Ecotoxicology (2007) 16:61–81 DOI 10.1007/s10646-006-0115-z

Crustacean endocrine toxicology: a review

Gerald A. LeBlanc

Accepted: 4 October 2006 / Published online: 18 January 2007 Springer Science+Business Media, LLC 2007

Abstract Crustaceans are major constituents to aqua- differentiation. Population studies have revealed dis- tic ecosystems that provide a variety of ecological and ruptions in crustacean growth, molting, sexual devel- economic services. Individual crustacean species are opment, and recruitment that are indicative of adept at occupying diverse niches and their success, in environmental endocrine disruption. However, envi- part, stems from neuro-endocrine signaling cascades ronmental factors other that pollution (i.e., tempera- that regulate physiology in response to environmental ture, parasitism) also can elicit these effects and and internal cues. Peptide hormones are major signal definitive causal relationships between endocrine dis- transducers in crustaceans. The crustacean hypergly- ruption in field populations of crustaceans and chem- cemic hormone family of peptides regulates various ical pollution is generally lacking. aspects of growth, reproduction, and metabolism. These peptides may function as the terminal hormone Keywords Crustacean Á Endocrine Á Ecdysteroid Á to regulate some physiological activities or may func- Terpenoid Á Methyl farnesoate Á Intersex tion as intermediates in a signaling cascade. Ecdyster- oids and terpenoids are two major classes of terminal signaling molecules in these cascades. Hormones from Introduction these two classes function independently or in concert to regulate various processes. Ecdysteroid signaling is The planet Earth is the domain of the arthropods and subject to toxicological disruption through distur- the aquatic environments are dominated by members bances in ecdysteroid synthesis or binding of toxicants of the arthropod subphylum Crustacea. Arthropod-like to the ecdysteroid receptor. Methyl farnesoate is the creatures (anomalocarids) first appear in the fossil major terpenoid hormone of crustaceans and also is record during the late Precambrian period. Early in the susceptible to disruption by environmental chemicals. subsequent Cambrian period (~500 million years ago), However, the methyl farnesoate signaling pathway is when speciation occurred at an unparalleled rate, poorly understood and only limited mechanistic con- crustacean-like trilobites emerged in abundance, firmation for disruption of this endocrine signaling accompanied by minute creatures of undisputed crus- pathway exists. Disruption of the ecdysteroid/terpe- tacean lineage (i.e., Bradoriid ostracods of the Chen- noid signaling pathways in crustaceans has been jiang fauna) (Hou and Bergstrom 1997) (Fig. 1). associated with aberrations in growth, metamorphosis, Crustaceans have undergone dynamic species radiation reproductive maturation, sex determination, and sex through evolutionary time (Brusca and Brusca 2003). The first decapods to appear in the fossil record were free swimming shrimp-like creatures of the Devonian G. A. LeBlanc (&) period (~400 million years ago). Benthic, lobster- Department of Environmental and Molecular Toxicology, shaped decapods appear in the record around the North Carolina State University, 27695-7633, Raleigh, NC, USA Triassic-Permian border (~250 million years ago) e-mail: [email protected] following the extinction of the previously abundant

123 62 G. A. LeBlanc

Fig. 1 Phylogeny of Anomalocarda contemporary crustacean classes

Agnostida (trilobites) Subphylum: Crustacea

Class: Remipedia

Class: Cephalocarida

Class: Branchiopoda

Class: Maxillopoda

Class: Malacostraca trilobites. The true crabs are late editions to the interact with neuro-endocrine signaling cascades, crustacean species repertoire, having appeared in the resulting in signal perturbations (Colborn and Clement fossil record roughly 1 million years ago (Cretaceous 1992). Such altered signaling can result in modifications period). to development, maturation, reproduction, and other Over 66,000 crustacean species are known to exist neuro-endocrine-regulated processes that hinder pop- today. Members of this subphylum (Phylum: Arthro- ulation sustainability (Spence et al. 1990). Continued poda) are largely aquatic, though a few species are exposure to such environmental contaminants can exclusively terrestrial (i.e., some Isopoda), while others result in the emergence of genetically modified popu- utilize both the aquatic and terrestrial environment lations that can tolerate this new environment (Huet (i.e., some Decapoda). Crustaceans are the major et al. 1996). The evolution of contaminant-tolerant constituent of zooplankton, either as minute species species is reasonably common among arthropods (i.e., (i.e., some Branchiopoda and Maxillopoda) or as larval insecticide-resistant insect populations; ffrench-Con- forms of larger species (i.e., Malacostraca). The largest stant et al. 2004) due to the high fecundity and short crustaceans include the American lobster (Homarus generation time of many arthropod species. However, americanus) which can attain a body length of ~1m just as trilobites and other species encompassing 85% and the giant spider crab (Macrocheira kaempferi) of the marine species succumbed to environmental which has a claw span of up to ~3 m (Barnes 1987). changes of the Permian period, adaptation to chemicals The diversity in crustacean morphology (Fig. 2) that interfere with neuro-endocrine signaling is likely reflects their success in occupying diverse ecological to be the exception rather than the rule among niches. Incumbent in this diversification has been the contemporary species. Herein, basic neuro-endocrine evolution of various strategies for detecting environ- signaling among crustaceans is reviewed along with mental cues and using these signals to regulate aspects documentation (both laboratory and field) of pertur- of survival (i.e., development, maturation, and repro- bations in such signaling by environmental influences. duction) that ensure population sustainability. Neuro- Finally, the potential utility of crustaceans to serve as endocrine pathways are critical to transducing these sentinels of environmental endocrine disturbance is signals (Beltz 1988). Changes in these signaling cas- discussed. cades through mutation may prove advantageous to a population by enhancing sustainability in a changing environment, resulting in the evolution of the popula- Crustacean physiology and systematics tion. More likely, such changes have no effect (neutral) or prove disadvantageous, conferring some liability to The ancestral pre-crustacean Anomalocarus exhibited sustainability, and are purged from the population little resemblance to any contemporary crustaceans gene pool. with its wing-like formation of swimming appendages Changes in signaling cascades also can occur due to and toothed, circular mouth (view a ‘‘swimming’’ environmental influences. Human activities over the Anomalocarus at http://www.calstatela.edu/faculty/ac- past half-decade have introduced a myriad of chemi- olvil/sediments.html). Morphologies diverged consid- cals into the environment that have the potential to erably among and even within the crustacean classes

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Fig. 2 The major morphotypes among the Crustacea. Significant variability in structure exists even within classes

though all contemporary crustaceans share several Critical body parts of crustaceans are (with a few common characteristics (Barnes 1987; Smith 2001; exceptions) shielded within a chitinous carapace. Brusca and Brusca 2003) (Fig. 2). The body of most Crustaceans typically possess one or two compound crustaceans is composed of three parts, a head (ceph- eyes and, depending upon the species and life stage, a alon), a thorax and an abdomen, though the thorax and simple eye call an ocellus. All crustaceans possess an abdomen are indistinguishable in some species and, open circulatory system. together, are often referred to as a trunk. Each body According to most contemporary phylogenic con- part consists of multiple segments and each segment structs, using both morphometric and DNA analyses, commonly bears a pair of appendages. Head segments modern crustaceans are segregated into five classes bear the first antennae (minor antennae or antenn- (Figs. 1, 2); although, inter- and intra-class relation- ules), second antennae (major antennae), mandibules, ships remain equivocal (Brusca and Brusca 2003; maxillules, and maxillae. The thorax and abdomen Koenemann and Jenner 2005). Crustaceans commonly bear appendages that contribute to various functions encountered by field biologists can typically be including movement, respiration, and reproduction. assigned to one of three classes: Branchiopoda,

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Maxillopoda, and Malacostraca. Basic body forms shrimp have a bivalved carapace along with concentric associated with the different crustacean classes are growth striations that confer significant overt resem- depicted in Fig. 2. blance to small bivalved mollusks (i.e., clams). Clam shrimp are typically bottom dwellers that filter organic Class: Branchiopoda particles from the water column or scrapped off of bottom substrates. Clam shrimps have limited swimming These small invertebrates are dominant crustacean mobility. residents of ephemeral habitats. Most are freshwater inhabitants, have a short life span, and are capable of Water fleas producing desiccation-resistant resting eggs. The trunk appendages of the branchiopods are used for respira- Water fleas are typically small ( < 3 mm) inhabitants tion, hence the name ‘‘gill feet.’’ Branchiopods have of freshwater environments, though some are as large distinct sexes and typically reproduce by cyclic parthe- as 2 cm (i.e., Leptodora kindtii). A few marine species nogenesis. However, males are not known to some have been described. The carapace folds (like a horse species (obligate parthenogens). Among the cyclic saddle) over the head and thoraxic regions. Depending parthenogens, sexual reproduction often results in the upon the species, many of the abdominal segments may production of resting eggs. be completely exposed or retractable. Some occupy the water column, using their second antennae as swim- Order: Anostraca (fairy shrimp, brine shrimp) ming appendages. Members of the genus Scapholeberis typically adhere to the surface film where they ‘‘skate’’ These small shrimp-like creatures are typically less just below the water surface. Water fleas feed mostly than 10 cm and most often less than 1 cm in length. by filtering phytoplankton and other small organic Fairy shrimp inhabit ephemeral ponds where annual matter from the water column. Bottom dwellers often desiccation of the habitat can result in significant scrape organic material from bottom substrates. A few elevations in concentrations of chemicals in the hab- species are predatory. A review of the use of Daphnia itat. Brine shrimp inhabit hypersaline lakes and marine magna as a model for the screening and testing of habitats. Most feed on organic debris in the water endocrine-disrupting chemicals in included in this issue column and scraped from surfaces. Some fairy shrimp (Tatarazako and Oda 2006). are predators. Class: Maxillopoda Order: Notostraca (tadpole shrimp) This widespread and morphologically diverse group Tadpole shrimp are typically 1–10 cm long and inhabit encompasses over 26,000 species (Brusca and Brusca inland waters of various salinities. Notostracans are not 2003). Most are small with reduced or absent abdo- known to be marine. Most inhabit ephemeral ponds mens and fused head and thorax regions. Some body and must contend with the same exposure issues segments may lack appendages. This class contains resulting from pond desiccation as described for the many parasitic crustaceans. Maxillopods include fairy shrimp. Most feed on organic matter associated the seed shrimp (Subclass: Ostracoda), barnacles with sediments, though some are predatory. (Infraclass: Cirripedia), and copepods (Subclass: Order: Diplostraca Copepoda).

The diplostracans are characterized by a bivalved Subclass: Ostracoda (seed shrimp) (clam shrimp) or folded (water flea) carapace that typically shields the entire body, though abdominal Ostracods are largely minute ( < 1 mm) crustaceans segments are exposed in some species. This order is that are widely distributed in marine and freshwater divided into four suborders encompassing clam shrimp environments. Members of one marine genera, Gigan- (Laevicaudata, Spinicaudata, and Cyclestherida) and tocpris, can attain lengths of up to 3 cm. The body of water fleas (Cladocera). the ostracod is enclosed by a bivalved, hinged carapace resulting in the common name of ‘‘mussel shrimp.’’ Clam shrimp The head region of seed shrimp dominates the body and the trunk is much reduced. Most ostracods are These are small to medium sized ( £ 2 cm) inhabitants bottom dwelling detritovores, though the giant seed of ephemeral and permanent freshwater habitats. Clam shrimp Gigantocpris is predatory. Ostracods are

123 Crustacean endocrine toxicology 65 dioecious and can reproduce either sexually or through have inhabited the terrestrial environment. Species parthenogenesis. range in size from ~0.5 to 500 mm in length. Isopods lack a carapace and are typically dorsally ventrally Infraclass: Cirripedia (barnacles) compressed. They are typically bottom dwellers where they crawl about in search of food. Isopods are diverse The barnacles are the lone group of sessile crustaceans. feeders and include herbivores, omnivores, detrito- These exclusively marine crustaceans spend most of vores, predators, and parasites. Most isopods are their adult life attached to rocks, shells, man-made dioecious with a few hermaphroditic forms. substrates, and other animals (turtle shells, whales, etc.). Some barnacles are truly parasitic. The carapace Order: Amphipoda of adult barnacles is covered with calcareous plates. Barnacles are suspension feeders and collect organic Amphipods inhabit both freshwater and marine hab- particles by extending and waving their feathery itats. They range in size from ~1 to 25 cm and share thoraxic appendages. Barnacles are typically hermaph- many physical characteristics with the isopods. A rodites that are capable of self-fertilization. notable difference among the two classes is that the most common amphipods tend to be laterally com- Subclass: Copepoda pressed and are typically better adapted to swimming than are isopods. Feeding strategies among amphipods This diverse group of crustaceans inhabits both marine are as diverse as among the isopods. Amphipods are and freshwater environments. Species are typically dioecious and reproduce sexually. small ( £ 10 mm) and often planktonic. Some parasitic forms are large (up to 25 cm). Most free-living cope- Order: Decapoda pods have a single, centrally located eye and filter feed on phytoplankton. Copepods are a major constituent Most decapods are marine, with a few freshwater to zooplankton and serve a vital role in the trophic- species (crayfish, prawns) and still fewer terrestrial transfer of nutrients. Copepods are dioecious and species (land crabs). Decapods are typically medium to reproduce sexually. A current review of the use of large sized crustaceans and include the largest crusta- copepods as a model for the screening and testing of cean species noted above. Decapods are the most endocrine-disrupting chemicals in included in this issue economically important of the crustaceans and are (Kusk and Wollenberger 2006). major constituents to invertebrate fisheries worldwide. Decapods can be free swimming or benthic crawlers Class: Malacostraca and many burrow into bottom sediments or beach sand. Decapods utilize the diverse feeding strategies The malacostracans encompass most of the larger, associated with other malacostracans. These crusta- more easily recognized, and economically important ceans are dioecious with strong sexual dimorphisms in crustaceans. Malacostracan crustaceans typically pos- some species (i.e., fiddler crabs). sess three distinguishable body parts—head, thorax, The Mysida (Mysid shrimp) represent an order of and abdomen. The head and thoraxic regions some- small shrimp-like malacostracan that holds prominence times fuse to form the cephalothorax, which is typically in endocrine toxicology as a recommended test species shielded by the carapace. There are over 40,000 (Verslycke et al. 2004) and are worthy of inclusion in identified species of malacostracans that, according to this discussion. Mysids range in size from roughly most classification schemes, are divided between the 2 mm to 8 cm. These organisms are largely marine subclasses Phyllocarida (primitive forms) and Eumal- pelagic or demersal though a few freshwater species acostraca (contemporary forms). Among the contem- exist. A current review of the use of mysids as a model porary forms are the orders: isopoda (isopods, pill for the screening and testing of endocrine-disrupting bugs, wood lice, and sow bugs), Amphipoda (amphi- chemicals is included in this issue (Verslycke et al. pods, scud, and sideswimmers), and Decapoda 2006). (shrimps, crabs, lobsters, and crayfish).

Order: Isopoda Crustacean endocrinology

The isopods consist of freshwater and marine species. Crustaceans have populated the marine, freshwater, They are also the most successful crustacean group to and terrestrial environments. Physiologies of these

123 66 G. A. LeBlanc organisms have evolved to suit their respective habitats (Chen et al. 2005). These hormones are produced in and respond to environmental cues in their environ- the X organ of malacostracan crustaceans and are ment that enhance their chances of survival and stored in and secreted from the sinus gland (Keller and reproduction. Crustaceans utilize a variety of neuro- Sedlmeier 1988). Both the X organ and sinus gland are endocrine signaling cascades to regulate their physiol- located in the eyestalk. Secretion of CHH hormones ogy. Some of these pathways are conserved among occurs in response to monoamine neurotransmitter various animal phyla. Others are unique to the (serotonin, dopamine) stimulation (Chen et al. 2003). Arthropoda. In recent years, toxicologists have recog- Neuro-signaling of CHH release occurs in response to nized that some endocrine pathways are highly sus- both environmental and endogenous cues (Beltz 1988). ceptible to aberrant signaling by environmental Crustacean hyperglycemic hormone family mem- chemicals resulting in various toxicities that can com- bers regulate diverse activities. Some are involved in promise population sustainability. These susceptible regulating carbohydrate metabolism to meet the endocrine pathways have been most extensively stud- energy needs of the organism in response to changing ied among the vertebrates. This information has environmental or physiological conditions. These are limited application to the Crustacea, since the domi- the hyperglycemic hormones from which the hormone nant, and perhaps most susceptible, signaling pathways family name was derived (Keller and Sedlmeier 1988). among the crustacean are mediated by ecdysteroids Molt-inhibiting hormone (MIH) negatively regulates and methyl farnesoate. These hormones are absent in ecdysteroid synthesis by the Y organ (see ecdysteroids the vertebrates. Thus, a general review of crustacean below) and serves as the linkage between neurological endocrinology is warranted in an effort to identify signaling and steroidal control of processes such as aspects of this system that are largely conserved among molting and embryo development (Chang 1985) animals as well as to recognize those aspects that are (Fig. 3). Mandibular organ-inhibiting hormone unique to this taxon. Most endocrine studies of the (MOIH) negatively regulates the secretion of methyl Crustacea have been performed with the malacostra- farnesoate from the mandibular organ and its associ- cans. While general observations and conclusions ated regulatory activities (see terpenoids below). drawn from studies of crabs, shrimp, crayfish, and Gonad-inhibiting hormone [GIH, also called vitelloge- lobsters can likely be applied to other crustacean taxa, nin-inhibiting hormone (VIH)] negatively regulates some aspects of endocrine signaling are clearly unique aspects of gonadal maturation (Aguilar et al. 1992; to some crustacean groups and readers are cautioned Huberman 2000). CHH family members often exhibit against extensive extrapolations of observations among mixed functions; for example two CHHs were identi- the crustacean classes. fied from American lobster (H. americanus) that contributed to regulating carbohydrate metabolism Peptide hormones (Chang et al. 1990). However, one hormone also contributed to the regulation of molting (Chang et al. Peptide hormones play an important role in regulating 1990); while, the other stimulated oocyte maturation various physiological processes in crustaceans. Peptide (Tensen et al. 1989). CHHs are divided into two hormones can serve as intermediates between neuro- subfamilies based upon amino acid sequence, struc- logical signaling (nerve transmission) and terminal tural characteristics, and functional divergence (Chen hormone signaling (i.e., steroid hormones) (Fig. 3)or et al. 2005). Subfamily 1 contains the hormones with as early, but terminal, hormones in a signaling cascade. hyperglycemic activity and subfamily 2 consist of the These peptide hormones are typically released from negative regulators (i.e., the inhibiting hormones) tissues in response to neurological stimulation, are (Chen et al. 2005). transported by the circulatory system to target sites, and bind receptors on the cell surface where they Androgenic gland hormone stimulate intracellular signal transduction pathways leading to the release of the next hormone in a cascade The androgenic gland is a diffuse tissue located in or directly regulating some physiological action. association with the terminal region of the male gamete ducts. This gland has thus far been identified Crustacean hyperglycemic hormone (CHH) family only in the malacostracan crustaceans (Isopoda, Deca- poda) and produces a peptide hormone: androgenic The crustacean hyperglycemic hormone (CHH) family gland hormone (AGH) (Sagi and Khalaila 2001). AGH of peptide hormones consists of hormones of ~80 has been definitively characterized in the isopods amino acids that share significant sequence similarity Armadillidium vulgare (Martin and Juchault 1999;

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Fig. 3 Deduced endocrine circuit in crustaceans that is suscep- The ecdysteroid receptor (EcR, black) regulates the expression tible to disruption by endocrine toxicants. Diagram depicts a of a variety of genes. This regulatory activity is controlled by the two-hormone circuit consisting of ecdysteroid (20E) and terpe- retinoid X receptor (RXR, gray). Unliganded retinoid receptor noid (MF) hormones inferred largely from studies in insects as (a) is permissive of ecdysteroid signaling. When bound by ligand, cited in the text. This regulatory circuit exists at both neuroen- the RXR can inhibit ecdysteroid signaling (b). The receptor docrine and endocrine levels. Black circles represent organs that HR38 (white), can bind the RXR (c) and repress ecdysteroid are responsive to one hormone and produce another hormone in signaling (Zhu et al. 2000). In addition to regulating steroid the circuit. Known signaling molecules (hormones) are identified signaling, the RXR can act independently to regulate gene in italics (MO monoamines, MIH molt-inhibiting hormone, 20E expression (i.e., induction of dhb2 and sex determining genes and 20-hydroxyecdysone, MOIH mandibular organ inhibiting hor- suppression of HR38). The proposed role of RXR in methyl mone, MF methyl farnesoate). Whether the circuit is opened (+) farnesoate signaling is based upon experiments performed in or closed (-) by the received signal is indicated. Arrows depict the insect systems with juvenile hormone (see text for details) direction of the signal. Receptor proteins are depicted as squares.

Okuno et al. 1999), Porcellio scaber and Porcellio of ovarian tissue to testicular tissue (LeBlanc et al. dilatatus (Ohira et al. 2003). The functional hormone 1999). AGH also negatively regulates vitellogenin from A. vulgare is a dimeric 8.7 kDa peptide consisting synthesis. Ablation of the androgenic gland from males of a 29 amino acid subunit (A chain) and a 44 amino stimulates vitellogenin synthesis by the fat body acid subunit (B chain) that are linked by two disulfide (Suzuki et al. 1990). Implantation of the androgen bridges. The hormone is translated as a single gene gland into females inhibits vitellogenin synthesis and product in which both subunits are linked by an ultimately causes functional sex reversal of the gonads additional peptide (C chain). As such, AGH resembles (Suzuki and Yamasaki 1997b). AGH seems to function members of the proinsulin superfamily of peptide as a sex differentiating factor in crustaceans but not a hormones (Martin and Juchault 1999; Okuno et al. sex determining factor. That is, administration of AGH 1999). The A and B chains are highly conserved among during sexual differentiation stimulates the develop- the three isopods from which the hormone has been ment of male sex characteristics; administration of characterized (Ohira et al. 2003). AGH to differentiated females causes reversal of sex Experiments involving hormone administration, characteristics; but administration of AGH to imma- androgenic gland implantation, or androgenic gland ture, undifferentiated animals has no effect on devel- ablation have revealed that the hormone is responsible opment of sex characteristics (Suzuki 1999). for general male sex differentiation (both primary and Compromised AGH signaling also has been associated secondary sex characteristics). This includes masculin- with intersex conditions in amphipods where males ization of pleopod and cheliped morphology, develop- exhibit normal masculine secondary sex characteristics ment of the male gonopore complexes and conversion along with a rudimentary brood chamber (Ford et al.

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2005). Internally, these intersex animals possess testes Ecdysteroids (predominantly 20-hydroxyecdysone) along with an oviduct. elicit their regulatory activity by binding to the ecdysteroid receptor (EcR, gene designation NR1H1, Ecdysteroids Fig. 3). Other NR1H group members include the liver- X receptor (LXR) and the farnesoid-X receptor (FXR) The ecdysteroids are signaling molecules that are well- of vertebrates. The EcR regulates gene transcription in characterized with respect to their role in regulating association with retinoid X receptor (RXR) (gene the molting process (ecdysis). Ecdysteroids are under designation NR2B; originally called ultraspiracle, Usp, the negative regulatory control of MIH (Fig. 3), which in insects). Upon ecdysteroid hormone binding the maintains ecdysteroid levels at low-circulating levels complex recruits co-activators that ultimately stimu- during the intermolt period. Just prior to molting, MIH lates gene transcription. Insect RXRs are capable of levels decrease resulting in a precipitous increase in binding terpenoid hormones (Fig. 5, discussed below) circulating ecdysteroid levels (Fig. 4). Ecdysis is trig- (Jones and Sharp 1997; Jones and Jones 2000; Jones gered by the subsequent decline in ecdysteroids back et al. 2001). When bound to juvenoid hormone, the to basal levels. Shedding of the old exoskeleton and its EcR/RXR complex attracts co-repressors that nega- replacement with a larger cuticle allows for growth of tively regulate gene transcription (Fig. 3) (Maki et al. the organism. 2004). The negative regulation of ecdysteroid activity Ecdysteroids also are necessary for normal embry- by binding of terpenoids to the EcR/RXR complex has onic development (Mu and LeBlanc 2002b, 2004b) and not yet been demonstrated in crustaceans. However, reproduction (Souty et al. 1982; Gunamalai et al. 2004). terpenoids have been shown to inhibit processes that Ecdysteroids, of maternal origin, are packaged into are under the positive control of ecdysteroids (Mu and eggs for use during early embryo development (Subr- LeBlanc 2004a). amoniam 2000). During latter stages of development, embryos appear capable of synthesizing ecdysteroids Terpenoids that orchestrate aspects of late embryo development (Mu and LeBlanc 2004b). Limited evidence also Terpenoids are a class of terminal hormones that suggests that ecdysteroids stimulate vitellogenesis dur- include 9-cis and all trans-retinoic acid, juvenile hor- ing ovarian oocyte maturation (Souty et al. 1982). mone (insects), and methyl farnesoate (crustaceans) While it is clear that ecdysteroid and vitellogenin levels (Fig. 5). The action of terpenoids in vertebrates is both progressively increase during ovarian maturation mediated by the nuclear receptors retinoic acid recep- (Suzuki et al. 1996; Jayasankar et al. 2002; Tseng et al. tor (RAR, gene designation NR1B), retinoic acid 2002), it is not clear at this time whether ecdysteroids orphan receptor (ROR, gene designation NR1F), and are responsible for the increase in vitellogenin levels. RXR (Mangelsdorf and Evans 1995; Stehlin-Gaon et al. 2003). The receptors that mediate the action of arthropod terpenoids are not currently known although RXR has been cloned from two decapod 250 crustaceans (Durica et al. 2002; Kim et al. 2005). Molt Molt Methyl farnesoate (Fig. 5) is a major terpenoid 200 hormone of crustaceans. This signaling molecule is the unexpoxidated form of juvenile hormone III of insects 150 (Fig. 5). Methyl farnesoate has been measured in over 30 species of crustaceans including decapods (crabs, 100 shrimp, etc.), cirripedes (barnacles), and anostracans (brine shrimp) (Laufer and Biggers 2001). This hor- 50 mone has many regulatory functions in crustaceans Ecdysone (pg/mg) that are associated with juvenile hormones in insects 0 (LeBlanc et al. 1999). Methyl farnesoate has been 25 30 35 40 45 50 55 60 implicated in reproductive maturation in decapods Age (hr) where it increases production of vitellogenin and eggs (Vogel and Borst 1989; Laufer and Biggers 2001; Fig. 4 Ecdysterone levels in juvenile daphnids at various times Rodriguez et al. 2002; Mak et al. 2005). Methyl between the first and second molt (Mu and LeBlanc 2004b). Data are presented as mean and SEM. Ecdysteroid levels were farnesoate also stimulates gonadal maturation (increased normalized to the wet tissue weight of the daphnids mass) of both males (testes) and female (ovaries)

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Fig. 5 Terpenoid compounds CH3 CH3 CH3 O and the functional mimic pyriproxyfen that have CH3 Methyl Farnesoate endocrine activity in H3C O crustaceans CH3 CH3 O H3C CH3 Juvenile Hormone III O H3C O

CH CH H3C CH3 3 3 COOH Retinoic Acid

CH3

CH3 O O N Pyriproxyfen

O decapods (Laufer et al. 1998; Reddy and Ramamurthi reproductive morphotype lacks these aggressive char- 1998; Kalavathy et al. 1999). acteristics (Laufer et al. 2005). Methyl farnesoate levels Effects of methyl farnesoate on larval development determine whether the male crayfish acquires the and metamorphosis have been noted in the barnacle reproductive morphotype upon molting. Low-circulat- Balanus amphitrite and the prawn Macrobrachium ing methyl farnesoate levels just prior to molting result rosenbergii, though observations among experiments in the acquisition or retention of the reproductive are not consistent and preclude establishing general morphotype upon molting; while high-methyl farneso- conclusions on the role of this hormone in crustacean ate levels result in acquisition or retention of the non- metamorphosis. In general, high-exposure concentra- reproductive morphotype. tions ( ‡ 1 lM) of methyl farnesoate stimulated meta- Branchiopod crustaceans are commonly cyclic morphosis of the barnacle (Yamamoto et al. 1997; parthenogens in which the population utilizes both Smith et al. 2000). However, 0.1 lM inhibited meta- asexual and sexual reproduction (Hebert 1978). Under morphosis in one study (Smith et al. 2000), while favorable environmental conditions, the population exposure levels as low as 0.01 lM stimulated meta- may exist largely as clonally reproducing females. morphosis in the other study (Yamamoto et al. 1997). Under specific environmental cues that forewarn of the In the decapod prawn, methyl farnesoate exposure demise of the habitat, the females will produce male concentrations ranging from 0.85 to 2.4 lM inhibited offspring. This is followed by the entry of the popu- larval growth and development resulting in the pro- lation into a sexual reproductive phase resulting in the duction of organisms with mixed larval and juvenile production of diapause, resting eggs. Among the physical traits (Abdu et al. 1998). In general, methyl daphnids (D. magna, D. pulex, D. pulicaria), high- farnesoate appears to stimulate larval development in methyl farnesoate levels in the maternal daphnid the cirripedes while inhibiting larval development in results in the production of male offspring (Olmstead the decapods. This conclusion is supported by exper- and LeBlanc 2002; Tatarazako et al. 2003; Rider et al. iments with methyl farnesoate mimics (discussed 2005). This sex-determining effect of the hormone is below). restricted to the latter stages of ovarian oocyte matu- Methyl farnesoate has clear regulatory roles in male ration (Olmstead and LeBlanc 2002). Once the eggs reproductive morphology and behavior in decapods are transferred to the brood chamber, sex is fixed. (Laufer et al. 1994; Laufer and Biggers 2001). Adult Historical observations have led to the conclusion that male crayfish Procambarus clarkii have two morpho- a branchiopod brood consist of either all male or all types. The reproductive morphotype has characteristic female offspring (Barker and Hebert 1986; Hobaek large chelae and spines on the ischiopodites. The non- and Larsson 1990). However, laboratory studies have

123 70 G. A. LeBlanc shown that intermediate levels of methyl farnesoate candidate genes induced by the methyl farnesoate will stimulate the production of mixed broods of receptor). offspring (Olmstead and LeBlanc 2002) and even 9-Cis retinoic acid is the prototypical ligand for the intersexed (bilateral gynandromorphic) individuals vertebrate RXR (Mangelsdorf and Evans 1995). RXR (A. W. Olmstead and G. A. LeBlanc, personal obser- is expressed in crustaceans (Durica and Hopkins 1996; vations). Methyl farnesoate has recently been shown to Kim et al. 2005; Wang et al. 2006)and9-cis retinoic be a sex determinant in cladoceran species of the acid has been measured in at least one decapod genuses Ceriodaphnia and Moina (Oda et al. 2005a). crustacean (Hopkins 2001). Endocrine signaling via 9- Male offspring also have been produced by adminis- cis retinoic acid/RXR has not been demonstrated in tration of methyl farnesoate to several suspect obligate crustaceans. However, the presence of the receptor and parthenogenetic lacustrine species of branchiopods in its putative hormone ligand argues that this hormone which males were previously unknown (Kim et al. signaling pathway is relevant in crustaceans. As in 2006). It is presently unknown whether this sex vertebrates (Chawla et al. 2001), 9-cis retinoic acid determining role of methyl farnesoate is common might stimulate transcriptional activity in crustaceans among other Branchiopoda and perhaps other crusta- that is associated with RXR homodimers. cean taxa. Retinoic acid signaling is important in the regula- A receptor that is responsible for transducing tion of limb development in vertebrates (Tabin 1991). methyl farnesoate signaling has not yet been identified. Similarly, evidence suggests that this signaling pathway RXR has been implicated as a receptor for juvenile also is involved in crustacean limb regeneration hormone in Drosophila (Jones and Sharp 1997; Jones (Hopkins 2001). 9-Cis retinoic acid and RXR are and Jones 2000; Jones et al. 2001; Xu et al. 2002; Maki present in regenerating crustacean limbs (Hopkins et al. 2004) and a similar role for crustacean RXR with 2001), RXR levels are elevated in the regenerating methyl farnesoate is possible considering the structural limb tissue by exogenous administration of 9-cis and functional similarity between juvenile hormone retinoic acid (Chung et al. 1998); and, administration and methyl farnesoate (Fig. 5). Juvenile hormone is of 9-cis retinoic acid to crabs during early limb capable of mimicking methyl farnesoate in at least regeneration disrupts the process in a manner sugges- some crustaceans (Tatarazako et al. 2003). However, tive of suppressed cellular differentiation and en- very high concentrations of juvenile hormone are hanced cellular proliferation (Hopkins and Durica required to activate insect RXR (Davey 2000). Fur- 1995). Taken together, these observations indicate that thermore, RXR-expressing insect cells that were retinoid signaling is important to normal limb regen- transfected with a reporter constructs containing a eration, and disruption of this signaling process by juvenoid response element have consistently failed to environmental endocrine disruptors could have dire show activation of the reporter gene with juvenile consequences to crustaceans. hormone treatment (Harmon et al. 1995; Baker et al. 2000). Vertebrate-type sex steroid hormones Recent evidence suggests that the crustacean methyl farnesoate receptor may function as a monomeric Several studies have shown responses of crustaceans to transcription factor following ligand activation. Treat- vertebrate-type steroid hormones. However, most of ment of daphnids (D. magna) with methyl farnesoate these studies were not designed to distinguish overt stimulated the binding of a factor (e.g., receptor) to a toxic responses from true endocrine signaling. Further, putative response element in the methyl farnesoate- the high-exposure concentrations often required to inducible hemoglobin 2 gene (Gorr et al. 2006). This elicit a response argues in favor of toxic effects of these response element consisted of a steroid/terpenoid compounds. Administration of vertebrate-type steroi- response element-like half-site adjacent to a 5¢ A/T- dal androgens (testosterone, androstenedione) to daph- rich extension. This binding site resembles response nids (D. magna) causes aberrant embryo development elements to monomeric nuclear receptors such as (LeBlanc 1999; LeBlanc et al. 2000, and discussed NGFB-I in mammals and HR38 in insects (Kozlova below). Co-administration of 20-hydroxyecdysone pro- et al. 1998; Meinke and Sigler 1999). Interestingly, tected embryos against this effect (Mu and LeBlanc RXR from jellyfish (Tripedalia cystophora) was shown 2002a). Further, testosterone interfered with the action to bind response elements as a monomer (Kostrouch of 20-hydroxyecdysone in an ecdysone-response insect et al. 1998). Perhaps, methyl farnesoate binds to cell line (Mu and LeBlanc 2002a). These observations crustacean RXR or a related receptor, which then indicated that the activity of testosterone in daphnids activates responsive genes as a monomer (see Fig. 3 for was mediated by its ability to interfere with ecdysteroid

123 Crustacean endocrine toxicology 71 signaling and not through an androgen signaling Aberrant peptide hormone signaling pathway. Steroidal estrogens (17b-estradiol, diethylstilbestrol, Hyperglycemia has been reported in several decapod 17a-ethinyl estradiol, estrone) have been reported to crustaceans following exposure to cadmium, naphtha- elicit various effects in crustaceans including altered lene, and DDT (Fingerman et al. 1981; Nagabhusha- gonadal development in Amphipoda (Segner et al. nam and Kulkarni 1981; Reddy et al. 1994, 1996). The 2003), decreased fecundity in Branchiopoda (Baldwin effects of these compounds on blood glucose levels et al. 1995), inhibition of naupliar development in were associated with changes in levels of CHH in the Maxillopoda (Andersen et al. 2001), and reduced eyestalks. Chemical-induced modification in eyestalk vitellin levels in Mysida (Ghekiere et al. 2006). How- peptide hormones and associated apical responses of ever, effects from chemical exposure in these experi- crustaceans are reviewed in detail elsewhere (Finger- ments are reported with no mechanistic support for the man et al. 1998). involvement of estrogen signaling. The lack of mech- anistic support for estrogen signaling in crustaceans Aberrant ecdysteroid signaling coupled with the apparent lack of an estrogen receptor in crustaceans (Thornton et al. 2003) argues that this Ecdysteroids regulate aspects of embryo development, signaling pathway is not relevant to this subphylum. growth (molting), and reproduction (perhaps vitellog- Progestogens (progesterone,17b-hydroxyprogester- enin synthesis). Accordingly, chemicals that interfere one,17a-hydroxyprogesterone) reportedly stimulate with ecdysteroid signaling have the potential to elicit ovarian maturation and vitellogenesis in decapod profound adverse effects on crustacean populations. crustaceans (Yano 1985, 1987; Quackenbush 1992; Chemicals with anti-ecdysteroidal activity in crusta- Zapata et al. 2003). Insufficient evidence exists to ceans have been identified that function as either ascribe this effect to direct progestogen signaling. ecdysteroid synthesis inhibitors or ecdysteroid receptor Although, this positive effect, which is similar to that antagonists. Chemicals with anti-ecdysteroidal activity observed with ecdysteroids and methyl farnesoate, include many of the classic estrogen receptor agonists warrants further study. of vertebrates (Table 1) and interaction with the estrogen receptor, to some degree, may be indicative of whether a chemical will interact with the ecdysteroid Toxicant-mediated endocrine disruption in crustaceans receptor or vice versa (Table 1). However, studies with ecdysteroid-responsive insect cells have demonstrated Exposure issues that non-steroidal EcR agonists are rare (Dinan et al.

With the exception of some malacostracans (i.e., some crabs, isopods), crustaceans inhabit the aquatic envi- Table 1 Anti-ecdysteroidal activity and estrogen receptor ago- ronment. Respiration occurs via gills or similar respi- nist activity of environmental chemicals as determined in cell- based assays (Dinan et al. 2001; NIEHS 2002) ratory appendages, and thus, aqueous uptake of chemicals across epithelial membranes is a major route Chemical EC50 (lM) of exposure. Many species are detritovores (i.e., Ecdysteroid receptor Estrogen receptor Cephalocarida, Ostracoda) and inhabit organic-rich sediments that can harbor significantly higher levels of Bisphenol A 100 0.3 Diethylphthalate 2,000 380 lipophilic chemicals than the surrounding water. These 4-Nonylphenol >1 1.0 organisms are subject to exposure both from pore Fluoranthene >50 - water, containing higher levels of chemical than in the Lindane 30 + general water column, as well a chemical associated 4,4¢-DDD >250 + 2,4¢-DDE >25 5,340 with the detritus. Bound chemicals can be rendered 4,4¢-DDE >25 3.0 bioavailable during digestion of the organic particles. 4,4¢DDT >25 1.0 Many Branchiopoda are restricted to ephemeral ponds Dieldrin >10 20 where exposure concentrations of chemicals can Reseratrol 10 - Zearalenone >25 0.002 increase as the pond desiccates. Finally, most crusta- ceans are mobile and can avoid exposure to noxious Greater than ( > ) values represent the highest concentration endocrine toxicants. However, mature cirripedes are evaluated at which a positive response was measured, but that response was less than 50%. A negative notation (-) indicates sessile and incapable of avoiding exposure to chemical that the compound elicited no response. A positive notation (+) exposures. indicates that a response was observed but not quantified

123 72 G. A. LeBlanc

2001). The binding of an environmental chemical to tions in ecdysteroid signaling in crustaceans by xeno- the EcR will more likely result in inhibition of biotics. Recent development of a crustacean EcR ecdysteroid signaling. reporter gene construct may stimulate screening efforts Ecdysteroid levels were significantly lowered in aimed at identifying chemicals that harbor this activity daphnid (D. magna) embryos isolated from maternal (Yokota et al. 2005). daphnids that were exposed to the agricultural fungi- Several studies have reported effects of environ- cide fenarimol (Mu and LeBlanc 2002b). Severely mental chemicals that are consistent with interference affected embryos experience early developmental with ecdysteroid signaling, though a precise mechanism arrest. Less severely impacted embryos exhibited of action was not established. The chemicals 4-nonyl- abnormalities in the development of the carapace and phenol (Shurin and Dodson 1997; LeBlanc et al. 2000), antennae that were indicative of disturbances in late propiconazole (Kast-Hutcheson et al. 2001), and embryo development. Effects of fenarimol on devel- bisphenol A (Mu et al. 2005), have elicited effects in opment were ameliorated if embryos were adminis- crustaceans consistent with anti-ecdysteroidal activity. tered 20-hydroxyecdysone during development The fungicides propiconazole (Ronis et al. 1998) and indicating that ecdysteroid deprivation due to fenari- fenarimol (Williams et al. 2000) inhibit cytochrome mol was responsible for the abnormal development. P450 (CYP) enzymes that are critical to ecdysteroid Direct incubation of isolated embryos with fenarimol synthesis. Both of these chemicals may inhibit ecdys- resulted only in the late developmental abnormalities. teroid synthesis through this enzyme inhibition. The 4- This suggests that the adverse effects on early embryo Nonylphenol is an antagonist of the insect ecdysteroid development observed during maternal exposure were receptor in vitro (Dinan et al. 2001). Bisphenol A was due to deprivation of maternally derived ecdysteroid to proposed to elicit anti-ecdysteroidal activity through a the embryo. Late developmental effects appeared to be receptor cross-talk mechanism (Mu and LeBlanc due to impaired de novo ecdysteroid synthesis by the 2004a; Mu et al. 2005). Thus, it is mechanistically embryos. plausible that all of these compounds elicit toxicity to Testosterone exposure causes abnormal embryo crustaceans via perturbations in ecdysteroid signaling. development of daphnids similar to that observed with fenarimol (LeBlanc et al. 2000). Administration of Aberrant methyl farnesoate signaling exogenous 20-hydroxyecdysone protected embryos against this toxicity of testosterone indicating that The elusive nature of the methyl farnesoate receptor testosterone interfered with normal ecdysteroid signal- has prevented direct evaluations of the ability of ing. Additional studies indicated that testosterone chemicals to bind the receptor as either agonists or elicited anti-ecdysteroidal activity by inhibiting the antagonists. However, assays have been developed EcR (Mu and LeBlanc 2002a, 2004b). with apical endpoints that are highly specific for methyl Among chemicals shown to elicit 20-hydroxyecdy- farnesoate signaling activity. Most notable with crus- sone-like activity in crustaceans are ponasterone A and taceans is the production of male offspring in bran- RH 5849. Ponasterone A is a steroid that was first chiopod daphnids (Wang et al. 2005). Reared under isolated from plants that has high-ecdysteroid activity appropriate conditions, daphnids will produce broods in insects (Nakanishi et al. 1966). Exposure of D. of entirely female offspring. Exposure to methyl magna to ponasterone A stimulated premature ecdysis farnesoate agonists will cause the production of male (Baldwin et al. 2001). However, molted carapaces were offspring with the incidence of males increasing in a not released from the organisms causing death. RH dose-dependent manner (Olmstead and LeBlanc 5849 is an insecticidal 20-hydroxyecdysone agonist. In 2003). Studies to date have shown that chemicals with the land crab Rhithropanopeus harrisii, RH 5849 methyl farnesoate agonist activity appear to be rare accelerated molting; and, in the barnacle B. amphitrite, with the exception of juvenile hormone-mimicking, RH 5849 enhanced attachment and metamorphosis of insect growth-regulating insecticides (Olmstead and the larvae (Clare et al. 1992). These experiments did LeBlanc 2003; Tatarazako et al. 2003; Oda et al. 2005b; not provide mechanistic support for the ecdysteroidal Wang et al. 2005). Some of these compounds (i.e., action of ponasterone A and RH 5849. However, fenoxycarb, pyriproxyfen; Fig. 5) are highly potent effects were consistent with ecdysteroid signaling agonists of methyl farnesoate and stimulate the pro- activity and suggest that chemicals with ecdysteroid duction of only male offspring at low-nanomolar activity in insects can stimulate ecdysteroid-regulated concentrations (Oda et al. 2005a; Rider et al. 2005). physiological processes in crustaceans. Relatively few Methyl farnesoate antagonist activity of chemicals studies have been performed that evaluated perturba- can be measured by exposing crustaceans to methyl

123 Crustacean endocrine toxicology 73 farnesoate to stimulate the endpoint being measured toxicity to field populations resulting in these outcomes (i.e., male offspring production in daphnids) and have intentionally been omitted from this review. assessing the ability of chemicals to block this action Effective biomarkers of endocrine disruption must be (Wang et al. 2005). Few results are presently available sufficiently specific to allow mechanistic inference to from such assays. Interestingly, dieldrin was shown to the cause of the measured disturbance. For example, reduce the proportion of males produced among high-vitellogenin levels in the blood of male oviparous daphnids that were naturally producing mixed-sex vertebrates is sufficiently specific to attribute the broods of daphnids (Dodson et al. 1999), presumably disturbance to estrogenic chemicals (Palmer et al. due to elevated endogenous methyl farnesoate levels, 1998). The endocrine system regulates many of the suggesting that this insecticide has anti-methyl farne- processes that differentiate males from females, larvae soate activity. However, dieldrin elicited a weak from juveniles, and reproductively active from repro- positive response in experiments specifically designed ductively senescent organisms. Either condition (i.e., to detect methyl farnesoate-like activity (i.e., using male versus female) is normal and would be expected daphnids that were producing only female offspring) in a population. As such, these conditions serve as poor (Wang et al. 2005). Thus, dieldrin appeared to block biomarkers of endocrine disruption. An exception to the action of low, endogenous levels of methyl farne- this premise is excess males in parthenogenic bran- soate, but in the absence of methyl farnesoate, elicited chiopod populations that normally exist predominantly a positive response. These results are consistent with as females. Sensitive and specific biomarkers of endo- dieldrin functioning as a mixed agonist/antagonist of crine disruption are those that identify abnormal the methyl farnesoate receptor (see LeBlanc 2003) for ‘‘intermediates’’ in a population (i.e., males with a discussion of mixed agonists/antagonists). female characteristics, larvae with juvenile character- A significant number of laboratory studies have istics). An increased incidence of such intermediates been performed with estuarine decapods and insect can be diagnostic of endocrine disruption, although the growth regulating insecticides (McKenney 2005). Rec- cause of the disruption (i.e., chemical exposure versus ognizing that these compounds function as methyl parasitic infection) requires further investigation. farnesoate mimics in crustaceans allows inference of Several candidate biomarkers of endocrine disruption the effects of compounds having this mechanism of in crustaceans are presented in Table 2. These bio- action on decapod crustaceans. Metamorphic success markers were selected because: (1) they occur in of decapods is among the most sensitive endpoints response to aberrant endocrine signaling, (2) they can affected by insect growth regulators. Delayed meta- be readily discerned in field populations, and (3) they morphosis or metamorphic abnormalities caused by are relatively rare in unaffected populations. exposure to insect growth regulators have been reported in shrimp, crabs, and lobster (Christiansen Intersex incidence et al. 1977a, b; Charmantier et al. 1988; McKenney and Celestial 1993; Celestial and McKenney 1994; Cripe The literature is replete with reports of intersex et al. 2003). Interestingly, insect growth regulators malacostracan decapods including the fiddler crab stimulated metamorphosis in cirripede barnacles Uca pugilator (Zou and Fingerman 2000), freshwater (Gomez et al. 1973) consistent with earlier suggestions crab Potamon fluviatile (Micheli 1991), snow crab that juvenoids have opposite regulatory effects on Chionoecetes opilio (Taylor 1986), blue crab Callinec- larval development/metamorphosis in decapods and tes sapidus (Johnson and Otto 1981), rock crab Cancer cirripedes. irroratus (Moriyasu et al. 1988), trapezoid crab Quad- rella coronata (Galil and Tom 1990), hermit crabs Clibanarius sp. (Turra 2004), lobster H. americanus Endocrine disruption in field populations (Chace and Moore 1959), crayfish Cherax quadricarin- atus (Sagi et al. 2002), burrowing crayfish Parastacus The attribution of endocrine toxicity to observed varicosus (Rudolph et al. 2001), freshwater crayfish disturbances in crustacean populations requires the Samastacus spinifrons (Rudolph 2002), mud shrimp identification of definitive biomarkers of such toxicity. Upogebia stellata (Pinn et al. 2001), penaeid shrimp Mortality, reduced fecundity, lowered recruitment, and Penaeopsis sp. (Farfante 1978), and prawn Pandalus impaired growth all might serve as indicators of borealis (Ivanov and Sokolov 1997). It must be noted endocrine disruption in crustacean; however, such that the South American crayfishes Parastacus and endpoints are indicative of adversity involving a variety Samastacus are reportedly sequential hermaphrodites of mechanisms. Accordingly, claims of endocrine and intersex individuals may represent normal transition

123 74 G. A. LeBlanc

Table 2 Candidate biomarkers of endocrine disruption in crus- location having a high incidence of intersex suggesting taceans that some environmental factor was responsible for the Biomarkera Hormone signal Sentinel high incidence of intersex at some localities. Sites disrupted deemed unaffected by this factor had incidences of intersex less than 1%. A survey of G. pulex populations Intersex individuals Androgenic gland Malacostraca hormone in the UK failed to reveal any relationship between Vitellogenin in males Androgenic gland Malacostraca intersex incidence and exposure to sewage effluent hormone (Gross et al. 2001). However, a highly significant Reduced egg Androgenic gland Malacostraca number of females collected from an effluent-contam- vitellogenin hormone Intersex individuals Methyl farnesoate Branchiopoda inated site had oocytes containing a reduced number of Excess males in Methyl farnesoate Branchiopoda yolk bodies and lipid globules, suggestive of reduced population vitellogenesis in these organisms (Gross et al. 2001). In Mixed sex broods of Methyl farnesoate Branchiopoda other studies, significant differences in vitellogenin-like offspring Larval-juvenile Methyl farnesoate Malacostraca protein levels were measured in amphipods Gammarus intermediates sp. collected from polluted sites (marinas, high-boat Larval-juvenile Methyl farnesoate Cirripedia traffic areas) as compared to those collected from intermediates pristine areas (Gajewski et al. 1996; Gagne et al. 2004). Incomplete molting Ecdysteroid All Reduced egg Ecdysteroid All Parasitic infections of amphipods with Microspori- vitellogenin dia have been associated with increased incidences of Altered cuticle Ecdysteroid All intersex (Ginsburger 1991; Kelly et al. 2004; Rodgers- constituents Gray et al. 2004). These parasites alter functional sex The indicated hormone signals represent the apical hormone in ratios in infected populations by feminizing males and an affected signaling pathway. Earlier hormones or receptors in thus favoring the transmission of the parasite to the signaling pathway may actually be affected by the disruptor offspring (Terry et al. 1998; Dunn et al. 2001; Kelly a Many endpoints alone may not be specifically diagnostic of et al. 2004). Similar parasitic feminization has been endocrine disruption but should be used in conjunction with other endpoints in a weight-of-evidence assessment observed among isopods infected with Wolbachia (Rigaud 1997; Rigaud and Moreau 2004). Intersex can result when the parasite only partially feminizes stages for these species (Rudolph et al. 2001; Rudolph the male (Rodgers-Gray et al. 2004). Thus, parasitic 2002). Intersex malacostracans are most often reported infection must be considered when evaluating the to be male, based upon the predominance of testicular cause of intersex within a malacostracan population. tissue and male reproductive track, with vestigial Among the Maxillopoda, intersex populations of female characteristics. These characteristics are con- freshwater and marine copepods have been reported. sistent with the intersex organisms being male with A population of Leptodiaptomus minutus was described compromised AGH signaling. Normal incidence of from a Canadian lake where 7% of the females (as intersex decapods is typically < 1%, excluding the determined by reproductive tract anatomy) displayed hermaphroditic crayfishes, which have a much higher male secondary sex characteristics that included male- incidence. Examples of secondary sex characteristics in like antennules and leg appendages (Sillett and Stem- intersex decapods are depicted in Fig. 6. berger 1998). Populations of the marine copepods Intersex also has been reported in populations of the Paramphiascella hyperborean, Stenhelia gibba, and malacostracan isopods and amphipods (Soutygrosset Halectinosoma sp. were identified in association with and Juchault 1987; Korczynski 1988; Dunn et al. 1994; sewage outfalls that had significantly elevated incidents Ford et al. 2005). Some intersex individuals have been of intersex (Moore and Stevenson 1991, 1994). This reported as having a largely male phenotype (Hastings similar aberration among different copepod species 1981); while, other have been described as having a inhabiting the same vicinity argued for the involvement predominately female phenotype (Ladewig et al. of some environmental factor in the occurrence of 2002). A study of amphipod Gammarus fossarum intersex. However, no clear relationship could be populations in five streams in Germany revealed established between exposure to sewage effluent and incidents of intersex ranging from 0.2 to 24% with no incidence of intersex (Moore and Stevenson 1994). discernible relationship between intersex incidence and Intersex has been noted among the Branchiopoda pollution (Jungmann et al. 2004). The incidence of including Cladocerans Daphnia sp. (Mitchell 2001) intersex increased among gammarids transplanted and Simocephalus sp. (Banta et al. 1939), brine from a location with a low incidence of intersex to a shrimp (Bowen and Hanson 1962), and fairy shrimp

123 Crustacean endocrine toxicology 75

Fig. 6 Secondary sex characteristics of Decapoda that can serve to detect intersex animals. The first pair of pleopods of male decapods is typically modified to transfer sperm to the gonopore of females(A). In females these pleopods are structured as swimmerettes. Intersex animals can have one modified pleopod (as depicted) or have pleopods that are intermediate between the male and female forms. The abdomen (also called the apron) of crabs is sexually dimorphic as depicted (B). Intersex crabs can have abdomens that exhibit male and female characteristics, often in a bilateral manner (depicted)

(Sassaman and Fugate 1997). Intersex branchiopods Molt frequency typically exhibit a bilateral division of sex character- istics (Fig. 7). Such bilateral gynandromorphism also Alterations in molt frequency can be highly indica- is commonly noted in reports of intersex among the tive of disruption of normal ecdysteroid signaling other crustacean genera (e.g., Johnson and Otto (Mu and LeBlanc 2002a, b). Molt frequency can be 1981; Taylor 1986; Micheli 1991) (Fig. 6). Experi- directly measured among isolated individuals of ments performed with D. magna have shown that frequently molting species (i.e., Branchiopoda). How- intersex individuals can arise when maternal daphnids ever, field evaluations of molt frequency among are exposed to marginal concentrations of methyl infrequent molters (i.e., Malacostracans) can be farnesoate (i.e., concentrations that produce mixed challenging. male/female broods) (Olmstead and LeBlanc, sub- The Avalon Peninsula (Newfoundland) snow crab mitted). We also have provided the first definitive (C. opilio) fishery collapsed in the 1980s (Taylor et al. evidence that environmental chemicals (insect 1994). Coincident with the demise of the population, growth-regulating insecticides) can increase the inci- was a reduction in the proportion of newly molted dence of intersex individuals in this species (Olmstead males along with a significant decrease in mean and LeBlanc, submitted). bottom temperatures in the region. Investigators In summary, the incidence of intersex individuals surmised that the colder than normal temperatures shows promise as a biomarker of environmental interrupted the molt cycle of the crab resulting in endocrine disruption among a wide range of crustacean reduced recruitment to the population. With contin- taxa. Intersex is considered abnormal in most species ued harvesting pressure, the population collapsed. with a typical background incidences of < 1%. It is a Supporting this assertion of causality, the adjacent definitive marker of disruption of endocrine signaling Bonavista Bay crab fishery did not experience a pathways involving sex determination (methyl farne- temperature decline during this time period and the soate signaling) and/or sex differentiation (AGH sig- crab fishery remained stable, both in terms of popu- naling). However, caution should be exercised when lation size and proportion of soft-shelled (newly interpreting the cause of intersex since environmental molted) individuals. The proportion of soft-shelled factors other then endocrine disrupting chemi- malacostracans within a population can serve as a cals—most notably parasitism—can be the cause. marker of integrity of the ecdysteroid-signaling path-

123 76 G. A. LeBlanc

Fig. 7 Secondary sex characteristics of Branchiopoda that can serve to detect intersex animals. Male commonly have enlarged first or second antennae that are modified for grasping females during mating. Diplostraca males typically have elongated first antennae; while, females have rudimentary first antennae (A, 1). Intersex animals sometimes have one elongated and one rudimentary antenna or may have two first antennae of intermediate length. Second antennae of Anostraca males are greatly enlarged and modified (B, arrow). The carapace edge of water fleas also is sexually dimorphic and can serve as a biomarker of intersex (A,2)

way assuming the normal incidence of soft-shelled Summary and conclusions individuals is known and against which deviations can be measured. Again, caution is advised as other The crustaceans encompass a wide range of morpho- environmental factors, for example temperature as logically diverse organisms that inhabit marine, fresh- noted in the above example, can affect this biomarker. water, and terrestrial environments. They hold key Exoskeleton composition may serve as a biomarker ecological roles in communities and have major eco- of molting when the normal infrequency of molting nomic importance as a food source to humans. The hampers direct assessments. Following molting, the endocrinology of crustaceans is similar to other ar- ratio of chitin to protein is low in the crustacean cuticle thropods with ecdysteroids and terpenoids having resulting in the characteristic soft-shell. The cuticle is major roles in regulating development, growth, matu- increasingly chitinized as it matures through post-molt, ration, and reproduction. Ample laboratory studies intermolt, and pre-molt stages and the ratio of chitin to have demonstrated the susceptibility of crustaceans to protein increases (Fig. 8). Characterization of normal environmental endocrine toxicants. Field studies have ratios of cuticle components such as, chitin:protein, revealed evidence of endocrine disruption among calcium:protein, etc., in species of interest could serve various crustacean populations, though the establish- as biomarkers of molt stage. Such information could ment of causality between these incidents of abnor- then be used to establish population trends in accel- mality and environmental chemicals is generally erated or delayed molting that may reflect exposure to lacking. The abundance of many crustacean popula- pro-ecdysteroidal or anti-ecdysteroidal agents. Chitin tions, their availability to sampling (i.e., commercial levels were measured in the cuticle of amphipods decapod crustacean catches, intertidal and freshwater (Gammarus sp.) collected from four intertidal areas sampling for isopods, amphipods, and branchiopods), that received high-boat activity and were compared to along with the existence of distinct biomarkers of a population collected from a pristine location (Gagne endocrine disruption (i.e., intersexuality; altered chem- et al. 2004). Chitin levels (not normalized to protein) ical composition of the cuticle) provide opportunities were significantly reduced in gammarids from three of to establish the degree to which environmental endo- the four high-traffic sites. Exposure of brine shrimp crine toxicants are impacting population sustainability. Artemia franciscana to 20-hdyroxyecdysone increased Several genes have been identified that are regulated chitin levels associated with these anostracans as by these endocrine signaling pathways in crustaceans compared to untreated animals (Gagne and Blaise including the hemoglobin gene hb2 (Rider et al. 2005), 2004). the receptor HR3 (El Haj et al. 1997), putative sex

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