Endocrine Regulation of the Reproduction in Crustaceans: Identiﬁcation of Potential Targets for Toxicants and Environmental Contaminants

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,KláraHilscherová1,4,RitaTriebskorn2,3,Heinz-R.Kohler¨ 2, Blahoslav Maršálek1,4 & Luděk Bláha1,4*

1Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX), Masaryk University, Kamenice 3, CZ-62500 Brno, Czech Republic; e-mail: [email protected] 2Animal Physiological Ecology Department, Eberhard-Karls University, T¨ubingen, Germany 3Steinbeis-Transferzentrum f¨ur Okotoxikologie¨ und Okophysiologie,¨ Rottenburg, Germany 4Academy 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 (1) Reproduction processes are generally sensitive to various endogenous and exogenous factors includ- Available data provide a clear evidence that crustaceans ing total energy pool (e.g., food supplies), seasonal are vulnerable to diverse chemicals and toxicants in variations and environmental conditions (including pol- the environment (James & Boyle 1998; Olmstead & lution), detoxification metabolism and also hormonal LeBlanc 2007; Verslycke et al. 2007), thus a range of regulations at various levels. Studies that examine re- crustacean species is often used in ecotoxicological responses of wildlife to complex polluted environment search. In particular, compounds and pollutants affect- often discussed developmental and reproduction toxi- ing endocrine homeostasis (i.e., endocrine disruptors) city. However, it may be often difficult to find direct are intensively studied also in crustaceans (Hutchin- links between unspecific effects and endocrine disrup- son 2002; LeBlanc 2007, Rodriguez et al. 2007). Se- tion caused by chemical pollution (Brian 2005; Zou rious reproductive disorders in crustaceans have been 2005). documented after exposure to such chemicals as in- (2) Other studies with crustaceans focus on known sects hormones and their mimics (Peterson et al. 2001) or suspected endocrine disruptors with the aim to ex- or vertebrate-like (xeno)hormones (Zou & Fingerman plore alteration of biological functions on biochemical 1997). Current approaches to study endocrine toxicol- and molecular levels. Several of such studies in crus- ogy of crustaceans include (1) studies with complex en- taceans add new information to mechanisms and path- vironmental mixtures with expected endocrine toxicity, ways previously described only in traditional insect and (2) controlled laboratory experiments with individ- and/or vertebrate models (Verslycke et al. 2002; Wu ual chemicals (suspected endocrine disruptors). et al. 2004; Kim et al. 2005a).

* Corresponding author

c 2008 Institute of Zoology, Slovak Academy of Sciences 140 E. Mazurová et al.

Sinus gland/X-organ MOIH, MIH, GIH/VIH GIH/VIH ?

Gonads MOIH ? Males: AGH ? MIH ? Serotonin ? Steroids (T) Dopamine? ? Mandibular organ Androgenic gland AGH MF Cerebral and Thoracic ganglia Y-organ Serotonin, Dopamine 20-HE MF ? CRUSTACEAN MF ?

ENDOCRINE SYSTEM ENDOCRINE Adults: MF ? Juveniles: MF ? Testosterone ?

Physiological Molting Development/ effects Ontogeny/Maturation

Adults: reproduction activity Male sex development, control male reproduction

Reproduction Reproduction cycle control; Adults: reproduction potency ? effects ? males in brood 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 crus- since they may also play a role in reproduction pro- tacean endocrinology (with special respect to reproduc- cesses. tion, see Fig. 1) with data from selected ecotoxicological studies. We have focused on reproduction related Neurohormones produced by X-organ effects, as it is one of the most important biological Sinus gland is a neurohemal organ located laterally on processes associated with species fitness and population optic ganglia in eyestalks and it is connected to X-organ survival. Rather than summarization of many ecotox- via a dense nervous tissue. Both organs are paired and icological case studies, we have aimed to identify gen- they serve as a key endocrine junction of central neu- eral physiological targets in crustaceans vulnerable to rosecretory signals (Withers 1992). exogenous xenobiotics including endocrine disruptors. The peptide hormones released from X-organ have The review covers following major areas related to re- diverse effects on growth and reproduction: molt- production physiology and toxicity in crustaceans: (i) inhibiting hormone (MIH) suppresses production of neuroendocrine control of reproduction, (ii) role of clas- ecdysteroids in Y-organ; mandibular-organ inhibiting sical endocrine glands in reproduction, and (iii) sex de- hormone (MOIH) down-regulates excretion of methyl termination in crustaceans. farnesoate (MF) from mandibular gland; and gonad inhibiting hormone (GIH, or according to some authors Neuroendocrine control of reproduction vitellogenin inhibiting hormone – VIH) represses ovarian maturation and testes growth (Chang 1993; Ohira To the present knowledge, neuroendocrine regulation et al. 1999). However, precise actions of separate neu- axis shares similar structure inside a broad Coelomates ropeptides are not yet fully understood since most of clade. In crustacean taxa, known secretory active sites the studies used whole organ homogenates or eyestalk include neurosecretory cells in cerebral and thoracic ablated animals (in which whole complex of sinus gland ganglion, sinus gland/X-organ complex, postcomissural and X-organ was removed). organ and pericardial organ (Cooke & Sullivan 1982). For example, the biochemical character of GIH is This chapter focuses first on the complex of sinus gland still a matter of discussion (Rodriguez et al. 2002a), and X-organ that were extensively studied, and they are and its structure seems to vary among species (Chaves functionally well characterized. Secondly, we summa- 2000). Therefore, factors with MIH/CHH/GIH effects rize functions of various biogenic amines in crustaceans were grouped into a peptide family of X-organ neuro- Ecotoxicology of crustacean reproduction 141 hormones but their precise structural variability and growth. This stimulation was further promoted by ad- detailed physiological effects remain to be investigated ditions of methionine enkephaline (M-ENK; a ligand of (Chang 1993). opioid receptor known to stimulate serotonin synthesis). Molt-inhibiting hormone (MIH) inhibits the pro- Contrary, oocyte growth was inhibited by naxolone, an duction of ecdysteroids by Y-organ affecting thus dura- opioid antagonist (Sarojini et al. 1997). However, it tion of the molting cycle and the onset of reproduction. seems that serotonin does not affect gonads directly. However, the study with the crab Carcinus maenas L., For example, methyl farnesoate (juvenile hormone ana- 1758 demonstrated that the sensitivity of Y-organ itself log that plays a role in crustacean molting and it to MIH is a more important factor than levels of MIH also stimulates gonadal maturation; see also discussion (Chung & Webster 2003). This was also supported by a below), and also other factors (named vitellogenesis- study with prawn Macrobrachium rosenbergii De Man, stimulating hormone VSH or vitellogenesis-stimulating 1879, where female eyestalk ablation resulted in short- ovarian hormone VSOH) were suggested as intermedi- ening of the reproduction molting period but levels of ates in serotonin-dependent gonadal stimulation (Ro- ecdysteroid (as well as vitellogenin) did not substan- driguez et al. 2002a). tially vary during molting stages (Okumura & Aida Some ecotoxicological studies also confirm that 2001). In vitro experiments with tissues of estuarine processes regulated by biogenic amines may become a crab Chasmagnathus granulata Dana, 1851 also showed target of selected environmentally relevant endocrine that heavy metals such as cadmium and copper inhibit disruptors. Fluoxetine (Prozac, selective serotonine re- release of GIH (Medesani et al. 2004). uptake inhibitor – SSRI) dosed in environmentally rel- In summary, functions of neuropeptides produced evant concentration 0.1 mg L−1 seriously inhibited by sinus gland/X-organ seem to be complex and highly fecundity of cladoceran Ceriodaphnia dubia Richard, variable among crustacean species, and full understand- 1894 (Brooks et al. 2003). Reduction of neonate num- ing to their biochemical regulatory pathways (and ef- bers was also observed during chronic exposure of C. fects on reproduction) will require further research. dubia to five SSRIs (fluoxetine, fluvoxamine, paroxe- tine, citalopram, sertraline) (Henry et al. 2004). On the Biogenic amines with neurohormonal function other hand, significant fecundity stimulations were ob- Biogenic amines – serotonine (5-hydroxytryptamin, 5- served in Daphnia sp.O.F.Müller, 1785 after chronic HT), dopamine and octopamine – were first detected exposures to fluoxetine (36 µgL−1,30day)(Flaherty in secretory cells in pericardial organ (Cooke & Sul- & Dodson 2005), and authors also reported serious de- livan 1982), and for a long time they have been con- velopmental defect and increased mortality when com- sidered only neurotransmitters in nervous synapses of bined with other drugs. Slight enhancement of fecun- crustaceans (for a review see, e.g., Cooke & Sullivan dity and significant growth reduction was reported in (1982)). Later, these amines (plus another one, proc- Hyalella azteca Saussure, 1858 exposed to fluoxetine in tolin) have been shown to act as neurohormones being sediment (Brooks et al. 2003). released into the hemolymph and having ‘adrenaline- Taken together, there are clear evidences that bio- like’ effects (Cooke & Sullivan 1982; Siwicki et al. 1987). genic amines may (indirectly) affect reproduction pro- Several studies suggest that biogenic amines are cesses in crustaceans, and their action may be nega- also able to alter reproduction processes in crustaceans, tively modulated by certain compounds (such as phar- and it seems that serotonin generally stimulates and maceuticals) polluting the environment (Kummerer dopamine inhibits reproduction potency. For example, 2004). it has been shown that serotonin caused significant stimulation of testes index and number of spermato- Role of classical endocrine glands in reproduc- cytes per testicular lobe in fiddler crab Uca pugilator tion Bosc, 1802 while dopamine (and its agonist 2-amino- 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, ADTN) All crustacean “classical” endocrine glands seem to retarded testicular development (Sarojini et al. 1995a). modulate reproduction. As also mentioned above, re- Testicular maturation was also promoted in red swamp production is sensitive to various non-specific factors crayfish Procambarus clarkii Girard, 1852 by injections (such as general environmental stress, detoxification of serotonin and spiperone (a dopamine antagonist) metabolism, energy budget etc.), and it may therefore (Sarojini et al. 1995b). Injection of spiperone also in- be complicated to directly relate reproduction changes creased gonadosomatic index and oocyte diameter in to chemical-induced endocrine disruption. This section females of P. clarkii (Rodriguez et al. 2002b). It has compiles the current knowledge about crustacean en- also been shown that serotonin induces ovarian matu- docrine glands and their hormones with available eco- ration and spawning in white shrimp Penaeus vannamei toxicological studies. Boone, 1931 (Vaca & Alfaro 2000). Regulatory role of biogenic amines during gonadal Y-organ development has been further supported by a study of Y-organ is histologically simple secretory organ located Sarojini et al. (1997). Authors exposed oocytes from P. on the head (near the base of the antennules) and it pro- clarkii to the thoracic ganglia (producing various bio- duces various hormones that regulate molting in crus- genic amines), and they observed stimulation of oocyte taceans. As mentioned in the previous section, its secre- 142 E. Mazurová et al. tory function is suppressed by molt-inhibiting hormone only RXR isoform with a specific hinge region in LBD (MIH) produced by X-organ (Withers 1992). However, domain (UpRXR+33) may hybridize with EcR, bind levels of MIH remain relatively constant during molt- to ecdysteroid hormone responsive elements on DNA ing cycle. Consequently, regulation of the sensitivity of and modulate expression of target genes (Durica et al. Y-organ to MIH seems to have an important role. In 2002). It is also known that the DBD domain of crus- fact, it has been shown that sensitivity of Y-organ to tacean RXR is closely related to insect ultraspiracle MIH significantly dropped in the premolt stage, and (USP) receptor, while LBD of RXR shares greater sim- it was restored again during later molt and postmolt ilarity to vertebrate RXRs rather than to USP (Wu et stages, but the factor that modulates the perceptiv- al. 2004). However, there are apparent functional dif- ity of Y-organ has not been described (Chung & Web- ferences between vertebrates and crustaceans as neither ster 2003). Factors such as alterations in cellular sec- 9-cis retinoic acid nor all-trans-retinoic acid (major ver- ond messengers (cAMP), modulations of steroidogenic tebrate retinoid receptor ligands) were able to activate enzymes or cholesterol uptake into Y-organ were sug- RXR in crustaceans (Peterson et al. 2001). gested to be involved (Spaziani et al. 1999). Thus, mod- Described ecdysteroid signalling may be affected ulations of some of these biochemical targets by xeno- by xenobiotics as documented by some ecotoxicologi- biotics may affect Y-organ secretory function and, con- cal studies. For example, male vertebrate sex steroid sequently, molting and reproduction in crustaceans. hormone testosterone (in relatively high concentrations Molting hormones produced by Y-organ in all 5–40 µM) interfered with 20-HE signalling in Daphnia Ecdysozoans (insects and crustaceans) chemically be- magna Straus, 1820 causing abnormal progeny devel- long to steroids. Similarly to vertebrates, choles- opment and prolongation of the first molting (Mu & terol (a steroid hormone precursor) is delivered to LeBlanc 2002). Testosterone (Mu & LeBlanc 2002) as Y-organ where steroidogenesis takes place on mito- well as three estrogens, bisphenol A, diethylphtalate chondria and microsomes (Spaziani et al. 1999). The and lindane (Dinan et al. (2001), antagonized effects (ecdy)steroidogenesis is a complicated network of en- of 20-HE in an insect (Drosophila) in vitro model sys- zymatic reactions, and it has not been completely de- tems indicating a ligand-competitive mechanism. Sim- scribed. In contrast to vertebrate hormones active molt- ilarly, other two xeno-ecdysteroids, strong fytoecdys- ing hormones (ecdysteroids) contain aliphatic side chain terone agonist Ponasterone A and synthetic ecdysone (Spaziani et al. 1999), but it may be expected that agonist bisacylhydrazine RG-102240, are potent lig- ecdysteroidogenesis would be sensitive to similar com- nads for EcR (that heterodimerizes with RXR) in U. pounds that modulate steroidogenesis in vertebrates. pugilator (Wu et al. 2004). Xenobiotics can also affect The most potent endogeneous ecdysteroid is 20- EcR levels in target cells as was documented for syn- hydroxyecdysone (20-HE). Its concentrations in the thetic juvenoids pyriproxifen and fenoxycarb that at- hemolymph are within µgL−1 concentrations in the in- tenuated expression of mRNA encoding EcR (and its termolt stage, and it may reach up to 100 µgL−1 in late heterodimer partner USP) in the insect in vitro model premolt/ecdysis/early postmolt stages as documented with Drosophila Kc cells (Mu & LeBlanc 2004). in M. rosenbergii (Okumura & Aida 2001). Other ecdys- A range of studies also examined effects of xeno- teroids have also been detected in hemolymph or other biotic on molting cycle duration and its frequency as tissues but usually in lower concentrations (Okumura documented for example with endosulfan and diethyl- & Aida 2001). stilbestrol in D. magna (Zou & Fingerman 1997) or Ecdysteroid action in target cells manifests via in- methomyl in crab Rhithropanopeus harrisii Gould, 1841 tracellular ecdysteroid receptor (EcR). EcR belongs to (Billinghurst et al. 2001). However, many of these ef- a nuclear receptor family (receptors acting as transcrip- fects may result from general stress (xenobiotic bur- tion factors), and it has a typical gene composition of den on overall metabolism) rather than direct chemical- five domains from which DNA-binding domain (DBD) induced endocrine disruption. It has been shown that and ligand-binding domain (LBD) are phylogenetically under chemical exposure, larval and juvenile stages of conserved. Analyses of these two domains in Uca pugi- shrimp Palaemonetes pugio Holthuis, 1949 can poorly lator (Durica et al. 2002) and Gecarcinus lateralis Frem- sustain a shift in energy allocation towards detoxifica- inville, 1835 (Kim et al. 2005a) revealed high similarity tion, which affected success in metamorphosis, and indi- to EcR from insects. Recent studies with U. pugilator rectly altered maturation and reproduction (McKenney also suggested temporal- and organ/tissue-specific dis- et al. 2004). Similarly, important role of overall fitness tribution of several EcR isoforms (Durica et al. 2002). for fecundity and molting frequency was demonstrated It may be deduced that various EcRs control specific also in multigeneration experiments with D. magna ex- responses in different organs and determine thus tissue posed to synthetic estrogen diethylstilbestrol (Baldwin sensitivity to ecdysteroids (Kim et al. 2005a; Zou 2005). et al. 1995). In the nucleus of responsive crustacean cells, ac- There is an increasing knowledge on the ecdys- tivated EcR hybridizes with another important nu- teroid signalling in crustaceans including the cross-talk clear receptor, retinoid X receptor (RXR) (Durica et with other endogenous hormones such as methyl far- al. 2002). RXR derived from fiddler crab U. pugila- nesoate or other juvenile hormone agonists (Tuberty tor (UpRXR) also exists in various isoforms (differing & McKenney 2005; LeBlanc 2007). However, a signif- in splicing hinge regions), and it has been shown that icant portion of the information has previously been Ecotoxicology of crustacean reproduction 143 derived from insect models. Although insects and crus- seem to exist in crustaceans (and it may also be also taceans are phylogenetically close Ecdysozoans, there involved in the effects of xenobiotics). are substantial differences in hormone regulations. In particular, molting strategy (persisting repeated molt- Gonads ing) plays a crucial role in the reproduction cycles in Gonads in male and female crustaceans crustaceans but not in insects, and this fact should be Most crustaceans are gonochorists, and the develop- carefully considered in physiological and ecotoxicologi- ment and function of female and male gonads is regu- cal studies. lated by different pathways and hormones (Charniaux- Cotton & Payen 1988). Mandibular organ Female gonadal development is controlled by sev- The paired mandibular organ (located more anteri- eral signals such as GIH/VIH from X-organ, unknown orly above the Y-organ) histologically consists of lipid- stimulating factor from central neural ganglia or MF secreting cells and another part where sesquiterpene (as discussed in previous section). For example, repro- methyl farnesoate (MF) is synthesized (Withers 1992). duction molting was induced in all eyestalk-excised fe- The physiological role of MF corresponds to the struc- males of M. rosenbergii, while about 31–49% control fe- turally similar juvenile hormone in insects which has males remained in non reproduction stages, and these been much better studied and characterized (Chang observations reveal inhibitory effects of GIH (Okumura 1993). Juveniod-like effects of MF (i.e., stagnation of & Aida 2001). Further, diameters of the oocytes of P. development and increased proportion of intermediate clarkii crayfish were significantly increased when incu- individuals with larval and post-larval characteristics) bated with the thoracic ganglia which confirms the pres- were experimentally revealed in developing larvae of ence of ovarian stimulating factor in nervous ganglia M. rosenbergii fed with MF-containing Artemia Leach, (Sarojini et al. 1997). The ovarian and reproduction 1819 vector (Abdu et al. 1998). Recent study of Olm- cycles in females are also closely correlated with molt- stead & LeBlanc (2007) also demonstrated interference ing, as it is known that ability of females to mate is of MF with processes of sex determination as exposures restricted to early postmolt period (at least in some of D. magna juveniles to MF resulted in abnormal de- species). Also embryogenesis is linked to molting cycle velopment (increased numbers of intersexual individu- as the first molting of oviposed females (females car- als). rying their fertilized eggs after copulation) occurs only In adults, MF has positive effects on gonadal de- after the embryos have fully hatched (Okumura & Aida velopment. It increased gonadosomatic index and en- 2001). hanced leucin incorporation into ovaries of P. clarkii In males (and masculinized gynogenetic individu- females (Rodriguez et al. 2002a) and stimulated ovar- als), androgenic gland protein hormone (AGH) is pro- ian growth of the same species (Laufer et al. 1998). duced by paired poorly differentiated tissue attached Some agens (such as juvenile hormone III or proges- to terminals of spermaducts. It has been shown that terone) were shown to suppress responses of ovaries AGH is essential for proper development of gonoducts to mandibular organ while estrogen 17β-estradiol had in malacostracan crustaceans (Katakura 1989), and it is stimulatory effect (Rodriguez et al. 2002a). MF also also responsible for male-type morphotype development induced vitellin incorporation into oocytes in Cherax in M. rosenbergii (Okumura & Hara 2004). Androgenic quadricarinatus Von Martens, 1868, and this process gland hormone also controls synthesis of vitellogenin seems to be mediated via protein kinase C α (PKCα) (precursor of a major egg protein) as documented in C. (Soroka et al. 2000). Other functions of MF (related to quadricarinatus (Sagi et al. 2002). The efforts to purify reproduction) may include determination of the male and characterize AGH succeeded only partially, and its sex morphotype as documented in freshwater prawn M. signalling pathways are not known (Okuno et al. 2001). rosenbergii (Okumura & Hara 2004). As mentioned above, molting persists into adult Sex steroid hormones and receptors stages in crustaceans (in contrast to insects), and it is It has been shown that crustaceans may also produce carefully coordinated with the reproduction cycle (at endogenous sex steroid hormones of vertebrate-type least in females). Therefore, MF is expected to play (Baldwin et al. 1995). For example, testosterone and a more important role during whole life cycle of crus- wide range of monohydroxylated and nonpolar testos- taceans than juvenile hormone in insects, and MF ac- terone metabolites were detected in whole body ho- tion must be closely coordinated with the signalling mogenates of mysid Neomysis integer Leach, 1815 (Ver- of ecdysteroids (Laufer et al. 1998). Nevertheless, close slycke et al. 2002). Sex specific differences in hormone parallels in juvenoid-like and ecdysteroid signalling ex- production were also observed (testosterone levels were ist between crustaceans and insects. In Drosophila,ju- five-fold higher in males and androstenedione was not venile hormone (and its agonist methoprene) activated detected in females Verslycke et al. 2002). Also a study transcription of some early ecdysone-inducible genes with shrimp Neocaridina denticulata Kemp, 1918 con- such as E75 orphan nuclear receptor (Dubrovsky et al. firmed the presence of estradiol and testosterone in 2004). Full-length cDNA of E75 was also found in crab whole body homogenates (Huang et al. 2004). However, G. lateralis (Kim et al. 2005b), and, correspondingly, di- direct regulatory role of steroid hormones in reproduc- rect signalling crosstalk between MF and ecdysteroids tion is not obvious in crustaceans as hemolymph lev- 144 E. Mazurová et al. els of testosetrone, 17α-hydroxytestosterone and 17β- mg L−1 diethylstilbestrol (DES) (Baldwin et al. 1995). estradiol did not significantly vary in females M. rosen- Various chemicals with xeno-hormonal potencies bergii during the ovarian cycle (Martins et al. 2007) elicit both stimulatory and inhibitory effects on crus- as well as in females of Marsupenaeus japonicus Bate, taceans’ reproduction that seems to differ from the 1888 (Okumura & Sakiyama, 2004). The cytochrome mechanisms and results observed in vertebrates. Recent P450 system is responsible for synthesis of steroid hor- international efforts aim to develop suitable test meth- mones and their metabolisation (formation of hydrox- ods for reproductive and developmental effects using ylated or dehydrogenated metabolites) in crustaceans various crustaceans such as D. magna (Tatarazako & but it seems to be different from the pathways known Oda 2007) or selected marine copepods (Kusk & Wol- in vertebrates (James & Boyle 1998). Further, enzymes lenberger 2007). However, various compounds being es- involved in ecdysteroidogenesis might also be involved trogenic in vertebrates tend to accelerate and promote in metabolism of other steroids but it has not been stud- reproductive activities in crustacean females while the ied in detail yet. same compounds negatively affect fecundity parameters In spite of several studies documenting presence (such as numbers of egg or development of embryos af- of steroid vertebrate-like sex hormones, the mech- ter fertilization). anisms of their action in crustaceans (and Proto- For example, in Corophium volutator Pallas, 1766 stomes in general) are only poorly understood. The fertility increased after treatment of 50 µgL−1 nonyl- presence of steroid receptors (SR) has been recognized phenol (Brown et al. 1999). Accelerated maturation was only in Deuterostomes, but recently Thornton (2004) also observed in Acartia tonsa Dana, 1849 after ex- proposed existence of ancestral steroid receptor (AnSR) posure to other estrogens 17β-estradiol and bisphenol also in predecessors (including Protostomes) about A (Andersen et al. 1999). In another study, only di- 600–1200 million years ago. Estrogen-related receptor ethylstilbestrol (DES), but not 17α-ethinylestradiol or (ERR) was isolated also from Drosophila melanogaster 17β-estradiol, enhanced proportion of Nitocra spinipes Meigen, 1830 (Thornton et al. 2003), and its pres- Boeck, 1865 females carrying nauplii (Breitholtz & ence was suggested in females of Gammarus fossarum Bengtsson 2001). However, as also discussed above, Koch, 1835 using a cross-reactivity assay with antibod- mechanisms involved in estrogen stimulatory effects are ies raised against highly conserved DBD domain of ver- complicated in crustaceans. For example, in vitro ef- tebrate ERα ortholog (Köhler et al. 2007). This study fects of 17α-hydroxyprogestrone and 17β-estradiol on has also shown that upon exposure to 10 µgL−1 of 17α- the ovary growth of P. clarkii manifested only when ethinylestradiol, ER-like protein in immature females ovaries were co/incubated with mandibulary gland tis- was induced to the levels comparable to adult female sue (Rodriguez et al. 2002a). G. fossarum, while adult females were less susceptible On the other hand, inhibitory effects of estro- to estrogen exposure (Köhler et al. 2007). gens were documented in studies with Tisbe battagliai Various studies documented effects of toxic chemi- Volkmann-Rocco, 1972 where the whole life cycle cals on several processes related to fecundity such as (i) exposures to estrone, 17α-ethinylestradiol and 17β- metabolism of sex steroid hormones, (ii) female repro- estradiol reduced numbers of nauplii (Hutchinson et ductive activities, (iii) fecundity (egg numbers) and em- al. 1999a). Correspondingly, 17α-ethinylestradiol and bryo development, (iv) production of vitellogenin and p-octylphenol (steroid and non-steroid estrogens) in- vitellin. hibited larval development of nauplii in Acartia tonsa Some studies described alteration of sex (Andersen et al. 2001) but inhibitory effects of nau- steroid metabolism by model compounds and xeno- pliar development were observed also after exposure to biotics in crustaceans. For example, acute exposure nonsteroidal anti-estrogen tamoxifen or anti-androgen of mysid Neomysis integer to testosterone (2 µgL−1) flutamide (Andersen et al. 2001). It seems that concen- increased level of 11β-hydroxytestosterone, and in- trations of both natural and synthetic estrogens (17β- ducedde novo synthesis of androstenedione (Verslycke estradiol and 4-nonylphenol) lower than 10 µgL−1 et al. 2002). Exposure of mysids to tributyltin chloride have no effects on larval development as documented (0.01–1 µgL−1) caused changes in hydroxylated testos- in the study of Billinghurst et al. (2001) with barnacle terone metabolites in whole body homogenates, and Elminius modestus Darwin, 1854. increased excretion of sulfated testosterone metabo- Vitellogenin (Vtg, the precursor of major egg lites (Verslycke et al. 2003). Relatively high concen- protein vitellin) is one of the most commonly employed tration of methoprene (100 µgL−1) significantly de- biomarkers of endocrine disruption in oviparous verte- creased glycosylated testosterone concentrations result- brates. Laboratory and field experiments documented ing in higher levels of male-specific sex hormones (ele- that estrogenic compounds increase plasma levels of vated metabolic androgenization ratio (Verslycke et al. Vtg in females and induce de novo synthesis of Vtg in 2004)). In the same study, opposite effect was found fish, birds etc. (Allner et al. 1999; Vethaak et al. 2002). after exposure to nonylphenol [decrease in endogenous These observations provoked studies of Vtg analogs in testosterone levels along with high glycosylation (Ver- invertebrates including crustaceans. slycke et al. 2004)]. Stimulation of glucuronyltransferase Vtg genes seem to be moderately conserved along and suppression of exogenous testosterone metaboliza- the phylogeny but vitellogenin isoforms detected among tion were also observed in D. magna treated with 0.5 invertebrates have different functionalities. In certain Ecotoxicology of crustacean reproduction 145 taxa they become involved for example in calcium or (i.e., ratio between males and females) in crustaceans ferric metabolism (Abdu et al. 2002; .Yokota et al. after the treatment with various xenobiotics (Watts et 2003). In crustaceans, vitellin protein has been purified al. 2002). Changes in sex ratio may affect reproduction and characterized in eggs from several species includ- success and may lead to unbalanced population growth. ing decapods, e.g., C. quadricarinatus (Sagi et al. 1996), Various mechanisms that determine sexual characteris- Palaemon elegans Rathke, 1837 (Sanders et al. 2005), tics were described among crustaceans but for majority M. rosenbergii (Lee et al. 1997)), copepod Amphias- of species, factors and genes determining male vs. fe- cus tenuiremis Brady et D. Robertson, 1875 (Volz & male phenotype are not known. Chandler 2004), barnacle Balanus amphitrite Darwin, Gonosomes. Sex heterochromosomes (gonosomes) 1854 (Billinghurst et al. 2000), mysid Neomysis inte- have been reported in crustaceans, and species with ger (Ghekiere et al. 2005), and amphipod Leptocheirus mammalian-like XY gonosomes were identified in all plumulosus Shoemaker, 1932 (Volz et al. 2002). Using subclades across crustacean taxon (Ginsburger-Vogel & polyclonal antibodies, synthesis of Vtg in hepatopan- Charniaux-Cotton 1982). Other sex chromosomal types creas of female C. quadricarinatus has been revealed are also common. For example, Cypria sp. Zenker, 1854 (Sagi et al. 2002). Interestingly, Vtg synthesis also oc- males display higher number of X chromosomes (XnY curs in intersex males after excision of androgenic gland chromosomal type), male unpaired gonosome (X0 chro- (Sagi et al. 2002). mosomal type) was observed in Ostracods, and bird- Similarly to vertebrate studies, modulations of Vtg like ZW sex determining system has been found inside by various xenobiotics were studied also in crustaceans. Copepoidae family (Ginsburger-Vogel & Charniaux- Slight elevations of vitellin level in P. elegans larvae Cotton 1982; Lecher et al. 1995). Therefore, there were observed after exposures to nonylphenol and 17β- is considerable variability in occurrence and types of estradiol (Sanders et al. 2005). Stimulations of vitellin gonosomes across crustacean taxons, and the sex deter- (here called cypris major protein) were also detected mination described for each particular species must be in naupliar and cypris larval stages of barnacle B. am- considered a species-specific, and its extrapolation to phitrite (Billinghurst et al. 2000). In females of P. pugio, other crustacean taxa is limited. pyrene (polycyclic aromatic hydrocarbon) exposures re- Autosomes. Besides gonosomes, other genes local- sulted in large eggs with elevated vitellin levels but also ized on different chromosomes (autosomes) seem to increased mortality during hatching (Oberdörster et al. modulate phenotypical sex characters. As an example, 2000). Authors deduced that lipophilic pyrene is stored some autosomal genes encoding colour were related to and transported along with vitellin into eggs where it feminization in Gammarus pulex L., 1758 (Hedgecock becomes toxic (Oberdörster et al. 2000). et al. 1982). Also current investigations with Daphnia Practical determination of vitellin as a biomarker pulex Leydig, 1860 revealed numerous genes that con- of endocrine disruption often uses antibody-based tech- tribute to phenotypic sex manifestation (Eads et al. niques such as western blotting or ELISA. However, its 2007). Phenotypic sex in crustaceans is also regulated routine application among crustaceans is complicated by previously discussed endocrine system and also other by low inter-specific cross-reactivity of anti-vitellin an- hormones such as 20–hydroxyecdysone, 20–HE, methyl tibodies. Alternative assessment of “alkali-labile phos- farnesoate, MF, or androgenic gland hormone, AGH. phates” (an indirect marker of vitellin) has been sug- Androgenic gland is an important sex-determining gested in mussels (Gagne & Blaise 2000), and it was factor in crustaceans (Katakura & Hasegawa 1983; applied also in shrimp N. denticulata (Huang et al. Suzuki 1999), and it is critical for male gonad devel- 2004). However, this method should be applied with opment during early ontogenesis (Ginsburger-Vogel & caution as alkali-labile phosphorus forms only a minor Charniaux-Cotton 1982). Eventual reversion of sexual part of the total phosphorus pool in crustacean vitel- phenotype is possible only during early ontogeny when logenin (Volz et al. 2002), and the presence of other male gonoducts are not yet fully formed. After full go- phosphorus sources can substantially affect the results nad differentiation, removal of androgenic gland leads (Stanton 1968). Another problem affecting interpreta- to only partial sex-reversal with intersex specimen de- tion of the toxicological studies may be a complexity velopment (Charniaux-Cotton 1960; Ford et al. 2005). of the natural processes during reproduction and early Gonadal life itself, spermatogenesis and reproduction development. For example, total proteins, the ratio of activity (and sex-determination of offspring) is then in- proteins to carbohydrates, composition of lipid classes fluenced by hormones such as 20–HE or MF (Okumura and fatty acids are known to naturally and dramatically & Hara 2004) but their effects seem to be highly dose- fluctuate during early ontogenesis (Nates & McKenney and time-dependent. 2000). These temporal changes must be well understood Daphnia pulex exposed to 1 and 10 µgL−1 20–HE and carefully considered when measuring responses of produced more male offspring but this effect was less other parameters (such as vitellin levels) to chemical evident at 100 µgL−1 (Peterson et al. 2001). Similarly, exposure (Billinghurst et al. 2000). occurrence of male nauplii increased in T. battagliai exposed only to 86.5 µgL−1 of 20–HE, while less pro- Sex determination nounced effects were recorded at lower or higher concentrations ranging 8.7–269 µgL−1 (Hutchinson et al. There are publications presenting shifts in sex ratio 1999b). Also MF (5–30 µgL−1) stimulated male occur- 146 E. Mazurová et al.

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) Daphnia sp. O.F. Müller, 1785 fluoxetine – FE, DC Flaherty & Dodson (2005) endosulfan, diethylstilbestrol – MO, SD Zou & Fingerman (1997) methyl farnesoate, pyriproxifen – MO, DC, Olmstead & LeBlanc (2007) SD, AG diethylstilbestrol – VSH Baldwin et al. (1995) 20-hydroxy-ecdysone – SD Peterson et al. (2001) testosterone – ES, MO Mu & LeBlanc (2002) SD Eads et al. (2007)

Malacostraca: Copepoda – copepods Acartia tonsa Dana, 1849 estradiol, bisphenol-A – DC Andersen et al. (1999) (xeno)estrogens, tamoxifen, ﬂutamide – FE Andersen et al. (2001) Bryocamptus zschokkei Schmeil, 1893 lindane – MO, FE Brown et al. (2003) Nitocra spinipes Boeck, 1865 (xeno)estrogens – FE Breitholtz & Bengtsson (2001) Tisbe battagliai Volkmann-Rocco, 1972 estrogens, antiestrogens, 20–hydroxy-ecdysone Hutchinson et al. (1999a, b) –FE,SD

Malacostraca: Amphipoda – amphipods Hyalella azteca Saussure, 1858 ﬂuoxetine – FE Brooks et al. (2003) androgens – ES Janer et al. (2005) Corophium volutator Pallas, 1766 nonylphenol – FE, SD Brown et al. (1999) Gammarus pulex L., 1758 estradiol – SD Watts et al. (2002) Gammarus fossarum Koch, 1836 bisphenol-A – OD Schirling et al. (2006)

Malacostraca: Decapoda – decapods Macrobrachium rosenbergii De Man, 1879 serotonine – DC Tangvuthipong & Damrongphol (2006) NHR, ES, M Okumura & Aida (2001) MF, AG Okumura & Hara (2004) VSH Martins et al. (2007) VLP Lee et al. (1997) Procambarus clarkii Girard, 1852 spiperone – SA, OD Sarojini et al. (1995a) Rodriguez et al. (2002b) naxolone – OD Sarojini et al. (1997) progesterone, 17beta-estradiol – OD Rodriguez et al. (2002a) NHR, BA, FE Fingerman (1997) Rhithropanopeus harrisii Gould, 1841 methomyl – MO Billinghurst et al. (2001) juvenile hormone agonists, fenoxycarb – DC Cripe et al. (2003) methoprene, pyriproxyfen, fenoxycarb – DC, McKenney (2005) FE Palaemonetes pugio Holthuis, 1949 juvenile hormone agonist, fenoxycarb, pyri- Tuberty & McKenney (2005) proxyfen – ES, DC, FE pyrene – VLP Oberd¨orster et al. (2000) methoprene, pyriproxyfen, fenoxycarb – DC, McKenney (2005) FE

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) and parasites (Ginsburger-Vogel 1991) may influence with the most sensitive period between 48–72 hours of manifestation of sex in individual organisms. Inter- neonates age. Similar effects have also been found for estingly, sensitivity of isopod and amphipod juveniles juvenile hormone antagonist methoprene that induced to photoperiod manipulation overlaps with the andro- exclusive production of males in D. pulex exposed to 10 genic gland development and differentiation (for re- and 100 µgL−1 (Peterson et al. 2001). Also a recent view see Ginsburger-Vogel & Charniaux-Cotton 1982). study of Olmstead and LeBlanc (2007) demonstrated It was also shown in the studies with Gammarus alteration of male sex determination in D. magna after duebeni Lilljeborg, 1851 that photoperiod manipu- exposures to MF. lation for parent organisms altered proportions of It was also shown that various epigenetic fac- males and females at F1 and F2 generations (Watt tors like temperature, photoperiod (Dunn et al. 2005) 1994). Ecotoxicology of crustacean reproduction 147

Knowledge about the sex determination in crus- Conclusion taceans is further complicated by generally high variability of genotypes, existence of sibling species, occur- In this review the endocrinology of crustaceans is put rence of subpopulations with different ploidy, presence together with published ecotoxicological data with at- of androgenetic females/gynogenetic males (Ginsbur- tempt to summarize the potential of xenobiotics to af- ger-Vogel 1989; Lecher et al. 1995; McCabe & Dunn fect reproduction of this subphyllum. Table 1 shows an 1997). Further, flexible sex modulation by range of ex- overview of model organisms from all major crustacen ogenous factors evolved in short term living species, classes along with selected experimental studies. Fol- e.g., Daphnia’s ameiotic phelogonic/amphiogonic par- lowing major findings and possible trends in the re- thenogenesis and sexual generation cycles (Innes 1997). search were identified: Therefore, the sensitivity of various genotypically differ- (1) The neurohormones and their effects on repro- ent groups within species related to sex determination duction were described in crustaceans. Modulations of to xenobiotics can vary enormously. their effects by man-made antidepressants were shown Vertebrate estrogenic hormones (17β-estradiol) as to alter also gonadal development, and further ecotoxi- well as synthetic xenoestrogens (17α-ethinylestradiol, cological research should explore ecological relevance of nonylphenol and diethylstilbestrol, DES) were the most these findings. often studied compounds with regard to sex modu- (2) Molting-related parameters (regulated by ecdy- lation in crustaceans. In the study of Watts et al. steroid hormones in crustaceans sharing some similar- (2002), mixed populations of G. pulex (consisting of ities with vertebrate sex hormones) were extensively neonates, juveniles and adults) were exposed to 17α- studied as a potential endpoint for endocrine disruptive ethinylestradiol at 0.1, 1 and 10 µgL−1 for a long chemicals. However, biochemical processes underlying term (100 days). Authors observed feminization ef- the link of molting with reproduction are still poorly fects (proportion of adult females increased in all characterized in crustaceans, and it is often difficult treatments), and there was also an increase in to- to discriminate whether the changes in molting result tal population size at 1 and 10 µgL−1 concentra- from endocrine disruption or non-specific toxicity. More tions. On the other hand, number of male adults specifically, assessment of ecdysteroid levels should be and praecopula guarding pairs did not differ among complemented by other information such as activities treatments (Watts et al. 2002). Also a study with in target tissues (ecdysteroid-sensitive cells). Gammarus fossarum showed inducibility of intersex in (3) Other potential biochemical targets for xeno- adults exposed for 8 weeks to surface water contain- biotics were recently discovered in crustaceans (e.g., ing higher levels of (xeno)estrogens, and authors sug- molecular data about ecdysteroid receptor, its homo/ gested that intersex specimen developed from males heterodimer hybridization with other receptors, affinity rather than females (Jungmann et al. 2004). Inter- to various exogeneous ligands, etc.), and these should sex in wildlife populations of Echinogammarus mari- be explored by further ecotoxicological studies. nus Leach, 1815 was also recently reported but no di- (4) Cross-links between signalling of ecdysteroids rect link to contamination was confirmed (Ford et al. (regulating molting) and methyl farnesoate (controlling 2007). development and maturation) have recently been de- On the other hand, there are numerous other scribed (Dubrovsky et al. 2004), and these processes as studies with estrogens that did not demonstrate pro- well as their modulations by contaminants should be nounced effects on sex determination in crustaceans. studied. Sex ratio was not altered by 17α-ethinylestradiol or (5) Some sex steroid hormones known from verte- 17β-estradiol in copepod N. spinipes (Breitholtz & brates (testosterone, progesterone) have been also re- Bengtsson 2001), 17α-ethinylestradiol, 17β-estradiol ported in crustaceans but their targets (steroid recep- and estrone in mysid T. battagliai (Hutchinson et al. tors) were not yet described. Vertebrate sex steroids 1999b), nor by nonylphenol in amphipod C. voluta- were shown to regulate gonadal development and an- tor (Brown et al. 1999) or Daphnia (Zou & Finger- tagonize ecdysteroid receptor signalling but full under- man 1997). In these studies, changes in other pa- standing of their action and possible modulations by rameters such as molting cycle duration, abnormal endocrine disruptors in crustaceans will require further development and progeny abundance were more ob- research attention. vious. Interestingly, the study of Zou & Fingerman (6) Determination of the sex in developing juve- (1997) showed slight (non-significant) stimulations of niles (and consequent modulation of the sex ratio in male offspring number in D. magna exposed to DES population) is a sensitive parameter that may be modu- or ‘estrogenic-like’ pesticide endosulfan (organochlorine lated by various xenobiotics (including “endocrine dis- cyclodiene). ruptors”) but also by environmental stress and non- However, data from the long term studies should specific toxicity. Understanding the physiology of sex always be carefully interpreted as our study (unpub- determination, the role of endocrine regulation in these lished data) documented that for example aggresivity of processes (including methyl farnesoate) will require fur- males towards females (resulting in higher female mor- ther studies. talities) may affect final numbers of surviving males, Although a significant portion of our knowledge females and eventual intersexual animals. about endocrine regulations in crustaceans has previ- 148 E. Mazurová et al. ously been derived with insect models, there is recently D.B. 2003. Waterborne and sediment toxicity of fluoxetine to a progressive development in the studies of crustacean select organisms. Chemosphere 52: 135–142. physiology and ecotoxicology (LeBlanc 2007) includ- Brown R.J., Conradi M. & Depledge M.H. 1999. 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