quality by feeding on algae (Dodson
and Hanazato, 1995).
The water flea Daphnia magna (Cladocera, Crusta-
cea) occupies an important position in aquatic food
webs because it is a major part of the diet of fish and
In general, daphnids reproduce by parthenogenesis,
in which females asexually produce genetically identical
female offspring. Because the parthenogenetic repro-
duction system provides rapid expansion of daphnid
populations, a daphnid population consists almost en-
tirely of females when resources are abundant. It is known
that daphnids switch their reproductive strategy from
parthenogenesis to gamogenesis (sexual reproduction) in
* Corresponding author. Address: National Institute for
Environmental Studies, 16-2 Onogawa, Tsukuba City, Ibaraki
305-8506, Japan. Tel.: +81-298-50-2887; fax: +81-298-50-2851.
E-mail address: tatarazako.norihisa@nies.go.jp (N. Tatara-
zako).
0045-6535/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0045-6535(03)00761-6

828
N. Tatarazako et al. / Chemosphere 53 (2003) 827–833
response to environmental changes such as depletion of
resources or crowding (Kleiven et al., 1992). After males
are produced, sexual reproduction can occur, which has
two major advantages. One is genetic recombination
(Deng and Lynch, 1996), leading to greater genetic di-
versity, which could improve a daphnid population’s
ability to adapt to changing environments. The other
advantage is the production of resting eggs that can
withstand severe environmental conditions, such as
freezing temperatures or drought.
[isopropyl(E,E)-(R,S)-11-methoxy-3,7,11-trimethyldo-
deca-2,4-dienoate] is a juvenile hormone (JH) analog
used to control mosquito populations by preventing
larvae from metamorphosing into adults (Arias and
Mulla, 1975). Insect JH analogs also include pyripr-
oxyfen and fenoxycarb (Fig. 1). Pyriproxyfen [phen-
oxyphenoxy(R,S)-2-(2-pyridyloxy)propyl ether] is a
juvenoid insect growth regulator used to control a wide
range of insect pests, including mosquitoes (Estrada and
Mulla, 1986) and chironomids (Ali et al., 1993). Fen-
oxycarb [ethyl 2-(4-phenoxyphenoxy)ethylcarbamate] is
a carbamate insect growth regulator. It is used in baits
for fire ants and for the control of fleas, mosquitoes,
cockroaches, butterflies, moths, and scale insects. It is
also used to control these pests on stored products (US
Environmental Protection Agency, 1983–1985).
Although the change in reproductive strategy in
daphnids is phenomenologically well known, little is
known concerning the neuroendocrine factors that link
the environmental stimuli to the ultimate biochemical
and physiological responses that initiate the production
of males (Baer and Owens, 1999). Recent studies have
shown that sex determination in invertebrates can be
influenced by toxicants. For example, exposure of
daphnid populations to atrazine in the laboratory alters
the sex ratio of the offspring (Dodson et al., 1999).
Methoprene stimulates male offspring production and
defers their production to later stages of the sexual re-
productive period, while inhibiting the production of
resting eggs and promoting the continuance of parthe-
nogenetic reproduction (Olmstead and LeBlanc, 2001).
Methylfarnesoate and juvenile hormone III are ju-
venile hormones in crustaceans and insects, respec-
tively (Laufer et al., 1993; Nijhout, 1994). Methoprene
High concentrations of JH and low concentrations
of molting hormone cause molting larvae to continue
growing as larval instars. JH has two primary effects on
insects: one is morphogenetic, that is, modulation of
larval growth and development and inhibition of pre-
mature metamorphosis (Riddiford, 1996); the other is a
gonadotrophic effect––regulation of several aspects of
reproductive physiology including vitellogenesis (Koe-
ppe and Ofengand, 1976).
Given that a JH analog, methoprene, affects the re-
productive system of Daphnia and that some insecticides
are JH analogs, it is natural that we should examine
O
Methylfarnesoate
O
O
O
O
Juvenile hormone III
O
O
Methoprene
Pyriproxyfen
O
O
O
N
O
O
O
O
Fenoxycarb
N
H
O
Fig. 1. Chemical structures of methylfarnesoate, juvenile hormone III, methoprene, pyriproxyfen, and fenoxycarb.

N. Tatarazako et al. / Chemosphere 53 (2003) 827–833
829
whether other JH analogs have the same effects on the
reproductive strategy of Daphnia. We found that
Daphnia exposed to JH and JH analogs produced more
males than normal, indicating that these chemicals can
be used to study the mechanisms involved in the tran-
sition from parthenogenesis to gamogenesis.
atilization of the test chemicals. The water quality (pH
and dissolved oxygen concentration) was measured
every 2 days (right after changing of water). A suspension
of 0.05 ml of Chlorella (4.3 · 108 cells mlꢀ1) was added to
each jar daily. Water hardness, pH, and dissolved oxy-
gen concentration were 75–85 mg lꢀ1, 7.0–7.5, and 80–
99%, respectively. The medium was changed every 2
days, and neonates were removed from the jar every day
and were counted by eye. The total number of neonates
born over 21 days at each concentration of test chemical,
as well as the total number born to the control group,
were calculated and compared. Neonates were raised in
beakers for several days until sexual differentiation.
They were then examined under a stereomicroscope to
determine their sex from the length of the primary an-
tennae (Olmstead and LeBlanc, 2000). The percentage of
male daphnids was then determined.
2. Materials and methods
Methylfarnesoate (all-trans, 90% pure) was provided
by Dr. H. Sonobe (Konan University, Kobe, Japan), and
it was synthesized by methylation of (2E,6E)-farnesoic
acid (which was a gift of Kuraray Co. Ltd., Tokyo, Japan)
with diazomethane according to a general methylation
procedure (Monson, 1971). The synthetic methylfarne-
soate was purified by silica gel chromatography (1.5%
diethyl ether in pentane) followed by reverse-phase high-
performance liquid chromatography (column; Wakosil
5C18, 150 mm · 4.6 mm, Wako, Japan) by isocratic elu-
tion with 70% acetonitrile in water. Juvenile hormone III
(technical grade, 75% pure) was obtained from ICN
Biomedicals (Irvine, CA, USA). Pyriproxyfen (technical
grade, 96.6% pure), fenoxycarb (technical grade, 96.6%
pure), and methoprene (technical grade, 96.6% pure) were
obtained from Wako Pure Chemical Industries Ltd.
(Osaka, Japan). Stock solutions of each chemical were
prepared at concentrations of 1 g lꢀ1 in ethanol (analytical
grade, Wako, Japan). Ethanol concentrations in all test
solutions were less than 0.1% v/v. Culture water was
prepared by filtering tap water through charcoal at room
temperature no more than 24 h before use.
After exposure to chemicals in experiments for 21
days, the experiments were continued for 8 days in
standard culture water (wash out experiment), to de-
termine whether the effects noted were reversible. That
is, if the sex ratio had been skewed toward males, would
those parent females revert to producing females if the
chemical was removed from their environment? At the
end of 21-day experiment, test animals were rinsed twice
with culture water. Sex ratios of neonates born during
the first 6 days and those born during the 7th and 8th
days were compared.
Only adult phase exposure experiment was conducted
to study how long the juvenoids take to induce male neo-
nates production. Adult daphnids (2–3 weeks old) carrying
embryos in the brood chamber were exposed to either
pyriproxyfen or fenoxycarb at the concentration of 300
ng lꢀ1. Sex ratios of neonates born during the first 2 days
and those born in the following 6 days were compared.
The differences in the total numbers of offspring
among treatments were analyzed by analysis of variance
and the Dunnett posthoc test (StatView, SAS Institute,
1998). The effects of chemicals on the sex ratio of the
offspring were analyzed by logistic regression analyses
with the maximum-likelihood method (PROC CAT-
MOD, SAS Institute, 1988). The sex ratios of offspring
in wash out experiment and only adult phase exposure
experiment were analyzed by Fisher’s exact test (Stat-
View, SAS Institute, 1998).
D. magna was obtained from the National Institute
for Environmental Studies (NIES), Tsukuba, Japan.
This culture has been maintained for more than 10
years. The strain of D. magna we used in the present
study produces fewer than 0.1% males under ideal ex-
perimental conditions. Our reproduction test was per-
formed for 21 days according to the methods for
survival and reproduction tests on D. magna proposed
by the OECD (1998). Females less than 24 h old were
used as the founding females in each test. They were
exposed to various concentrations of the test substance
according to the OECD test conditions, then fed and
observed daily for 21 days. Cultures were kept in an
incubator at a temperature of 24 1 ꢁC and a photo-
period of 14 h light/10 h dark. Six nominal concentra-
tions of each test chemical, including a culture water
control, were prepared by dilution with fresh culture
water. All 21-day experiments were conducted with a
dilution factor of 3 except for methoprene, in which the
factor was set to 2. Eight replicate glass jars (100 ml),
each containing an individual D. magna female in 50 ml
of media, were used for each concentration. Thus, 48
glass jars and daphnids were used to test each chemical.
The jars were covered with Teflon caps to prevent vol-
3. Results
3.1. Juvenile hormone and its analogs reduce the number
of offspring produced
In the present study, five juvenoids (methylfarneso-
ate, juvenile hormone III, methoprene, pyriproxyfen,
and fenoxycarb) were tested for their effects on fecundity
and sex determination in D. magna. Both pyriproxyfen

830
N. Tatarazako et al. / Chemosphere 53 (2003) 827–833
and fenoxycarb reduced the total number of offspring
at relatively low concentrations, 10–30 ng lꢀ1 (Fig. 2). In
contrast, methylfarnesoate, juvenile hormone III, and
methoprene showed significant reduction of reproduc-
tion only at much higher concentrations (Fig. 2). Thus,
the effect of juvenoids on Daphnia fecundity covered a
wide range of chemical concentrations, although they
have similar concentration-dependent effects.
In the control group, only female neonates were pro-
duced. The lowest concentration at which production of
males was observed for the other JH agonists was
3.7 · 103, 3.3 · 105, and 1.3 · 105 ng lꢀ1 for methylfarne-
soate, juvenile hormone III, and methoprene, respec-
tively, and was 100–1000 times as high as that for
pyriproxyfen and fenoxycarb (Fig. 3).
3.3. Wash out experiment and only adult phase exposure
experiment
3.2. Juvenile hormone agonists skew the sex ratio toward
males
In wash out experiment, when exposure of Daphnia
to methylfarnesoate, juvenile hormone III, pyriproxy-
fen, fenoxycarb, or methoprene was stopped after 21
days and the same individuals were then reared in
standard culture water, male neonates were not pro-
duced after 7–8 days (Table 1). In contrast, when adult
daphnids that had been exposed to neither fenoxycarb
nor pyriproxyfen were treated with 300 ng lꢀ1 of fen-
Interestingly, at the concentration of 100 ng lꢀ1, py-
riproxyfen and fenoxycarb began to induce the pro-
duction of males (Fig. 3). The sex ratio (males per total
number of neonates) increased in a concentration-
dependent manner, and almost all neonates produced
were males when the parent females were exposed to
either pyriproxyfen or fenoxycarb at 1000 ng lꢀ1 (Fig. 3).
120
Methylfarnesoate
100
80
60
40
20
0
Pyriproxyfen
*
*
*
*
Methoprene
*
JH III
104
Fenoxycarb
101 102
103
105
106
107
Concentration (ng/l)
Fig. 2. Mean total numbers of neonates born per female, represented as relative value to control group in each experiment. Total
number of neonates averaged for control groups in five experiments was 201 30 (mean SD). Asterisks indicate the lowest juvenoid
concentration at which reproduction was significantly lower than in the control (Dunnett test, P < 0:05).
1.0
Methylfarnesoate
Pyriproxyfen
JH III
0.5
Fenoxycarb
Methoprene
0
101
102
103
104
105
106
107
Concentration (ng/l)
Fig. 3. Relationship between chemical concentration and sex ratio of offspring. Each curve was fitted by logistic regression analysis.
Regression coefficients for slopes were all statistically significant ðP < 0:001Þ.

N. Tatarazako et al. / Chemosphere 53 (2003) 827–833
831
Table 1
Recovery of sex ratio from effects of five juvenoidsa
Chemicals
Day 1–6
Male
Day 7–8
Male
Female
Female
Pyriproxyfen
Fenoxycarb
209
208
132
35
1645
974
0
0
0
0
0
242
490
*
*
*
*
*
Methylfarnesoate
Juvenile hormone III
Methoprene
3645
1443
1040
1704
490
23
852
a Exposure to chemicals was stopped after 21 days. The total numbers of males and females across all juvenoid concentrations except
for the control during 2 periods after stopping exposure are shown. Asterisks indicate a significant difference in sex ratios between the 2
periods (Fisher’s exact test, P < 0:001).
Table 2
Induction of male neonates when adult daphnids were exposed to pyriproxyfen or fenoxycarba
Chemicals
Day 1–2
Male
Day 3–8
Male
Female
Female
Pyriproxyfen
Fenoxycarb
0
0
90
164
99
69
0
36
*
*
a Five adult daphnids were treated with 300 ng lꢀ1 of pyriproxyfen or fenoxycarb. The total numbers of males and females born to
five daphnids are shown. Asterisks indicate a significant difference in sex ratios between the 2 periods (Fisher’s exact test, P < 0:001).
oxycarb or pyriproxyfen, after 3 days, the sex ratio of
the offspring changed from 0, indicating all of them are
female, to 0.66–1.0 (Table 2).
are consistent with their observations in that we found
that methoprene stimulated the production of males.
Most of the experiments, however, were conducted
under experimental conditions which were arranged so
that male neonates would appear in the control (Dodson
et al., 1999; Olmstead and LeBlanc, 2000, 2001; Peterson
et al., 2001). Therefore, it is impossible to distinguish
between two possibilities; i.e., male production was ini-
tiated by test chemicals or male production primarily
induced by other environmental factors was just accel-
erated by test chemicals. Few previous reports mention
the induction of almost all male offspring under envi-
ronmental conditions which do not induce any male in
the absence of chemicals. Only recently, juvenoid hor-
mone methylfarnesoate has been found to be a male sex
determinant in daphnids (Olmstead and LeBlanc, 2002).
They demonstrated that methylfarnesoate causes adult
daphnids to switch from the production of female to the
production of male offspring, which is consistent with
our results.
4. Discussion
In the present study, we found that five juvenoids
increased the percentage of males produced by exposed
D. magna. Since the skewed sex ratio toward male on
exposure to the chemicals was accompanied by reduced
reproduction, there is a possibility that female-biased
mortality rate gives rise to male-biased sex ratio. How-
ever, this possibility is rejected because daphnids usually
produce only females under these assay conditions (i.e.,
fewer than 0.1% males for the strain we used) and male
neonates have never been observed in the controls in the
present study. The skew in the sex ratio by juvenoids in
the present study was caused not by different mortality
rates between males and females but by a functional
change to produce males by any signals.
The five chemicals, which induced male production in
the present study, have a feature in common; i.e., they
are all juvenoids. Among the five juvenoids, two IGR,
pyriproxyfen and fenoxycarb, which induced male pro-
duction at a relatively low concentration, have phen-
oxyphenyl group. We also tested the possibility that the
phenoxyphenyl group contained in pyriproxyfen and
fenoxycarb is related to the switch to male production.
However, some pesticides containing a phenoxyphenyl
structure, such as fenpropathrin, cyhalothrin, permeth-
rin, halfenprox, and ethofenprox, were not effective in
inducing male offspring (data not shown).
Several papers report an increase in the ratio of male
neonates under certain conditions (Dodson et al., 1999;
Olmstead and LeBlanc, 2000, 2001; Peterson et al.,
2001). Olmstead and LeBlanc (2001) reported that
methoprene suppressed male production in D. magna
early in the sexual reproductive phase but significantly
increased the percentage of male neonates produced later
in the sexual reproductive phase, a period when the male
production ratio is normally low. Our experiment was
performed under different experimental conditions, i.e.,
non-crowded and high food condition, but our results

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N. Tatarazako et al. / Chemosphere 53 (2003) 827–833
From the results obtained here and previous study by
logical role of invertebrates in global aquatic systems
and the apparent ubiquitous presence of forms of JH in
these species as a hormone, it is important to determine
whether the results observed in daphnids are more uni-
versal. For the conservation of aquatic ecosystems, it is
necessary to consider the influence of these insecticides
on the full range of non-target species.
Olmstead and LeBlanc (2002), pyriproxyfen, fenoxycarb,
and other JH agonists may act on molecular targets re-
sponsible for switching on male production. Although
the mechanisms associated with male production are not
clear, the chemicals used in our study can be useful as
probes for studying these mechanisms. It is clear that JH
and its receptor have some decisive role in male pro-
duction, because all juvenoids induced male production.
Besides, a decrease in reproduction of D. magna by ex-
posure to juvenoids in the present study suggests that
reproductive potency is related to the change in repro-
ductive strategy and have some mechanisms in common.
Molecular cloning of the JH receptor and studies ex-
amining the binding affinity of these chemicals should
help to elucidate the effects we observed.
Acknowledgements
We are grateful to Dr. Louis Guillette, University of
Florida, for his encouragement and for critical reading
of the manuscript. We thank Ms. Ryoko Abe and Mr.
Masaaki Koshio for the assistance in the laboratory.
Special thanks are due to Dr. H. Sonobe, who provided
us with the synthesized methylfarnesoate. This work was
supported in part by a Grant-in-Aid for Scientific Re-
search of Priority Area (A) from the Ministry of Edu-
cation, Sports, Culture, Science and Technology of
Japan and by grants from the Ministry of Environment.
The results in wash out experiment indicate that the
effect of the five juvenoids disappears in a week (Table
1). Under our experimental conditions, eggs in a brood
chamber take about 2 days to be released from the
mother. It suggests that eggs in the ovary at the end of
exposure of the test chemicals are under the influence of
juvenoid. It also implies that the effect of juvenoids to
D. magna is not permanent.
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The time necessary for producing males was also con-
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dicates that the sex determination occurs at the stage when
eggs are in the ovary. Our results support Olmstead and
LeBlanc (2002), reporting that sex determination occurred
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Norihisa Tatarazako got his Ph.D. degree in Agriculture, from
the University of Tokyo, Bunkyo-ku, Tokyo, Japan in No-
vember 2002. During April 1988–March 2001, he was an R&D
engineer at Oji Pulp and Paper Co. Ginza, Tokyo, Japan. At
this time, he studied about pulp and paper mill effluents and
developed biological tests to evaluate environmental pollution.
From April 2001 to till date, he is an R&D engineer at the
National Institute for Environmental Studies, Tsukuba, Iba-
raki, Japan. As a member and R&D engineer, he is studying
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