Biologia 63/2: 139—150, 2008
Section Zoology
DOI: 10.2478/s11756-008-0027-x
Review
Endocrine regulation of the reproduction in crustaceans:
Identification of potential targets for toxicants and environmental
contaminants
Edita Mazurová1, Klára Hilscherová1,4, Rita Triebskorn2,3, Heinz-R. Köhler2,
Blahoslav Maršálek1,4 & Luděk Bláha1,4*
1
Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX), Masaryk University, Kamenice 3,
CZ-62500 Brno, Czech Republic; e-mail: [email protected]
2
Animal Physiological Ecology Department, Eberhard-Karls University, Tübingen, Germany
3
Steinbeis-Transferzentrum für Ökotoxikologie und Ökophysiologie, Rottenburg, Germany
4
Academy of Sciences of the Czech Republic, Institute of Botany, Brno, Czech Republic
Abstract: Progress in ecotoxicological research documents that crustaceans are highly vulnerable to diverse chemicals and
toxicants in the environment. In particular, pollutants affecting endocrine homeostasis in crustaceans (i.e., endocrine disruptors) are intensively studied, and serious reproductive disorders have been documented. In this review, current knowledge
about the endocrine regulation of the crustacean reproduction is put together with the published ecotoxicological data with
an attempt to summarize the potential of xenobiotics to affect crustacean reproduction. Following gaps and trends were
identified: (1) Studies are required in the field of neurohormone (serotonin and dopamine) regulation of the reproduction and
possible modulations by environmental toxicants such as antidepressant drugs. (2) Molting-related parameters (regulated
by ecdysteroid hormones) are closely coordinated with the development and reproduction cycles in crustaceans (cross-links
with methyl farnesoate signalling), and their susceptibility to toxicants should be studied. (3) Other biochemical targets
for xenobiotics were recently discovered in crustaceans and these should be explored by further ecotoxicological studies
(e.g., new information about ecdysteroid receptor molecular biology). (4) Some sex steroid hormones known from vertebrates (testosterone, progesterone) have been reported in crustaceans but knowledge about their targets (crustacean steroid
receptors) and signalling is still limited. (5) Determination of the sex in developing juveniles (affecting the sex ratio in
population) is a sensitive parameter to various xenobiotics (including endocrine disruptors) but its modulation by general
environmental stress and non-specific toxicity should be further studied.
Key words: crustaceans; reproduction; endocrine disruption; sex determination; contaminant; ecotoxicology
Introduction
Available data provide a clear evidence that crustaceans
are vulnerable to diverse chemicals and toxicants in
the environment (James & Boyle 1998; Olmstead &
LeBlanc 2007; Verslycke et al. 2007), thus a range of
crustacean species is often used in ecotoxicological research. In particular, compounds and pollutants affecting endocrine homeostasis (i.e., endocrine disruptors)
are intensively studied also in crustaceans (Hutchinson 2002; LeBlanc 2007, Rodriguez et al. 2007). Serious reproductive disorders in crustaceans have been
documented after exposure to such chemicals as insects hormones and their mimics (Peterson et al. 2001)
or vertebrate-like (xeno)hormones (Zou & Fingerman
1997). Current approaches to study endocrine toxicology of crustaceans include (1) studies with complex environmental mixtures with expected endocrine toxicity,
and (2) controlled laboratory experiments with individual chemicals (suspected endocrine disruptors).
(1) Reproduction processes are generally sensitive
to various endogenous and exogenous factors including total energy pool (e.g., food supplies), seasonal
variations and environmental conditions (including pollution), detoxification metabolism and also hormonal
regulations at various levels. Studies that examine responses of wildlife to complex polluted environment
often discussed developmental and reproduction toxicity. However, it may be often difficult to find direct
links between unspecific effects and endocrine disruption caused by chemical pollution (Brian 2005; Zou
2005).
(2) Other studies with crustaceans focus on known
or suspected endocrine disruptors with the aim to explore alteration of biological functions on biochemical
and molecular levels. Several of such studies in crustaceans add new information to mechanisms and pathways previously described only in traditional insect
and/or vertebrate models (Verslycke et al. 2002; Wu
et al. 2004; Kim et al. 2005a).
* Corresponding author
c 2008 Institute of Zoology, Slovak Academy of Sciences
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CRUSTACEAN
ENDOCRINE SYSTEM
Sinus gland/X-organ
MOIH, MIH, GIH/VIH
GIH/VIH ?
MOIH ?
MIH ?
Serotonin ?
Dopamine?
?
Mandibular organ
MF
Y-organ
20-HE
Gonads
Steroids (T)
Males: AGH ?
Androgenic gland
AGH
Cerebral and Thoracic ganglia
Serotonin, Dopamine
MF ?
MF ?
Adults: MF ?
Juveniles: MF ?
Testosterone ?
Physiological
effects
Development/
Ontogeny/Maturation
Molting
Adults: reproduction activity
control
Reproduction
effects
Reproduction cycle control;
? males in brood
Male sex development,
male reproduction
Adults: reproduction potency ?
Juveniles: delay in maturation ?
Fig. 1. The diagram of endocrine system in crustaceans with specific regard to regulation of reproduction. The individual glands and
their active substances are described in the upper part, physiological and reproduction endpoints are depicted below. The complex
of sinus gland and X-organ downregulates gonads directly by GIH/VIH (Gonads/Vitellogenesis Inhibiting Hormone), and it also
regulates Y-organ by MIH (Molt-Inhibiting Hormone) and mandibular organ by MOIH (Mandibular Organ Inhibiting Hormone). Also
the products of cerebral and thoracic ganglia (biogenic amines serotonin and dopamine) were found to affect gonadal activity but the
mechanisms are not known (dashed lines/arrows). Mandibular organ produces methyl farnesoate (MF) that regulates gonads directly
(variable effects depending on the maturity status), and it also indirectly affects molting (probably via signaling of 20-hydroxyecdysone,
20-HE). Further, product of the androgenic gland (Androgenic Gland Hormone, AGH) affects gonads and promotes development of
male sexual characteristics. Competition for receptor sites has been discovered also between testosterone (T) and 20-HE.
This review combines current knowledge on crustacean endocrinology (with special respect to reproduction, see Fig. 1) with data from selected ecotoxicological studies. We have focused on reproduction related
effects, as it is one of the most important biological
processes associated with species fitness and population
survival. Rather than summarization of many ecotoxicological case studies, we have aimed to identify general physiological targets in crustaceans vulnerable to
exogenous xenobiotics including endocrine disruptors.
The review covers following major areas related to reproduction physiology and toxicity in crustaceans: (i)
neuroendocrine control of reproduction, (ii) role of classical endocrine glands in reproduction, and (iii) sex determination in crustaceans.
Neuroendocrine control of reproduction
To the present knowledge, neuroendocrine regulation
axis shares similar structure inside a broad Coelomates
clade. In crustacean taxa, known secretory active sites
include neurosecretory cells in cerebral and thoracic
ganglion, sinus gland/X-organ complex, postcomissural
organ and pericardial organ (Cooke & Sullivan 1982).
This chapter focuses first on the complex of sinus gland
and X-organ that were extensively studied, and they are
functionally well characterized. Secondly, we summarize functions of various biogenic amines in crustaceans
since they may also play a role in reproduction processes.
Neurohormones produced by X-organ
Sinus gland is a neurohemal organ located laterally on
optic ganglia in eyestalks and it is connected to X-organ
via a dense nervous tissue. Both organs are paired and
they serve as a key endocrine junction of central neurosecretory signals (Withers 1992).
The peptide hormones released from X-organ have
diverse effects on growth and reproduction: moltinhibiting hormone (MIH) suppresses production of
ecdysteroids in Y-organ; mandibular-organ inhibiting
hormone (MOIH) down-regulates excretion of methyl
farnesoate (MF) from mandibular gland; and gonad inhibiting hormone (GIH, or according to some authors
vitellogenin inhibiting hormone – VIH) represses ovarian maturation and testes growth (Chang 1993; Ohira
et al. 1999). However, precise actions of separate neuropeptides are not yet fully understood since most of
the studies used whole organ homogenates or eyestalk
ablated animals (in which whole complex of sinus gland
and X-organ was removed).
For example, the biochemical character of GIH is
still a matter of discussion (Rodriguez et al. 2002a),
and its structure seems to vary among species (Chaves
2000). Therefore, factors with MIH/CHH/GIH effects
were grouped into a peptide family of X-organ neuroUnauthenticated
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Ecotoxicology of crustacean reproduction
hormones but their precise structural variability and
detailed physiological effects remain to be investigated
(Chang 1993).
Molt-inhibiting hormone (MIH) inhibits the production of ecdysteroids by Y-organ affecting thus duration of the molting cycle and the onset of reproduction.
However, the study with the crab Carcinus maenas L.,
1758 demonstrated that the sensitivity of Y-organ itself
to MIH is a more important factor than levels of MIH
(Chung & Webster 2003). This was also supported by a
study with prawn Macrobrachium rosenbergii De Man,
1879, where female eyestalk ablation resulted in shortening of the reproduction molting period but levels of
ecdysteroid (as well as vitellogenin) did not substantially vary during molting stages (Okumura & Aida
2001). In vitro experiments with tissues of estuarine
crab Chasmagnathus granulata Dana, 1851 also showed
that heavy metals such as cadmium and copper inhibit
release of GIH (Medesani et al. 2004).
In summary, functions of neuropeptides produced
by sinus gland/X-organ seem to be complex and highly
variable among crustacean species, and full understanding to their biochemical regulatory pathways (and effects on reproduction) will require further research.
Biogenic amines with neurohormonal function
Biogenic amines – serotonine (5-hydroxytryptamin, 5HT), dopamine and octopamine – were first detected
in secretory cells in pericardial organ (Cooke & Sullivan 1982), and for a long time they have been considered only neurotransmitters in nervous synapses of
crustaceans (for a review see, e.g., Cooke & Sullivan
(1982)). Later, these amines (plus another one, proctolin) have been shown to act as neurohormones being
released into the hemolymph and having ‘adrenalinelike’ effects (Cooke & Sullivan 1982; Siwicki et al. 1987).
Several studies suggest that biogenic amines are
also able to alter reproduction processes in crustaceans,
and it seems that serotonin generally stimulates and
dopamine inhibits reproduction potency. For example,
it has been shown that serotonin caused significant
stimulation of testes index and number of spermatocytes per testicular lobe in fiddler crab Uca pugilator
Bosc, 1802 while dopamine (and its agonist 2-amino6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, ADTN)
retarded testicular development (Sarojini et al. 1995a).
Testicular maturation was also promoted in red swamp
crayfish Procambarus clarkii Girard, 1852 by injections
of serotonin and spiperone (a dopamine antagonist)
(Sarojini et al. 1995b). Injection of spiperone also increased gonadosomatic index and oocyte diameter in
females of P. clarkii (Rodriguez et al. 2002b). It has
also been shown that serotonin induces ovarian maturation and spawning in white shrimp Penaeus vannamei
Boone, 1931 (Vaca & Alfaro 2000).
Regulatory role of biogenic amines during gonadal
development has been further supported by a study of
Sarojini et al. (1997). Authors exposed oocytes from P.
clarkii to the thoracic ganglia (producing various biogenic amines), and they observed stimulation of oocyte
141
growth. This stimulation was further promoted by additions of methionine enkephaline (M-ENK; a ligand of
opioid receptor known to stimulate serotonin synthesis).
Contrary, oocyte growth was inhibited by naxolone, an
opioid antagonist (Sarojini et al. 1997). However, it
seems that serotonin does not affect gonads directly.
For example, methyl farnesoate (juvenile hormone analog that plays a role in crustacean molting and it
also stimulates gonadal maturation; see also discussion
below), and also other factors (named vitellogenesisstimulating hormone VSH or vitellogenesis-stimulating
ovarian hormone VSOH) were suggested as intermediates in serotonin-dependent gonadal stimulation (Rodriguez et al. 2002a).
Some ecotoxicological studies also confirm that
processes regulated by biogenic amines may become a
target of selected environmentally relevant endocrine
disruptors. Fluoxetine (Prozac, selective serotonine reuptake inhibitor – SSRI) dosed in environmentally relevant concentration 0.1 mg L−1 seriously inhibited
fecundity of cladoceran Ceriodaphnia dubia Richard,
1894 (Brooks et al. 2003). Reduction of neonate numbers was also observed during chronic exposure of C.
dubia to five SSRIs (fluoxetine, fluvoxamine, paroxetine, citalopram, sertraline) (Henry et al. 2004). On the
other hand, significant fecundity stimulations were observed in Daphnia sp. O.F. Müller, 1785 after chronic
exposures to fluoxetine (36 µg L−1 , 30 day) (Flaherty
& Dodson 2005), and authors also reported serious developmental defect and increased mortality when combined with other drugs. Slight enhancement of fecundity and significant growth reduction was reported in
Hyalella azteca Saussure, 1858 exposed to fluoxetine in
sediment (Brooks et al. 2003).
Taken together, there are clear evidences that biogenic amines may (indirectly) affect reproduction processes in crustaceans, and their action may be negatively modulated by certain compounds (such as pharmaceuticals) polluting the environment (Kummerer
2004).
Role of classical endocrine glands in reproduction
All crustacean “classical” endocrine glands seem to
modulate reproduction. As also mentioned above, reproduction is sensitive to various non-specific factors
(such as general environmental stress, detoxification
metabolism, energy budget etc.), and it may therefore
be complicated to directly relate reproduction changes
to chemical-induced endocrine disruption. This section
compiles the current knowledge about crustacean endocrine glands and their hormones with available ecotoxicological studies.
Y-organ
Y-organ is histologically simple secretory organ located
on the head (near the base of the antennules) and it produces various hormones that regulate molting in crustaceans. As mentioned in the previous section, its secreUnauthenticated
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142
tory function is suppressed by molt-inhibiting hormone
(MIH) produced by X-organ (Withers 1992). However,
levels of MIH remain relatively constant during molting cycle. Consequently, regulation of the sensitivity of
Y-organ to MIH seems to have an important role. In
fact, it has been shown that sensitivity of Y-organ to
MIH significantly dropped in the premolt stage, and
it was restored again during later molt and postmolt
stages, but the factor that modulates the perceptivity of Y-organ has not been described (Chung & Webster 2003). Factors such as alterations in cellular second messengers (cAMP), modulations of steroidogenic
enzymes or cholesterol uptake into Y-organ were suggested to be involved (Spaziani et al. 1999). Thus, modulations of some of these biochemical targets by xenobiotics may affect Y-organ secretory function and, consequently, molting and reproduction in crustaceans.
Molting hormones produced by Y-organ in all
Ecdysozoans (insects and crustaceans) chemically belong to steroids. Similarly to vertebrates, cholesterol (a steroid hormone precursor) is delivered to
Y-organ where steroidogenesis takes place on mitochondria and microsomes (Spaziani et al. 1999). The
(ecdy)steroidogenesis is a complicated network of enzymatic reactions, and it has not been completely described. In contrast to vertebrate hormones active molting hormones (ecdysteroids) contain aliphatic side chain
(Spaziani et al. 1999), but it may be expected that
ecdysteroidogenesis would be sensitive to similar compounds that modulate steroidogenesis in vertebrates.
The most potent endogeneous ecdysteroid is 20hydroxyecdysone (20-HE). Its concentrations in the
hemolymph are within µg L−1 concentrations in the intermolt stage, and it may reach up to 100 µg L−1 in late
premolt/ecdysis/early postmolt stages as documented
in M. rosenbergii (Okumura & Aida 2001). Other ecdysteroids have also been detected in hemolymph or other
tissues but usually in lower concentrations (Okumura
& Aida 2001).
Ecdysteroid action in target cells manifests via intracellular ecdysteroid receptor (EcR). EcR belongs to
a nuclear receptor family (receptors acting as transcription factors), and it has a typical gene composition of
five domains from which DNA-binding domain (DBD)
and ligand-binding domain (LBD) are phylogenetically
conserved. Analyses of these two domains in Uca pugilator (Durica et al. 2002) and Gecarcinus lateralis Freminville, 1835 (Kim et al. 2005a) revealed high similarity
to EcR from insects. Recent studies with U. pugilator
also suggested temporal- and organ/tissue-specific distribution of several EcR isoforms (Durica et al. 2002).
It may be deduced that various EcRs control specific
responses in different organs and determine thus tissue
sensitivity to ecdysteroids (Kim et al. 2005a; Zou 2005).
In the nucleus of responsive crustacean cells, activated EcR hybridizes with another important nuclear receptor, retinoid X receptor (RXR) (Durica et
al. 2002). RXR derived from fiddler crab U. pugilator (UpRXR) also exists in various isoforms (differing
in splicing hinge regions), and it has been shown that
E. Mazurová et al.
only RXR isoform with a specific hinge region in LBD
domain (UpRXR+33) may hybridize with EcR, bind
to ecdysteroid hormone responsive elements on DNA
and modulate expression of target genes (Durica et al.
2002). It is also known that the DBD domain of crustacean RXR is closely related to insect ultraspiracle
(USP) receptor, while LBD of RXR shares greater similarity to vertebrate RXRs rather than to USP (Wu et
al. 2004). However, there are apparent functional differences between vertebrates and crustaceans as neither
9-cis retinoic acid nor all-trans-retinoic acid (major vertebrate retinoid receptor ligands) were able to activate
RXR in crustaceans (Peterson et al. 2001).
Described ecdysteroid signalling may be affected
by xenobiotics as documented by some ecotoxicological studies. For example, male vertebrate sex steroid
hormone testosterone (in relatively high concentrations
5–40 µM) interfered with 20-HE signalling in Daphnia
magna Straus, 1820 causing abnormal progeny development and prolongation of the first molting (Mu &
LeBlanc 2002). Testosterone (Mu & LeBlanc 2002) as
well as three estrogens, bisphenol A, diethylphtalate
and lindane (Dinan et al. (2001), antagonized effects
of 20-HE in an insect (Drosophila) in vitro model systems indicating a ligand-competitive mechanism. Similarly, other two xeno-ecdysteroids, strong fytoecdysterone agonist Ponasterone A and synthetic ecdysone
agonist bisacylhydrazine RG-102240, are potent lignads for EcR (that heterodimerizes with RXR) in U.
pugilator (Wu et al. 2004). Xenobiotics can also affect
EcR levels in target cells as was documented for synthetic juvenoids pyriproxifen and fenoxycarb that attenuated expression of mRNA encoding EcR (and its
heterodimer partner USP) in the insect in vitro model
with Drosophila Kc cells (Mu & LeBlanc 2004).
A range of studies also examined effects of xenobiotic on molting cycle duration and its frequency as
documented for example with endosulfan and diethylstilbestrol in D. magna (Zou & Fingerman 1997) or
methomyl in crab Rhithropanopeus harrisii Gould, 1841
(Billinghurst et al. 2001). However, many of these effects may result from general stress (xenobiotic burden on overall metabolism) rather than direct chemicalinduced endocrine disruption. It has been shown that
under chemical exposure, larval and juvenile stages of
shrimp Palaemonetes pugio Holthuis, 1949 can poorly
sustain a shift in energy allocation towards detoxification, which affected success in metamorphosis, and indirectly altered maturation and reproduction (McKenney
et al. 2004). Similarly, important role of overall fitness
for fecundity and molting frequency was demonstrated
also in multigeneration experiments with D. magna exposed to synthetic estrogen diethylstilbestrol (Baldwin
et al. 1995).
There is an increasing knowledge on the ecdysteroid signalling in crustaceans including the cross-talk
with other endogenous hormones such as methyl farnesoate or other juvenile hormone agonists (Tuberty
& McKenney 2005; LeBlanc 2007). However, a significant portion of the information has previously been
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derived from insect models. Although insects and crustaceans are phylogenetically close Ecdysozoans, there
are substantial differences in hormone regulations. In
particular, molting strategy (persisting repeated molting) plays a crucial role in the reproduction cycles in
crustaceans but not in insects, and this fact should be
carefully considered in physiological and ecotoxicological studies.
Mandibular organ
The paired mandibular organ (located more anteriorly above the Y-organ) histologically consists of lipidsecreting cells and another part where sesquiterpene
methyl farnesoate (MF) is synthesized (Withers 1992).
The physiological role of MF corresponds to the structurally similar juvenile hormone in insects which has
been much better studied and characterized (Chang
1993). Juveniod-like effects of MF (i.e., stagnation of
development and increased proportion of intermediate
individuals with larval and post-larval characteristics)
were experimentally revealed in developing larvae of
M. rosenbergii fed with MF-containing Artemia Leach,
1819 vector (Abdu et al. 1998). Recent study of Olmstead & LeBlanc (2007) also demonstrated interference
of MF with processes of sex determination as exposures
of D. magna juveniles to MF resulted in abnormal development (increased numbers of intersexual individuals).
In adults, MF has positive effects on gonadal development. It increased gonadosomatic index and enhanced leucin incorporation into ovaries of P. clarkii
females (Rodriguez et al. 2002a) and stimulated ovarian growth of the same species (Laufer et al. 1998).
Some agens (such as juvenile hormone III or progesterone) were shown to suppress responses of ovaries
to mandibular organ while estrogen 17β-estradiol had
stimulatory effect (Rodriguez et al. 2002a). MF also
induced vitellin incorporation into oocytes in Cherax
quadricarinatus Von Martens, 1868, and this process
seems to be mediated via protein kinase C α (PKCα)
(Soroka et al. 2000). Other functions of MF (related to
reproduction) may include determination of the male
sex morphotype as documented in freshwater prawn M.
rosenbergii (Okumura & Hara 2004).
As mentioned above, molting persists into adult
stages in crustaceans (in contrast to insects), and it is
carefully coordinated with the reproduction cycle (at
least in females). Therefore, MF is expected to play
more important role during whole life cycle of crustaceans than juvenile hormone in insects, and MF action must be closely coordinated with the signalling
of ecdysteroids (Laufer et al. 1998). Nevertheless, close
parallels in juvenoid-like and ecdysteroid signalling exist between crustaceans and insects. In Drosophila, juvenile hormone (and its agonist methoprene) activated
transcription of some early ecdysone-inducible genes
such as E75 orphan nuclear receptor (Dubrovsky et al.
2004). Full-length cDNA of E75 was also found in crab
G. lateralis (Kim et al. 2005b), and, correspondingly, direct signalling crosstalk between MF and ecdysteroids
143
seem to exist in crustaceans (and it may also be also
involved in the effects of xenobiotics).
Gonads
Gonads in male and female crustaceans
Most crustaceans are gonochorists, and the development and function of female and male gonads is regulated by different pathways and hormones (CharniauxCotton & Payen 1988).
Female gonadal development is controlled by several signals such as GIH/VIH from X-organ, unknown
stimulating factor from central neural ganglia or MF
(as discussed in previous section). For example, reproduction molting was induced in all eyestalk-excised females of M. rosenbergii, while about 31–49% control females remained in non reproduction stages, and these
observations reveal inhibitory effects of GIH (Okumura
& Aida 2001). Further, diameters of the oocytes of P.
clarkii crayfish were significantly increased when incubated with the thoracic ganglia which confirms the presence of ovarian stimulating factor in nervous ganglia
(Sarojini et al. 1997). The ovarian and reproduction
cycles in females are also closely correlated with molting, as it is known that ability of females to mate is
restricted to early postmolt period (at least in some
species). Also embryogenesis is linked to molting cycle
as the first molting of oviposed females (females carrying their fertilized eggs after copulation) occurs only
after the embryos have fully hatched (Okumura & Aida
2001).
In males (and masculinized gynogenetic individuals), androgenic gland protein hormone (AGH) is produced by paired poorly differentiated tissue attached
to terminals of spermaducts. It has been shown that
AGH is essential for proper development of gonoducts
in malacostracan crustaceans (Katakura 1989), and it is
also responsible for male-type morphotype development
in M. rosenbergii (Okumura & Hara 2004). Androgenic
gland hormone also controls synthesis of vitellogenin
(precursor of a major egg protein) as documented in C.
quadricarinatus (Sagi et al. 2002). The efforts to purify
and characterize AGH succeeded only partially, and its
signalling pathways are not known (Okuno et al. 2001).
Sex steroid hormones and receptors
It has been shown that crustaceans may also produce
endogenous sex steroid hormones of vertebrate-type
(Baldwin et al. 1995). For example, testosterone and a
wide range of monohydroxylated and nonpolar testosterone metabolites were detected in whole body homogenates of mysid Neomysis integer Leach, 1815 (Verslycke et al. 2002). Sex specific differences in hormone
production were also observed (testosterone levels were
five-fold higher in males and androstenedione was not
detected in females Verslycke et al. 2002). Also a study
with shrimp Neocaridina denticulata Kemp, 1918 confirmed the presence of estradiol and testosterone in
whole body homogenates (Huang et al. 2004). However,
direct regulatory role of steroid hormones in reproduction is not obvious in crustaceans as hemolymph levUnauthenticated
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els of testosetrone, 17α-hydroxytestosterone and 17βestradiol did not significantly vary in females M. rosenbergii during the ovarian cycle (Martins et al. 2007)
as well as in females of Marsupenaeus japonicus Bate,
1888 (Okumura & Sakiyama, 2004). The cytochrome
P450 system is responsible for synthesis of steroid hormones and their metabolisation (formation of hydroxylated or dehydrogenated metabolites) in crustaceans
but it seems to be different from the pathways known
in vertebrates (James & Boyle 1998). Further, enzymes
involved in ecdysteroidogenesis might also be involved
in metabolism of other steroids but it has not been studied in detail yet.
In spite of several studies documenting presence
of steroid vertebrate-like sex hormones, the mechanisms of their action in crustaceans (and Protostomes in general) are only poorly understood. The
presence of steroid receptors (SR) has been recognized
only in Deuterostomes, but recently Thornton (2004)
proposed existence of ancestral steroid receptor (AnSR)
also in predecessors (including Protostomes) about
600–1200 million years ago. Estrogen-related receptor
(ERR) was isolated also from Drosophila melanogaster
Meigen, 1830 (Thornton et al. 2003), and its presence was suggested in females of Gammarus fossarum
Koch, 1835 using a cross-reactivity assay with antibodies raised against highly conserved DBD domain of vertebrate ERα ortholog (Köhler et al. 2007). This study
has also shown that upon exposure to 10 µg L−1 of 17αethinylestradiol, ER-like protein in immature females
was induced to the levels comparable to adult female
G. fossarum, while adult females were less susceptible
to estrogen exposure (Köhler et al. 2007).
Various studies documented effects of toxic chemicals on several processes related to fecundity such as (i)
metabolism of sex steroid hormones, (ii) female reproductive activities, (iii) fecundity (egg numbers) and embryo development, (iv) production of vitellogenin and
vitellin.
Some studies described alteration of sex
steroid metabolism by model compounds and xenobiotics in crustaceans. For example, acute exposure
of mysid Neomysis integer to testosterone (2 µg L−1 )
increased level of 11β-hydroxytestosterone, and inducedde novo synthesis of androstenedione (Verslycke
et al. 2002). Exposure of mysids to tributyltin chloride
(0.01–1 µg L−1 ) caused changes in hydroxylated testosterone metabolites in whole body homogenates, and
increased excretion of sulfated testosterone metabolites (Verslycke et al. 2003). Relatively high concentration of methoprene (100 µg L−1 ) significantly decreased glycosylated testosterone concentrations resulting in higher levels of male-specific sex hormones (elevated metabolic androgenization ratio (Verslycke et al.
2004)). In the same study, opposite effect was found
after exposure to nonylphenol [decrease in endogenous
testosterone levels along with high glycosylation (Verslycke et al. 2004)]. Stimulation of glucuronyltransferase
and suppression of exogenous testosterone metabolization were also observed in D. magna treated with 0.5
E. Mazurová et al.
mg L−1 diethylstilbestrol (DES) (Baldwin et al. 1995).
Various chemicals with xeno-hormonal potencies
elicit both stimulatory and inhibitory effects on crustaceans’ reproduction that seems to differ from the
mechanisms and results observed in vertebrates. Recent
international efforts aim to develop suitable test methods for reproductive and developmental effects using
various crustaceans such as D. magna (Tatarazako &
Oda 2007) or selected marine copepods (Kusk & Wollenberger 2007). However, various compounds being estrogenic in vertebrates tend to accelerate and promote
reproductive activities in crustacean females while the
same compounds negatively affect fecundity parameters
(such as numbers of egg or development of embryos after fertilization).
For example, in Corophium volutator Pallas, 1766
fertility increased after treatment of 50 µg L−1 nonylphenol (Brown et al. 1999). Accelerated maturation was
also observed in Acartia tonsa Dana, 1849 after exposure to other estrogens 17β-estradiol and bisphenol
A (Andersen et al. 1999). In another study, only diethylstilbestrol (DES), but not 17α-ethinylestradiol or
17β-estradiol, enhanced proportion of Nitocra spinipes
Boeck, 1865 females carrying nauplii (Breitholtz &
Bengtsson 2001). However, as also discussed above,
mechanisms involved in estrogen stimulatory effects are
complicated in crustaceans. For example, in vitro effects of 17α-hydroxyprogestrone and 17β-estradiol on
the ovary growth of P. clarkii manifested only when
ovaries were co/incubated with mandibulary gland tissue (Rodriguez et al. 2002a).
On the other hand, inhibitory effects of estrogens were documented in studies with Tisbe battagliai
Volkmann-Rocco, 1972 where the whole life cycle
exposures to estrone, 17α-ethinylestradiol and 17βestradiol reduced numbers of nauplii (Hutchinson et
al. 1999a). Correspondingly, 17α-ethinylestradiol and
p-octylphenol (steroid and non-steroid estrogens) inhibited larval development of nauplii in Acartia tonsa
(Andersen et al. 2001) but inhibitory effects of naupliar development were observed also after exposure to
nonsteroidal anti-estrogen tamoxifen or anti-androgen
flutamide (Andersen et al. 2001). It seems that concentrations of both natural and synthetic estrogens (17βestradiol and 4-nonylphenol) lower than 10 µg L−1
have no effects on larval development as documented
in the study of Billinghurst et al. (2001) with barnacle
Elminius modestus Darwin, 1854.
Vitellogenin (Vtg, the precursor of major egg
protein vitellin) is one of the most commonly employed
biomarkers of endocrine disruption in oviparous vertebrates. Laboratory and field experiments documented
that estrogenic compounds increase plasma levels of
Vtg in females and induce de novo synthesis of Vtg in
fish, birds etc. (Allner et al. 1999; Vethaak et al. 2002).
These observations provoked studies of Vtg analogs in
invertebrates including crustaceans.
Vtg genes seem to be moderately conserved along
the phylogeny but vitellogenin isoforms detected among
invertebrates have different functionalities. In certain
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Ecotoxicology of crustacean reproduction
taxa they become involved for example in calcium or
ferric metabolism (Abdu et al. 2002; .Yokota et al.
2003). In crustaceans, vitellin protein has been purified
and characterized in eggs from several species including decapods, e.g., C. quadricarinatus (Sagi et al. 1996),
Palaemon elegans Rathke, 1837 (Sanders et al. 2005),
M. rosenbergii (Lee et al. 1997)), copepod Amphiascus tenuiremis Brady et D. Robertson, 1875 (Volz &
Chandler 2004), barnacle Balanus amphitrite Darwin,
1854 (Billinghurst et al. 2000), mysid Neomysis integer (Ghekiere et al. 2005), and amphipod Leptocheirus
plumulosus Shoemaker, 1932 (Volz et al. 2002). Using
polyclonal antibodies, synthesis of Vtg in hepatopancreas of female C. quadricarinatus has been revealed
(Sagi et al. 2002). Interestingly, Vtg synthesis also occurs in intersex males after excision of androgenic gland
(Sagi et al. 2002).
Similarly to vertebrate studies, modulations of Vtg
by various xenobiotics were studied also in crustaceans.
Slight elevations of vitellin level in P. elegans larvae
were observed after exposures to nonylphenol and 17βestradiol (Sanders et al. 2005). Stimulations of vitellin
(here called cypris major protein) were also detected
in naupliar and cypris larval stages of barnacle B. amphitrite (Billinghurst et al. 2000). In females of P. pugio,
pyrene (polycyclic aromatic hydrocarbon) exposures resulted in large eggs with elevated vitellin levels but also
increased mortality during hatching (Oberdörster et al.
2000). Authors deduced that lipophilic pyrene is stored
and transported along with vitellin into eggs where it
becomes toxic (Oberdörster et al. 2000).
Practical determination of vitellin as a biomarker
of endocrine disruption often uses antibody-based techniques such as western blotting or ELISA. However, its
routine application among crustaceans is complicated
by low inter-specific cross-reactivity of anti-vitellin antibodies. Alternative assessment of “alkali-labile phosphates” (an indirect marker of vitellin) has been suggested in mussels (Gagne & Blaise 2000), and it was
applied also in shrimp N. denticulata (Huang et al.
2004). However, this method should be applied with
caution as alkali-labile phosphorus forms only a minor
part of the total phosphorus pool in crustacean vitellogenin (Volz et al. 2002), and the presence of other
phosphorus sources can substantially affect the results
(Stanton 1968). Another problem affecting interpretation of the toxicological studies may be a complexity
of the natural processes during reproduction and early
development. For example, total proteins, the ratio of
proteins to carbohydrates, composition of lipid classes
and fatty acids are known to naturally and dramatically
fluctuate during early ontogenesis (Nates & McKenney
2000). These temporal changes must be well understood
and carefully considered when measuring responses of
other parameters (such as vitellin levels) to chemical
exposure (Billinghurst et al. 2000).
Sex determination
There are publications presenting shifts in sex ratio
145
(i.e., ratio between males and females) in crustaceans
after the treatment with various xenobiotics (Watts et
al. 2002). Changes in sex ratio may affect reproduction
success and may lead to unbalanced population growth.
Various mechanisms that determine sexual characteristics were described among crustaceans but for majority
of species, factors and genes determining male vs. female phenotype are not known.
Gonosomes. Sex heterochromosomes (gonosomes)
have been reported in crustaceans, and species with
mammalian-like XY gonosomes were identified in all
subclades across crustacean taxon (Ginsburger-Vogel &
Charniaux-Cotton 1982). Other sex chromosomal types
are also common. For example, Cypria sp. Zenker, 1854
males display higher number of X chromosomes (Xn Y
chromosomal type), male unpaired gonosome (X0 chromosomal type) was observed in Ostracods, and birdlike ZW sex determining system has been found inside
Copepoidae family (Ginsburger-Vogel & CharniauxCotton 1982; Lecher et al. 1995). Therefore, there
is considerable variability in occurrence and types of
gonosomes across crustacean taxons, and the sex determination described for each particular species must be
considered a species-specific, and its extrapolation to
other crustacean taxa is limited.
Autosomes. Besides gonosomes, other genes localized on different chromosomes (autosomes) seem to
modulate phenotypical sex characters. As an example,
some autosomal genes encoding colour were related to
feminization in Gammarus pulex L., 1758 (Hedgecock
et al. 1982). Also current investigations with Daphnia
pulex Leydig, 1860 revealed numerous genes that contribute to phenotypic sex manifestation (Eads et al.
2007). Phenotypic sex in crustaceans is also regulated
by previously discussed endocrine system and also other
hormones such as 20–hydroxyecdysone, 20–HE, methyl
farnesoate, MF, or androgenic gland hormone, AGH.
Androgenic gland is an important sex-determining
factor in crustaceans (Katakura & Hasegawa 1983;
Suzuki 1999), and it is critical for male gonad development during early ontogenesis (Ginsburger-Vogel &
Charniaux-Cotton 1982). Eventual reversion of sexual
phenotype is possible only during early ontogeny when
male gonoducts are not yet fully formed. After full gonad differentiation, removal of androgenic gland leads
to only partial sex-reversal with intersex specimen development (Charniaux-Cotton 1960; Ford et al. 2005).
Gonadal life itself, spermatogenesis and reproduction
activity (and sex-determination of offspring) is then influenced by hormones such as 20–HE or MF (Okumura
& Hara 2004) but their effects seem to be highly doseand time-dependent.
Daphnia pulex exposed to 1 and 10 µg L−1 20–HE
produced more male offspring but this effect was less
evident at 100 µg L−1 (Peterson et al. 2001). Similarly,
occurrence of male nauplii increased in T. battagliai exposed only to 86.5 µg L−1 of 20–HE, while less pronounced effects were recorded at lower or higher concentrations ranging 8.7–269 µg L−1 (Hutchinson et al.
1999b). Also MF (5–30 µg L−1 ) stimulated male occurUnauthenticated
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E. Mazurová et al.
146
Table 1. Examples of crustacean representatives and studies related to reproduction endocrinology and/or reproduction toxicity.
Taxon
Examples of studies
References
Maxillopoda: Cladocera – water fleas
Ceriodaphnia dubia Richard, 1894
SSRIs* – FE
Brooks et al. (2003)
Henry et al. (2004)
Flaherty & Dodson (2005)
Zou & Fingerman (1997)
Olmstead & LeBlanc (2007)
Daphnia sp. O.F. Müller, 1785
fluoxetine – FE, DC
endosulfan, diethylstilbestrol – MO, SD
methyl farnesoate, pyriproxifen – MO, DC,
SD, AG
diethylstilbestrol – VSH
20-hydroxy-ecdysone – SD
Baldwin et al. (1995)
Peterson et al. (2001)
testosterone – ES, MO
SD
Mu & LeBlanc (2002)
Eads et al. (2007)
estradiol, bisphenol-A – DC
(xeno)estrogens, tamoxifen, flutamide – FE
lindane – MO, FE
(xeno)estrogens – FE
estrogens, antiestrogens, 20–hydroxy-ecdysone
– FE, SD
Andersen et al. (1999)
Andersen et al. (2001)
Brown et al. (2003)
Breitholtz & Bengtsson (2001)
Hutchinson et al. (1999a, b)
Corophium volutator Pallas, 1766
Gammarus pulex L., 1758
Gammarus fossarum Koch, 1836
fluoxetine – FE
androgens – ES
nonylphenol – FE, SD
estradiol – SD
bisphenol-A – OD
Brooks et al. (2003)
Janer et al. (2005)
Brown et al. (1999)
Watts et al. (2002)
Schirling et al. (2006)
Malacostraca: Decapoda – decapods
Macrobrachium rosenbergii De Man, 1879
serotonine – DC
Procambarus clarkii Girard, 1852
NHR, ES, M
MF, AG
VSH
VLP
spiperone – SA, OD
Tangvuthipong & Damrongphol
(2006)
Okumura & Aida (2001)
Okumura & Hara (2004)
Martins et al. (2007)
Lee et al. (1997)
Sarojini et al. (1995a)
Rodriguez et al. (2002b)
Sarojini et al. (1997)
Rodriguez et al. (2002a)
Fingerman (1997)
Billinghurst et al. (2001)
Cripe et al. (2003)
McKenney (2005)
Malacostraca: Copepoda – copepods
Acartia tonsa Dana, 1849
Bryocamptus zschokkei Schmeil, 1893
Nitocra spinipes Boeck, 1865
Tisbe battagliai Volkmann-Rocco, 1972
Malacostraca: Amphipoda – amphipods
Hyalella azteca Saussure, 1858
Rhithropanopeus harrisii Gould, 1841
Palaemonetes pugio Holthuis, 1949
naxolone – OD
progesterone, 17beta-estradiol – OD
NHR, BA, FE
methomyl – MO
juvenile hormone agonists, fenoxycarb – DC
methoprene, pyriproxyfen, fenoxycarb – DC,
FE
juvenile hormone agonist, fenoxycarb, pyriproxyfen – ES, DC, FE
pyrene – VLP
methoprene, pyriproxyfen, fenoxycarb – DC,
FE
Tuberty & McKenney (2005)
Oberdörster et al. (2000)
McKenney (2005)
Explanations: BA – biogenic amines, DC – developmental changes, ES – ecdysteroids, FE – fecundity, MF – methyl farnesoate, MO –
molting, NHR – neurohormonal regultion (including eyestalkablation experiments), OD – ovarian development, SA – spermatogenesis
alteration, SD – sex determination, * SSRIs – selective serotonine reuptake inhibitors, VLP – vitellogenin-like proteins, VSH – vertebrate
sex hormones.
rence in D. pulex offspring (Olmstead & Leblanc 2002)
with the most sensitive period between 48–72 hours of
neonates age. Similar effects have also been found for
juvenile hormone antagonist methoprene that induced
exclusive production of males in D. pulex exposed to 10
and 100 µg L−1 (Peterson et al. 2001). Also a recent
study of Olmstead and LeBlanc (2007) demonstrated
alteration of male sex determination in D. magna after
exposures to MF.
It was also shown that various epigenetic factors like temperature, photoperiod (Dunn et al. 2005)
and parasites (Ginsburger-Vogel 1991) may influence
manifestation of sex in individual organisms. Interestingly, sensitivity of isopod and amphipod juveniles
to photoperiod manipulation overlaps with the androgenic gland development and differentiation (for review see Ginsburger-Vogel & Charniaux-Cotton 1982).
It was also shown in the studies with Gammarus
duebeni Lilljeborg, 1851 that photoperiod manipulation for parent organisms altered proportions of
males and females at F1 and F2 generations (Watt
1994).
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Ecotoxicology of crustacean reproduction
Knowledge about the sex determination in crustaceans is further complicated by generally high variability of genotypes, existence of sibling species, occurrence of subpopulations with different ploidy, presence
of androgenetic females/gynogenetic males (Ginsburger-Vogel 1989; Lecher et al. 1995; McCabe & Dunn
1997). Further, flexible sex modulation by range of exogenous factors evolved in short term living species,
e.g., Daphnia’s ameiotic phelogonic/amphiogonic parthenogenesis and sexual generation cycles (Innes 1997).
Therefore, the sensitivity of various genotypically different groups within species related to sex determination
to xenobiotics can vary enormously.
Vertebrate estrogenic hormones (17β-estradiol) as
well as synthetic xenoestrogens (17α-ethinylestradiol,
nonylphenol and diethylstilbestrol, DES) were the most
often studied compounds with regard to sex modulation in crustaceans. In the study of Watts et al.
(2002), mixed populations of G. pulex (consisting of
neonates, juveniles and adults) were exposed to 17αethinylestradiol at 0.1, 1 and 10 µg L−1 for a long
term (100 days). Authors observed feminization effects (proportion of adult females increased in all
treatments), and there was also an increase in total population size at 1 and 10 µg L−1 concentrations. On the other hand, number of male adults
and praecopula guarding pairs did not differ among
treatments (Watts et al. 2002). Also a study with
Gammarus fossarum showed inducibility of intersex in
adults exposed for 8 weeks to surface water containing higher levels of (xeno)estrogens, and authors suggested that intersex specimen developed from males
rather than females (Jungmann et al. 2004). Intersex in wildlife populations of Echinogammarus marinus Leach, 1815 was also recently reported but no direct link to contamination was confirmed (Ford et al.
2007).
On the other hand, there are numerous other
studies with estrogens that did not demonstrate pronounced effects on sex determination in crustaceans.
Sex ratio was not altered by 17α-ethinylestradiol or
17β-estradiol in copepod N. spinipes (Breitholtz &
Bengtsson 2001), 17α-ethinylestradiol, 17β-estradiol
and estrone in mysid T. battagliai (Hutchinson et al.
1999b), nor by nonylphenol in amphipod C. volutator (Brown et al. 1999) or Daphnia (Zou & Fingerman 1997). In these studies, changes in other parameters such as molting cycle duration, abnormal
development and progeny abundance were more obvious. Interestingly, the study of Zou & Fingerman
(1997) showed slight (non-significant) stimulations of
male offspring number in D. magna exposed to DES
or ‘estrogenic-like’ pesticide endosulfan (organochlorine
cyclodiene).
However, data from the long term studies should
always be carefully interpreted as our study (unpublished data) documented that for example aggresivity of
males towards females (resulting in higher female mortalities) may affect final numbers of surviving males,
females and eventual intersexual animals.
147
Conclusion
In this review the endocrinology of crustaceans is put
together with published ecotoxicological data with attempt to summarize the potential of xenobiotics to affect reproduction of this subphyllum. Table 1 shows an
overview of model organisms from all major crustacen
classes along with selected experimental studies. Following major findings and possible trends in the research were identified:
(1) The neurohormones and their effects on reproduction were described in crustaceans. Modulations of
their effects by man-made antidepressants were shown
to alter also gonadal development, and further ecotoxicological research should explore ecological relevance of
these findings.
(2) Molting-related parameters (regulated by ecdysteroid hormones in crustaceans sharing some similarities with vertebrate sex hormones) were extensively
studied as a potential endpoint for endocrine disruptive
chemicals. However, biochemical processes underlying
the link of molting with reproduction are still poorly
characterized in crustaceans, and it is often difficult
to discriminate whether the changes in molting result
from endocrine disruption or non-specific toxicity. More
specifically, assessment of ecdysteroid levels should be
complemented by other information such as activities
in target tissues (ecdysteroid-sensitive cells).
(3) Other potential biochemical targets for xenobiotics were recently discovered in crustaceans (e.g.,
molecular data about ecdysteroid receptor, its homo/
heterodimer hybridization with other receptors, affinity
to various exogeneous ligands, etc.), and these should
be explored by further ecotoxicological studies.
(4) Cross-links between signalling of ecdysteroids
(regulating molting) and methyl farnesoate (controlling
development and maturation) have recently been described (Dubrovsky et al. 2004), and these processes as
well as their modulations by contaminants should be
studied.
(5) Some sex steroid hormones known from vertebrates (testosterone, progesterone) have been also reported in crustaceans but their targets (steroid receptors) were not yet described. Vertebrate sex steroids
were shown to regulate gonadal development and antagonize ecdysteroid receptor signalling but full understanding of their action and possible modulations by
endocrine disruptors in crustaceans will require further
research attention.
(6) Determination of the sex in developing juveniles (and consequent modulation of the sex ratio in
population) is a sensitive parameter that may be modulated by various xenobiotics (including “endocrine disruptors”) but also by environmental stress and nonspecific toxicity. Understanding the physiology of sex
determination, the role of endocrine regulation in these
processes (including methyl farnesoate) will require further studies.
Although a significant portion of our knowledge
about endocrine regulations in crustaceans has previUnauthenticated
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148
ously been derived with insect models, there is recently
a progressive development in the studies of crustacean
physiology and ecotoxicology (LeBlanc 2007) including complex genomic studies (Eads et al. 2007), and
there is a recent expansion of genomic toxicology also
in crustaceans (Colbourne et al. 2005). Recent findings also indicate high diversity within crustaceans,
and consequently, data derived with a single species
should carefully be interpreted as well as their extrapolations to other crustaceans. Numerous studies documented that chemicals (including endocrine disruptors
in vertebrates) affect reproduction-related processes in
crustaceans which may have broader ecological consequences. However, generalization of the findings and
simple interpretations as “endocrine disruption” should
always be critically evaluated.
Acknowledgements
Research of the ecotoxicology of endocrine disrupting chemicals in RECETOX is supported by Ministry of Education, C.R. (Project INCHEMBIOL No. 0021622412) and the
ECODIS project (6th EC FWP, No. 518043-1).
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Received August 31, 2007
Accepted October 20, 2007
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