Stress signaling: coregulation of hemoglobin and male sex determination through a terpenoid signaling pathway in a crustacean
1 Department of Environmental and Molecular Toxicology, North Carolina State
University, Raleigh, NC 27695-7633, USA
2 Division of Hematology, Department of Medicine, Brigham and Women's
Hospital, Harvard Medical School, Boston, MA 02115, USA
* Author for correspondence (e-mail: ga_leblanc{at}ncsu.edu)
Accepted 13 October 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cladocera, juvenoid, endocrine disruption, evolution, nuclear receptor, Daphnia magna.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The terpenoid hormone methyl farnesoate is emerging in crustaceans as a
major hormone responsible for transducing environmental signals. Methyl
farnesoate is synthesized by the mandibular organ of Decapod crustaceans
(Laufer et al., 1987) and its
secretion is negatively regulated by members of the crustacean hyperglycemic
hormone (CHH) family of neuropeptides that are synthesized by the
X-organ/sinus gland complex (Liu and
Laufer, 1996
). Methyl farnesoate is structurally similar to
juvenoid hormones of insects and retinoid hormones of vertebrates, and its
activity is probably mediated through interaction with a nuclear receptor.
Methyl farnesoate has been associated with a variety of physiological
processes in crustaceans related to reproduction, including testicular
maturation (Kalavathy et al.,
1999), ovarian development
(Reddy and Ramamurthi, 1998
),
vitellogenesis (Vogel and Borst,
1989
) and mating behavior
(Laufer et al., 1993
).
Reproductive maturation in crustaceans also is subject to various
environmental signals (Benzie,
1997
). Whether some of these signals are transduced within the
organism by methyl farnesoate is not currently known.
The freshwater crustacean Daphnia magna utilizes two reproductive
strategies that are regulated by environmental signals. Daphnids typically
reproduce asexually by parthenogenesis
(Hebert, 1978;
Lynch and Gabriel, 1983
).
Diploid female offspring that are genetically identical to their mother are
generally produced during asexual reproduction. Asexual reproduction provides
for rapid expansion of the population, in a favorable environment. In response
to environmental signals such as a drop in food quantity or quality, a
decrease in photoperiod, and crowding
(Carvalho and Hughes, 1983
;
Klevien et al., 1992
; Stross
and Hill,
1968a
,b
)
daphnids enter a sexual reproductive phase by producing males and sexually
responsive females. These animals mate; the fertilized, diapause eggs are
encased in a protective ephippium, and these eggs are released into the
environment. These diapause eggs can resist desiccation or freezing and can
hatch decades following release (Hebert,
1978
). In addition, the hydrophobic ephippium facilitates
dispersal of the eggs through air currents or adherence to migrating
species.
We recently discovered that methyl farnesoate is a male sex determinant in
daphnids (Olmstead and LeBlanc,
2002). Exposure of maternal organisms with maturing oocytes in the
ovaries to methyl farnesoate causes the oocytes to develop into males. This
discovery suggests that a methyl farnesoate signaling cascade is responsible
for transducing environmental signals that are responsible for the switch from
asexual to sexual reproduction. In a search for environmental signals that
stimulate male offspring production via the methyl farnesoate
signaling pathway, we observed that females, stimulated to produce male
offspring, often develop a distinct copper color. We speculated that this
color change may represent increased hemoglobin accumulation in these
organisms (Hoshi et al.,
1977
). In the present study, we tested the hypothesis that male
offspring and hemoglobin production are coregulated by the same signaling
pathway. Results demonstrate the existence of an environmental stress response
in daphnids that is mediated by terpenoid hormones. Results also identify
potential means for identifying the terpenoid receptors of arthropods and
provide insight into mechanisms responsible for the development of asexual
populations of daphnids.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hormone treatment
Daphnids were exposed to the terpenoid hormone methyl farnesoate (Echelon
Biosciences, Salt Lake City, UT, USA), and the analogs pyriproxyfen (Chem
Service, West Chester, PA, USA), methoprene (Chem Service) and fenoxycarb
(Chem Service). This group of compounds is referred to as terpenoid hormones
in this study, based upon common activity to the terpene methyl farnesoate,
while recognizing that pyriproxyfen and fenoxycarb lack isoprene units
characteristic of terpenes. Daphnids (juveniles for hemoglobin induction and
gravid adults for male sex determination) were isolated from the cultures and
individually housed in 50 ml beakers containing 40 ml of culture medium. For
each experiment, 5-10 daphnids were exposed to the desired concentration of
chemical and 5-10 daphnids were exposed to medium without chemical. Daphnids
were provided food twice daily (1.4x107 cells of algae and 50
µl fish food suspension for D. magna, 5.6x106
cells of algae and 20 µl fish food suspension for D.
pulex/pulicaria). Daphnids were maintained under the same conditions as
described for culturing. Hemoglobin levels were measured after 48 h.
For male offspring production, daphnids were maintained under these
conditions and were transferred to new medium every 2-3 days until the release
of the third brood of offspring. Sex of the individual offspring in this brood
was determined. Third broods were evaluated to ensure that the maternal
organisms had been exposed to the hormone during the critical period of
ovarian oocyte maturation when sex of the offspring is determined
(Olmstead and LeBlanc, 2002).
Methyl farnesoate was dissolved in methanol and the analogs were dissolved in
ethanol for delivery to the daphnid media. Carrier solvent was also added to
the control medium at the same concentration present in the respective hormone
treatment, and concentrations in the final solutions never exceeded 0.02%.
Hemoglobin analyses
Spectrophotometry
Hemoglobin content of individual D. magna was determined according
to van Dam et al. (1995) with
modifications. Individual adult daphnids were homogenized in 600 µl of
distilled water by sonication for 5 s using a Vitra-CellTM hand-held
sonicator (Sonics & Materials Inc., Danbury, CT, USA). Particles were
pelleted by centrifugation at 10 000 g. A sample (400 µl)
of the supernatant was used to measure hemoglobin content. The remainder of
the supernatant was used to measure protein content
(Bradford, 1976
). Standard
solutions of bovine hemoglobin (Sigma, St Louis, MO, USA; 1.0-60 µg
hemoglobin in 400 µl) were used to generate standard curves. 24 µl of
0.10% KCN was added to each 400 µl sample. Samples were incubated for 5 min
at room temperature, then absorbance (E) was measured at 380, 415 and 440 nm.
Absorbances at 380 and 440 nm were used to discern background absorbance
flanking the absorbance peak (415 nm) of oxygenated hemoglobin. Absorbance due
to hemoglobin was calculated as:
E415-[(E380+E440)/2]. Hemoglobin absorbance
values were converted to µg hemoglobin using the standard curve. Final
hemoglobin values associated with individual daphnids were either normalized
to the protein content of the homogenate or presented as µg hemoglobin per
daphnid.
Hemoglobin color scoring
The small size of D. pulex/pulicaria and juvenile D.
magna precluded analyses of hemoglobin levels in individual animals.
Therefore, a colorimetric procedure was devised and validated using adult
D. magna as a means of estimating hemoglobin content of individuals
that were too small for direct spectrophotometric analyses. Color of daphnids
as related to hemoglobin content was numerically scored using a narrative
description of the color associated with the gonadal/visceral region and
respiratory appendages (Table
1) in combination with direct comparison to a color-gradient card,
which facilitated judgment of the intensity of color. The color gradient card
was computer generated (Photoshop, Adobe, San Jose, CA, USA) and depicted
shades of copper that encompassed the range exhibited by the daphnids. The
transparency of daphnids allowed for direct microscopic color scoring of
individuals without harming the organisms. Thus, changes in relative
hemoglobin levels could be monitored in the same organism over time. For
method validation, daphnids were scored for level of coloration using a
dissecting microscope (10x magnification), then hemoglobin content of
the individual daphnids was quantified by spectrophotometry. The relationship
between the two measures of hemoglobin was determined
(Fig. 1A,B). Coefficient of
variation between different technicians scoring the same daphnids was <15%.
Coefficient of variation of standard curves that described the relationship
between color score and true hemoglobin level (as µg per daphnid) deduced
by different technicians using different animals was <35% with respect to
both slope and x intercept. This approach was considered
semi-quantitative, as variables such as size of the organisms would be likely
to impact the scoring interpretation. This approach was only used to judge
relative differences in hemoglobin levels in relation to hormone treatment.
The method proved highly effective for assessing relative hemoglobin in
individuals for such comparisons within these limitations.
|
|
Electrophoresis
Increases in pigmented protein (i.e. hemoglobin) with pyriproxyfen
treatment were assessed following native protein polyacrylamide gel
electrophoresis. 17-20 adult D. magna were homogenized as described
above, following exposure to either 3 nmol l-1 pyriproxyfen or
carrier solvent without pyriproxyfen. Protein content of the supernatant was
determined (Bradford, 1976) and
a volume of supernatant containing 350 µg protein was subjected to
electrophoretic separation according to Laemmli
(1970
) with the following
modifications. Electrophoresis was performed using a Mighty SmallTM
electrophoresis apparatus (Bio-Rad, Hercules, CA, USA) with a 5% running gel
and a 4% stacking gel. Both gels were prepared at a pH 8.8. Sodium dodecyl
sulfate was not added to any of the gels or buffers. Electrophoresis was
performed for 30 min at 4°C and 200 V. Following electrophoresis, gel
images were digitized with a digital imager (model 640BU, Acer CM, City of
Industry, CA, USA).
mRNA analyses
Hemoglobin mRNA levels were quantified by real-time RT-PCR. 30 control or
hormone-exposed daphnids were crushed to a fine powder using a pestle and
mortar containing liquid nitrogen. RNA was isolated from the powdered
preparation using the SV Total RNA Isolation System (Promega, Madison, WI,
USA). The RNA yield was determined by absorbance at 260 nm and purity was
measured by the 260/280 nm ratio. Integrity of RNA was confirmed by
agarose/formaldehyde gel electrophoresis
(Sambrook et al., 1989). RNA
was converted to cDNA using the Promega ImProm-IITM Reverse Transcription
System with oligo (dT) primers.
The sequences for D. magna hemoglobin and actin genes were
accessed through GenBank. Primers were designed for the highly specific
non-homologous untranslated region (UTR) of hemoglobin hb2 gene. The
primers were designed using ABI Primer Express Software (Applied Biosystems,
Foster City, CA, USA) and the primer option with the lowest penalty (26.5) was
selected. The hemoglobin primers were: forward
5'-TCCTCTGACGACCTGGACTCAT-3', reverse
5'-CCATTAGCCGAGGTTGAAATTG-3'. Constitutively expressed actin was
used as a control for the reaction and to normalize hemoglobin results among
samples (forward 5'-CCTGAGCGCAAATACTCCGT-3', and reverse
5'-CAGAGAGGCCAAGATGGAGC-3'). All primers were acquired from Qiagen
(Valencia, CA, USA) and were reconstituted in TE buffer (1 mol l-1
Tris, 0.5 mol l-1 EDTA, pH 8.0). The hemoglobin hb2 gene
product was selected for analyses because this gene is primarily responsible
for the increase in hemoglobin mRNA in response to terpenoid signaling
(Gorr et al., 2004).
Real-time RT-PCR was carried out using the ABI PRISM® 7000 Sequence
Detection System with SYBR® Green PCR Mastermix on MicroAmp® Optical
96-well Reaction Plates equipped with ABI PRISMTM Optical Adhesive Covers
(Applied Biosystems). The following default parameters were used: 1 cycle of
50°C for 2 min; 1 cycle of 95°C for 10 min; 40 cycles of 95°C for
15 s followed by 60°C for 1 min; 1 cycle of 4°C for .
Dissociation curves were routinely generated with amplification products
using the protocol provided by the instrument manufacturer. A single melting
peak was consistently present following each amplification, indicating that
only a single product was amplified in each well. Relative hemoglobin mRNA
levels in the starting samples were discerned by the threshold cycle (Ct). The
Ct is the PCR cycle at which a statistically significant increase in
amplification product is detected (Bustin,
2000). Relative hemoglobin mRNA levels in the samples were
determined by dividing the Ct derived with hemoglobin primers by the Ct
derived with actin primers.
Sequence identity of the PCR product also was established. Amplification product was generated for sequencing using a Bio-Rad iCycler with the same cDNA and primers that were used in real-time RT-PCR. Amplification was performed using Promega PCR Master Mix with the same thermal profile as was used for real-time RT-PCR. A single amplification product was detected following electrophoresis in a 3.5% agarose gel. This product was excised from the gel and sequenced (Seqwright, Houston, TX, USA). The derived sequence was entered into an NCBI nucleotide Blast search. Significant matches were made with `Daphnia magna dhb2 mRNA for hemoglobin' (E value=2e-18) and `Daphnia magna hemoglobin gene cluster (dhb3, dhb1 and dhb2 genes)' (E value=4e-17), accession numbers AB021136 and AB021134, respectively. No significant homology was determined between the amplification product and any other gene product.
Male sex determination
Neonatal male and female daphnids were distinguished based upon the longer
first antennae of males as determined under 10x magnification
(Olmstead and LeBlanc, 2000).
Neonatal daphnids identified as male using this criterion mature into
completely differentiated males that exhibit normal male reproductive
behaviors (G.A.L., personal observations).
Statistics
Significant differences were evaluated using ANOVA and Dunnett's multiple
comparison test when comparing several treatments to a control. Paired
comparisons were evaluated using Student's t-test. Significance was
established at P<0.05 and all analyses were performed using JMP
statistical software (SAS Institute, Cary, NC, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The increase in hemoglobin levels with terpenoid treatment was further evaluated using the potent inducer pyriproxyfen (Fig. 4). Exposure of daphnids to pyriproxyfen significantly elevated levels of pigment (Fig. 4A) that was specifically associated with an electrophoretically distinct protein (Fig. 4B) that shared spectral characteristics with hemoglobin (Fig. 4C). Pyriproxyfen also elevated levels of hemoglobin hb2 mRNA as measured by real-time RT-PCR (Fig. 4C). Taken together, these results demonstrate that the increase in pigmentation in response to terpenoid hormones represents elevated hemoglobin levels. Elevated hemoglobin levels were evident within the first 24 h of exposure to pyriproxyfen and levels continued to increase linearly over at least the next 24 h (Fig. 5).
|
|
Responsiveness of daphnid clones to terpenoid hormones
Eleven clonal populations of the D. pulex/pulicaria
species complex, along with our laboratory stock of D. magna, were
evaluated for responsiveness to methyl farnesoate with respect to hemoglobin
accumulation and male sex determination. Five populations of D.
pulex/pulicaria were observed in culture to produce males and six were
considered non-male producers, based upon extensive observation. Methyl
farnesoate treatment elevated hemoglobin levels in all five clones of male
producers, along with the laboratory stock of D. magna
(Table 2). All but one clone
(MP5) also produced male offspring in response to methyl farnesoate
(Table 2). Incontrast, none of
the non-male producing clones produced male offspring in response to methyl
farnesoate and only one clone (NP1) produced hemoglobin in response to the
hormone. The general coresponsiveness of hemoglobin accumulation and male sex
determination among the various clones to methyl farnesoate provides further
evidence that these processes are regulated by a common signaling pathway.
Clones NP2 through NP6 are apparently defective in some component of this
pathway. In contrast, the induction of hemoglobin but not male offspring
production by methyl farnesoate with clones MP5 and NP1 suggests that these
clones are defective in sex-determining genes downstream from the methyl
farnesoate signaling pathway.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hemoglobin levels are typically elevated in daphnids in response to low
dissolved oxygen (Kimura et al.,
1999; Kobayashi and Hoshi,
1982
) through the action of the hypoxia signaling pathway
(Bunn and Poyton, 1996
;
Nambu et al., 1996
). Hypoxia
inducible factor (HIF) is a transcriptional activator that responds to hypoxia
by binding to hypoxia response elements (HRE) located in the promoter region
of responsive genes (Bunn and Poyton,
1996
). The hypoxia-activated hemoglobin hb2 gene of
Daphnia magna is flanked by several HREs that are able to confer
robustly induced and HIF-dependent gene transcription in response to low
oxygen levels (Gorr et al.,
2004
). The induction of hemoglobin confers increased tolerance of
the daphnids to low environmental dissolved oxygen levels, allowing the
organisms to survive this stress (Pirow et
al., 2001
).
Male offspring are produced by daphnids in response to environmental
stressors such as reduced food and crowding
(Hobaek and Larsson, 1990;
Olmstead and LeBlanc, 2001
).
Other environmental signals, such as changes in photoperiod, are also known to
stimulate the production of male offspring
(Stross and Hill, 1968a
).
Environmental signals that stimulate male offspring production are typically
associated with the onset of seasonal conditions (i.e. pond desiccation or
freezing) that will prove inhospitable to the population. The introduction of
males into the population allows for sexual reproduction which is typically
associated with the generation of drought or freezing-resistant diapause eggs.
This reproductive strategy allows for survival of the population during
periods of adversity.
The environmental signal to produce male offspring is transduced, within
the organism, by a terpenoid signaling pathway
(Fig. 6; Olmstead and LeBlanc, 2002;
Tatarazako et al., 2003
). The
results of the present study demonstrate that hemoglobin levels are regulated
by this same terpenoid signaling pathway in addition to the hypoxia/HIF
signaling pathway. Male sex determination required only a pulse of methyl
farnesoate during a critical period of ovarian oocyte maturation
(Olmstead and LeBlanc, 2002
).
In contrast, hemoglobin levels increase with increasing duration of elevated
hormone levels (Fig. 5). Thus,
environmental factors that stimulate a sustained elevation in hormone levels
would be likely to impact both sex determination and hemoglobin levels, while
transient increases in hormone levels would significantly impact only sex
determination. Taken together, environmental factors may exist that impact
hemoglobin synthesis only (sustained low oxygen), male offspring production
only (those causing a terpenoid hormone pulse), or both processes (those
causing a sustained increase in terpenoid hormone). Studies are underway to
identify putative environmental signals that costimulate both processes.
|
Zeis et al. (2004) recently
reported that hemoglobin concentration in daphnids increased with increasing
temperature. Mitchell (2001
)
noted a low incidence of sexually ambiguous offspring when daphnids were
reared at 30°C. Elevated temperature may prove to be an environmental
signal that stimulates both hemoglobin induction and male sex determination
through the common signaling pathway. Preliminary experiments in our
laboratory support this premise. However, elevated temperature may stimulate
different signaling pathways, resulting in multiple outcomes. For example,
oxygen saturation decreases with increasing water temperature, which may
stimulate hemoglobin production via the hypoxia signaling pathway.
Increased temperature may also adversely impact the uptake or assimilation of
nutrients resulting in male production via the terpenoid signaling
pathway.
The minimum components to the putative terpenoid signaling pathway described in this study would consist of the hormone (i.e. methyl farnesoate), its receptor (i.e. methyl farnesoate receptor) and response elements on responsive genes (i.e. hemoglobin genes, sex determining genes; Fig. 6). Components upstream of the responsive genes would be common components to the pathway. Five of the six non-male producing clones of daphnids evaluated produced neither male offspring nor hemoglobin in response to methyl farnesoate treatment. This complete lack of responsiveness suggests that these clones are deficient in some common component of the signaling pathway. Since methyl farnesoate was provided to the organisms, hormone was not deficient in the treated organisms. Thus the likely common component that was deficient in these organisms is the methyl farnesoate receptor. In contrast, two of the evaluated clones produced hemoglobin in response to methyl farnesoate but did not produce males. This would suggest that the common components to the signaling pathway are intact but that these clones are deficient in some terminal sex-determining genes. These variously deficient clones may prove highly useful in future studies aimed at identifying the methyl farnesoate receptor as well as genes involved in sex determination.
The regulation of male offspring production by methyl farnesoate implies
that daphnids possess sex determining genes, such as sex-1 in C.
elegans (Carmi et al.,
1998), dsx in Drosophila
(Yi and Zarkower, 1999
), or
csd in the honeybee (Beye et al.,
2003
). Some of the sex-determining genes of daphnids, along with
the methyl farnesoate-responsive hemoglobin genes, may possess
cis-acting regulatory elements that interact with the putative methyl
farnesoate receptor. We have identified a functional cis element in
the promoter of the hemoglobin hb2 gene of D. magna that
binds nuclear factor(s) present in methyl farnesoate-treated daphnids (G.A.L.
and T.A.G., manuscript in preparation). This element resembles binding motifs
of several mammalian orphan receptors, most notably NGFI-B
(Wilson et al., 1991
), SF-1
(Wilson et al., 1993
), and RZR
(Carlberg et al., 1994
)
proteins, all of which bind as monomers to their target DNA.
The coregulation of hemoglobin levels and male offspring production by a
common signaling pathway suggests some survival advantage to this phenomenon.
Daphnids commonly inhabit temporary ponds
(Dudycha, 2004), and
environmental signals that forewarn the complete desiccation of the habitat
during summer may stimulate the terpenoid signaling pathway. Thus, this early
signal of impending habitat loss would provide sufficient time for entry of
the population into the sexual phase of the reproductive cycle.
Desiccation-resistant diapause eggs would result from the sexual reproduction
allowing for survival of the population. Increased hemoglobin may occur in
response to this signal to meet the respiratory requirement of sexual
reproduction in a habitat experiencing decreasing oxygen-carrying capacity due
to increasing temperature. Future studies may reveal that the methyl
farnesoate signaling pathway induces specific hemoglobin subunits that have
increased oxygen affinity in elevated temperature environments
(Lamkemeyer et al., 2003
).
Lastly, the results of the present study provide insight into the evolution
of non-male-producing populations of daphnids. As cyclic parthenogens, daphnid
populations can benefit from rapid population growth during periods of
environmental stasis, yet can resort to sexual reproduction to survive periods
of environmental change. Costs of sexual reproduction to the population are
significant since males contribute no offspring to the population, and sexual
reproduction produces few offspring relative to the numbers produced clonally
(Innes et al., 2000;
Korpelainen, 1992
). The
benefit of sexual reproduction is genetic exchange among individuals coupled
to diapause and dispersion (Hebert,
1978
; Rispe and Pierre,
1998
). Sexual reproduction may be crucial to the survival of
populations inhabiting environments that are periodically rendered
inhospitable due to complete desiccation, freezing, etc. A mutation that
disables the terpenoid stress signaling pathway, such as a mutation in the
methyl farnesoate receptor gene, in a population of daphnids inhabiting a
marginally variable environment (i.e. a habitat where environmental signals
stimulate sexual reproduction, but where sexual reproduction/diapause egg
production is not necessary for the survival of the population) would be
selected for as these mutant daphnids would not pay the costs associated with
male production. Such a marginally variable environment may exist, for example
where water temperatures become significantly elevated but the habitat never
completely desiccates. Considering that all of the methyl farnesoate analogs
used in this study have commercial application as insecticides raises
questions as to whether the introduction of such chemicals into the
environment could result in the establishment of artificial marginal
environments causing genetic drift from cyclic parthenogenic to non-male
producing parthenogenic populations.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Benzie, J. A. H. (1997). A review of the effect of genetics and environment on the maturation and larval quality of the giant tiger prawn Penaeus monodon. Aquaculture 155, 69-85.[CrossRef]
Beye, M., Hasselmann, M., Fondrk, M. K., Page, R. E. and Omholt, S. W. (2003). The gene csd is the primary signal for sexual development in the honeybee and encodes an SR-Type protein. Cell 114,419 -429.[Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Bronson, F. H. (1985). Mammalian reproduction: an ecological perspective. Biol. Reprod. 32, 1-26.[Abstract]
Bunn, H. F. and Poyton, R. O. (1996). Oxygen
sensing and molecular adapatation to hypoxia. Physiol.
Rev. 76,839
-885.
Bustin, S. A. (2000). Absolute quantification
of mRNA using real-time reverse transcription polymerase chain reaction
assays. J. Mol. Endocrinol.
25,169
-193.
Carlberg, C., van Huijsduijnen, R. H., Staple, J. K., DeLamarter, J. F. and Becker-Andre, M. (1994). RZRs, a new family of retinoid-related orphan receptors that function as both monomer and homodimers. Mol. Endocrinol. 8, 757-770.[Abstract]
Carmi, I., Kopczynski, J. B. and Meyer, B. J. (1998). The nuclear hormone receptor SEX-1 is an X-chromosome signal that determines nematode sex. Nature 396,168 -173.[CrossRef][Medline]
Carvalho, G. and Hughes, R. (1983). The effect of food availability, female culture-density, and photoperiod on ephippia production in Daphnia magna Straus (Crustacea: Cladocera). Freshwater Biol. 13,37 -46.
Dudycha, J. L. (2004). Mortality dynamics of Daphnia in contrasting habitats and their role in ecological divergence. Freshwater Biol. 49,505 -514.[CrossRef]
Gorr, T. A., Cahn, J. D., Yamagata, H. and Bunn, H. F.
(2004). Hypoxia-induced synthesis of hemoglobin in the crustacean
Daphnia magna is hypoxia-inducible factor-dependent. J.
Biol. Chem. 279,36038
-36047.
Hebert, P. D. N. (1978). The population biology of Daphnia (Crustacea, Daphnidae). Biol. Rev. 53,387 -426.
Hobaek, A. and Larsson, P. (1990). Sex determination in Daphnia magna. Ecology 71,2255 -2268.
Horie, Y., Kanda, T. and Mochida, Y. (2000). Sorbitol as an arrester of embryonic development in diapausing eggs of the silkworm, Bombyx mori. J. Insect Physiol. 46,1009 -1016.[CrossRef][Medline]
Hoshi, T., Yahagi, N. and Watanabe, T. (1977). Studies on physiology and ecology of plankton. Further studies on O2 response to haemoglobin of Daphnia magna in vivo. Sci. Rep. Niigata Univ. Ser. D 11, 7-13.
Innes, D. J., Fox, C. J. and Winsor, G. L. (2000). Avoiding the cost of males in obligately asexual Daphnia pulex (Leydig). Proc. R. Soc. Lond. B 267,991 -997.[CrossRef][Medline]
Kalavathy, T., Mamatha, P. and Sreenivasula Reddy, P. (1999). Methyl farnesoate stimulates testicular growth in the freshwater crab Oziotelphusa senex senes fabricius. Naturwissenschaften 86,394 -395.[CrossRef]
Kimura, S., Tokishita, S. I., Ohta, T., Kobayashi, M.,
Kobayashi, M. and Yamagata, H. (1999). Heterogeneity and
differential expression under hypoxia of two-domain hemoglobin chains in the
water flea, Daphnia magna. J. Biol. Chem.
274,10649
-10653.
Klevien, O., Larsson, P. and Hobaek, A. (1992). Sexual reproduction in Daphnia magna requires three stimuli. Oikos 65,197 -206.
Kobayashi, M. and Hoshi, T. (1982). Relationship between the haemoglobin concentration of Daphnia magna and the ambient oxygen concentration. Comp. Biochem. Physiol. 72A,247 -249.[CrossRef]
Korpelainen, H. (1992). Lowered female reproductive effort as an indicator for increased male production and sexuality in Daphnia (Crustacea: Cladocera). Invert. Reprod. Dev. 22,281 -290.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Lamkemeyer, T., Zeis, B. and Paul, R. J. (2003). Temperature acclimation influences temperature-related behavious as well as oxygen-transport physiology and biochemistry in the water flea Daphnia magna. Can. J. Zool. 81,237 -249.[CrossRef]
Laufer, H., Borst, D., Baker, F. C., Carrasco, C., Sinkus, M., Reuter, C. C., Tsai, L. W. and Schooley, D. A. (1987). Identification of a juvenile hormone-like compound in a crustacean. Science 235,202 -205.
Laufer, H., Ahl, J. S. B. and Sagi, A. (1993). The role of juvenile hormones in crustacean reprodution. Amer. Zool. 33,365 -374.
Liu, L. and Laufer, H. (1996). Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hyperglycemic effects from the spider crab, lbinia emarginata. Arch. Insect Biochem. Physiol. 32,375 -385.[CrossRef]
Lynch, M. and Gabriel, W. (1983). Phenotypic evolution and parthenogenesis. Am. Nat. 122,745 -764.[CrossRef]
Mitchell, S. E. (2001). Intersex and male development in Daphnia magna. Hydrobiologia 442,145 -156.[CrossRef]
Nambu, J. R., Chen, W., Hu, S. and Crews, S. T. (1996). The Drosophila melanogaster similar bHLH-PAS gene encodes a protein related to human hypoxia-inducible factor 1 alpha and Drosophila single-minded. Gene 172,249 -254.[CrossRef][Medline]
Olmstead, A. W. and LeBlanc, G. A. (2000). Effects of endocrine-active chemicals on the development of sex characteristics of Daphnia magna. Environ. Toxicol. Chem. 19,2107 -2113.
Olmstead, A. W. and LeBlanc, G. A. (2001). Temporal and quantitative changes in sexual reprodutive cycling of the cladoceran Daphnia magna by a juvenile hormone analog. J. Exp. Zool. 290,148 -155.[CrossRef][Medline]
Olmstead, A. W. and LeBlanc, G. A. (2002). The juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna. J. Exp. Zool. 293,736 -739.[CrossRef][Medline]
Olmstead, A. W. and LeBlanc, G. A. (2003). Insecticidal juvenile hormone analogs stimulate the production of male offspring in the crustacean Daphnia magna. Environ. Health Perspect. 111,919 -924.[Medline]
Pirow, R., Baumer, C. and Paul, R. J. (2001). Benefits of haemoglobin in the cladoceran Daphnia magna. J. Exp. Biol. 204,3425 -3441.[Medline]
Reddy, P. S. and Ramamurthi, R. (1998). Methyl farnesoate stimulates ovarian maturation in freshwater crab Oziotelphusa senex senex Fabricius. Curr. Sci. 74, 68-70.
Rispe, C. and Pierre, J. S. (1998). Coexistence between cyclical parthenogens, obligate parthenogens, and intermediates in a fluctuating environment. J. Theor. Biol. 195,97 -110.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Steger, R. W. and Bartke, A. (1996). Environmental modulation of neuroendocrine function. In Handbook of Endocrinology, vol. I (ed. G. H. Gass and H. M. Kaplan), pp. 121-156. New York: CRC Press.
Stross, R. G. and Hill, J. C. (1968a). Photoperiod control of winter diapause in freshwater crustacean Daphnia. Biol. Bull. 134,176 -198.
Stross, R. G. and Hill, J. C. (1968b). Diapause induction in Daphnia requires two stimuli. Science 150,1462 -1464.
Tatarazako, N., Oda, S., Watanabe, H., Morita, M. and Iguchi, T. (2003). Juvenile hormone agonists affect the occurrence of male Daphnia. Chemosphere 53,827 -833.[CrossRef][Medline]
van Dam, R. A., Barry, M. J., Ahokas, J. T. and Holdway, D. A. (1995). Toxicity of DTPA to Daphnia carinata as modified by oxygen stress and food limitation. Ecotoxicol. Environ. Safety 31,117 -126.[CrossRef][Medline]
Vogel, J. M. and Borst, D. W. (1989). Spider crab yolk protein: Molecular characterization and the effects of methyl farnesoate (MF) on its hemolymph levels. Amer. Zool. 29, 49A.
Wilson, T. E., Fahrner, T. J., Johnston, M. and Milbrandt, J. (1991). Identification of the DNA binding site of NGF1-B by genetic selection in yeast. Science 252,1296 -1300.[Medline]
Wilson, T. E., Farhner, T. J. and Milbrandt, J. (1993). The orphan receptors NGF1-B and steroidogenic factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol. Cell. Biol. 13,5794 -5804.[Abstract]
Yamashita, O. (1996). Diapause hormone of the silkworm, Bombyx mori: structure, gene expression and function. J. Insect Physiol. 42,669 -679.[CrossRef]
Yi, W. and Zarkower, D. (1999). Similarity of
DNA binding and transcriptional regulation by Caenorhabditis elegans
MAB-3 and Drosophila melanogaster DSX suggests conservation of sex
determining mechanisms. Development
126,873
-881.
Zeis, B., Lamkemeyer, T. and Paul, R. J. (2004). Molecular adaptation of Daphnia magna hemoglobin. Micron 35,47 -49.[CrossRef][Medline]
Related articles in JEB: