Endocrine Disruption in Crustaceans Due to Pollutants: A Comprehensive Review

Mechanisms of Endocrine Disruption in Crustaceans

• Physiological Levels of Endocrine Disruption:

  • Disruption of Hormone Secretion: Endocrine-disrupting compounds (EDCs) can directly alter, inhibit, or stimulate the secretion of hormones by acting on cellular mechanisms regulating hormone synthesis or release from endocrine glands.
  • Interference with Hormone-Receptor Interactions: EDCs can function directly as receptor agonists or antagonists by binding to specific hormone receptors. Indirectly, EDCs can alter signal transduction pathways downstream of receptor binding, modifying the ultimate physiological response.
  • Modification of Hormone Metabolism: EDCs can alter the biotransformation and excretion rates of circulating hormones in organs such as the hepatopancreas or liver.

• Chemical Classes of Invertebrate Endocrine Disruptors:

  • Organic Contaminants: Alkylphenols (which exhibit estrogenic activity), polychlorinated biphenyls (PCBs such as Aroclor 1242), chlorinated pesticides (e.g., endosulfan, heptachlor, lindane), herbicides (e.g., atrazine), and petroleum hydrocarbons (e.g., naphthalene).
  • Natural and Synthetic Estrogens: Steroids present in sewage effluents, such as $17\beta\text{-estradiol}$, estrone, diethylstilbestrol (DES), and $17\alpha\text{-ethinylestradiol}$.
  • Heavy Metals and Organometallics: Metals such as cadmium ($\text{Cd}$), copper ($\text{Cu}$), mercury ($\text{Hg}$), zinc ($\text{Zn}$), and lead ($\text{Pb}$). Organometallics such as tributyltin (TBT) induce imposex in female gastropods by inhibiting cytochrome P450-dependent aromatase (preventing testosterone conversion to estradiol) and suppressing testosterone excretion.

• Screening and Bioassay Methodologies:

  • Drosophila melanogaster Ecdysteroid Receptor Bioassay: Uses an ecdysteroid-responsive hemocyte cell line (BII) to test xenobiotic agonist/antagonist activity against the ecdysteroid receptor. Cell proliferation serves as the measured endpoint.
  • In Vivo Life-Cycle Assays: Assays using copepods such as Tisbe battagliai (covering the full $21\,\text{day}$ life cycle) measure sublethal effects on growth, development, and reproduction.
  • Integration of Bioassays: Discrepancies exist between in vitro receptor binding and in vivo physiological outcomes due to physiological factors like organismal uptake restrictions, metabolic degradation, and active excretion.

Overview of Crustacean Endocrinology

• Neuroendocrine Control Organs:

  • X-Organ–Sinus Gland Complex: The primary neuroendocrine center of crustaceans. The X-organ is situated within the medulla terminalis of the eyestalk (or in homologous head central nervous system structures in species without stalked eyes) and projects axons to the neurohemal sinus gland located between the medulla externa and medulla interna.
  • Neuropeptide Family: Molt-inhibiting hormone (MIH), gonad-inhibiting hormone (GIH), mandibular organ-inhibiting hormone (MOIH), and crustacean hyperglycemic hormone (CHH) belong to a single structurally related peptide family exhibiting functional cross-reactivity.
  • Additional Sinus Gland Secretions: Neurodepressing hormone (NDH) and pigment-migrating hormones regulating epithelial chromatophores and distal retinal pigments in ommatidia.

• Endocrine Control of Molting:

  • Y-Organs: Paired non-neural endocrine structures (homologous to insect prothoracic organs) that synthesize and secrete ecdysone.
  • Peripheral Conversion: Ecdysone is converted in peripheral tissues into the active molting hormone $20\text{-hydroxyecdysone}$.
  • Role of MIH: MIH inhibits ecdysone synthesis in Y-organs by repressing intracellular cyclic adenosine monophosphate ($\text{cAMP}$) levels. A drop in circulating MIH at premolt triggers ecdysone secretion. Y-organs become unresponsive to MIH during the second half of premolt.
  • Role of Methyl Farnesoate (MF): MF is an unepoxidated sesquiterpenoid (analogous to insect juvenile hormone III, JHIII) secreted by paired mandibular organs, which are under inhibitory control by MOIH. MF directly stimulates Y-organs to secrete ecdysone. Administration of JHIII induces molting in adult crayfish Procambarus clarkii.
  • Hemolymphatic Calcium Modulation: Calcium levels in the hemolymph directly modulate the ecdysone secretion rate by Y-organs.
  • Diagrammatic representation of crustacean molting control:

• Endocrine Control of Reproduction:
  • Female Reproductive Pathways:
  • GIH directly targets oocytes, repressing vitellogenin uptake from hemolymph during secondary vitellogenesis and suppressing ovarian hormone secretion.
  • Ovarian Hormones: Peptidic ovarian hormones and steroid hormones ($17\beta\text{-estradiol}$, progesterone, $17\alpha\text{-hydroxyprogesterone}$) correlate with vitellogenic cycles. $17\alpha\text{-hydroxyprogesterone}$ directly stimulates ovarian growth in vivo and in vitro.
  • Gonad-Stimulating Hormone (GSH): Secreted by non-eyestalk neuroendocrine nodes including the brain and thoracic ganglia. Serotonin ($5\text{-HT}$) stimulates GSH release, while dopamine represses GSH secretion. Neurotransmitters also regulate GIH release from eyestalks. A ventral nerve cord CHH isoform is hypothesized to act as GSH.
  • Reproductive Role of MF and Ecdysteroids: Dietary or pellet-administered MF/JHIII stimulates ovarian maturation in Procambarus clarkii and Chasmagnathus granulata. Ecdysteroids promote ovarian proliferation or post-molt vitellogenesis in penaeid shrimp.
  • Male Reproductive Pathways:
  • Androgenic Glands: Paired structures attached to the vas deferens that secrete androgenic gland hormone (AGH).
  • Regulation by AGH: AGH dictates primary male gonad differentiation, spermatogenesis, and secondary male sexual character development. Eyestalk GIH and thoracic/brain GSH regulate AGH secretion rather than directly targeting testes.
  • Comparative diagrammatic model of female and male reproductive regulation:

• Carbohydrate and Osmoregulatory Dynamics:
  • CHH Functions: CHH elevates hemolymphatic glucose concentrations by activating glycogen phosphorylase in the hepatopancreas. CHH also mediates water uptake during ecdysis and promotes early vitellogenic oocyte enlargement.

Effects of Endocrine-Disrupting Compounds on Molting and Growth

• Estrogenic and Xenobiotic Disruptors of Ecdysis:

  • Daphnia magna Molt Delay: Exposure of neonate D. magna to diethylstilbestrol (DES), endosulfan, Aroclor 1242, or diethyl phthalate significantly delays molting.
  • Epidermal Chitobiase Inhibition: DES, endosulfan, Aroclor 1242, and diethyl phthalate inhibit epidermal chitobiase in the fiddler crab Uca pugilator. Chitobiase is an ecdysone-regulated enzyme responsible for partial digestion of the chitinous exoskeleton during premolt; its inhibition indicates ecdysteroid receptor blockade.
  • Anti-Ecdysteroidal Steroids and Fungicides: Testosterone delays molting in early instars of D. magna without altering circulating ecdysone levels, acting as a direct antagonist at the ecdysteroid receptor (confirmed in Drosophila melanogaster cell lines). Conversely, the agricultural fungicide fenarimol depresses circulating ecdysone titers in D. magna.
  • Copepod Naupliar Arrest: $17\alpha\text{-ethinylestradiol}$, p-octylphenol, tamoxifen, and flutamide inhibit naupliar development to the copepodite stage in Acartia tonsa, potentially by elevating the ecdysone concentration threshold required for metamorphosis.
  • Receptor Screening Outcomes: Screening of 80 environmental pollutants on Drosophila melanogaster BII cells identified bisphenol A, diethyl phthalate, and lindane as potent low-concentration ecdysone receptor antagonists.
  • Organochlorine Pesticides: Heptachlor delays molt onset in larval Homarus americanus, accompanied by reduced circulating ecdysteroids and induction of cytochrome P450 enzymes (CYP45).

• Molt Acceleration and Pesticides:

  • Emamectin Benzoate: Exposure to emamectin benzoate (a sea lice treatment in aquaculture) induces precocious molting in female American lobsters (Homarus americanus). As a GABAergic agent, emamectin benzoate represses the secretion of the inhibitory neuropeptide MIH from the sinus gland.
  • Heavy Metal Modulation: Chronic copper exposure shortens the intermolt period in juvenile Chasmagnathus granulata.

• Heavy Metal Inhibition of Ecdysis:

  • Cadmium Premolt Arrest: Exposure of adult Chasmagnathus granulata to cadmium from early premolt causes complete molting inhibition. Crabs are arrested specifically at the $D_1$ premolt stage prior to the physiological ecdysteroid surge.
  • Mechanism of Cadmium Toxicity: Cadmium does not disrupt calcium resorption from the cuticle, nor does it act via eyestalk neuropeptides (inhibition occurs identically in eyestalk-ablated crabs). Exposure initiated after the peak in ecdysteroid titer does not prevent ecdysis, demonstrating that cadmium selectively represses ecdysone synthesis/secretion in Y-organs, potentially via calcium channel blockade.

• Juvenoid Agents and Somatic Growth Dynamics:

  • Methoprene Activity: Nanomolar exposure to methoprene (an insect juvenile hormone analog) decreases molt frequency in D. magna through juvenoid receptor interaction. JHIII inhibits naupliar development in copepods but stimulates ecdysis in adult crayfish.
  • Somatic Growth Uncoupling: Antiandrogens (cyproterone), surfactants (cetyltrimethylammonium bromide), cadmium, and copper reduce somatic growth in D. magna without altering intermolt duration. Reductions in somatic growth must be differentiated from endocrine disruption, as toxicant-induced feeding inhibition and metabolic stress demands can cause identical growth retardation.

Effects of Endocrine-Disrupting Compounds on Reproduction

• Sex Determination and Sexual Differentiation:

  • Cladoceran Plasticity: Environmental stressors (temperature extremes, starvation, crowding) trigger parthenogenetic Daphnia eggs to switch from female to male differentiation during a critical window approximately $1\,\text{h}$ before egg deposition.
  • Hormonal Dimorphism Control: Methoprene exposure produces all-female broods in Daphnia pulex, whereas $20\text{-hydroxyecdysone}$ exposure yields all-male broods. Methyl farnesoate induces male offspring production in Daphnia magna.
  • Atrazine Effects: Embryonic exposure of Daphnia pulicaria to $0.5\,\text{mg/L}$ atrazine shifts sex determination toward male production.
  • Alterations of Secondary Sex Characters: Exposure of young D. magna to DES or methoprene increases female abdominal process size, whereas exposure to the androgen androstenedione increases male first antenna length.

• Reproductive Output in Copepods and Amphipods:

  • Steroid Sensitivity Thresholds: In Tisbe battagliai, estrone, $17\beta\text{-estradiol}$, and $17\alpha\text{-ethinylestradiol}$ show no-observed-effect concentrations (NOECs) for survival and reproduction that are more than two orders of magnitude higher than $20\text{-hydroxyecdysone}$, causing no sex ratio shift. Nonylphenol at $62\,\mu\text{g/L}$ causes high mortality ($6\%$ survival to maturity) without altering sex ratios.
  • Reproductive Hyper-Stimulation: Exposure of Acartia tonsa to $20\text{--}23\,\mu\text{g/L}$ of $17\beta\text{-estradiol}$ or bisphenol A increases reproductive output, whereas non-estrogenic 2,3-dichlorophenol causes no effect.
  • Amphipod Morphological Alterations: Gammarus pulex exposed to nonylphenol exhibits increased fertility, lower survival, and enlarged male second antennae (implicating androgenic gland disruption). Exposure of G. pulex to $0.1\,\mu\text{g/L}$ $17\alpha\text{-ethinylestradiol}$ increases female sex ratio bias and overall population size.

• Heavy Metal Disruption of Ovarian Development:

  • Uca pugilator Response: A $2\,\text{week}$ cadmium exposure inhibits ovarian growth in intact Uca pugilator but has no effect on eyestalk-ablated crabs. In vitro co-incubation of ovarian tissue with eyestalk tissue demonstrates that cadmium stimulates GIH secretion from sinus glands.
  • Chasmagnathus granulata Response: Cadmium and copper inhibit ovarian growth in eyestalk-ablated Chasmagnathus granulata. In vitro assays reveal that cadmium and copper reduce $^3\text{H}\text{-leucine}$ incorporation into ovarian proteins in media supplemented with MF or $17\alpha\text{-hydroxyprogesterone}$, proving metal interference with hormone signal transduction pathways within ovarian tissue.
  • Counteracting Endocrine Mechanisms: In intact C. granulata, cadmium and copper concurrently inhibit GIH secretion, which counteracts the peripheral ovarian suppression and conceals net phenotypic changes in intact animals.

Effects on Glycemia and Physiological Homeostasis

• Stress Response vs. Endocrine Disruption:

  • CHH Dynamics: Acute elevated hemolymphatic glucose is a standard physiological stress response mediated by CHH release during environmental perturbations (emersion, starvation, osmotic shifts).

• Short-Term Heavy Metal and Hydrocarbon Exposure:

  • Acute Hyperglycemia: Exposure of intact Procambarus clarkii and Uca pugilator to cadmium or naphthalene causes hyperglycemia; eyestalk-ablated animals show no glucose rise.
  • Differential Neuroendocrine Mechanisms: Injection of eyestalk extracts from cadmium-exposed U. pugilator into recipient crabs produces reduced hyperglycemia compared to control extracts (indicating cadmium inhibits CHH synthesis/accumulation). In contrast, extracts from naphthalene-exposed crabs cause enhanced hyperglycemia (indicating naphthalene stimulates CHH synthesis).
  • Heavy Metal Kinetics in Palaemon elegans: Exposure to mercury, cadmium, copper, zinc, or lead induces hyperglycemia peaking at $3\,\text{h}$ post-exposure and returning to baseline by $8\,\text{h}$. Copper and zinc trigger hyperglycemia across broad concentrations, whereas mercury, cadmium, and lead elicit responses strictly at intermediate concentrations.

• Subchronic Heavy Metal Exposure:

  • Hypoglycemic Responses: A $2\,\text{week}$ subchronic exposure of intact Chasmagnathus granulata to cadmium or copper induces dose-dependent hypoglycemia. Copper elicits hypoglycemia at concentrations $2.5\text{-fold}$ lower than cadmium. Eyestalk-ablated crabs show no hypoglycemic response.
  • Mechanisms of Subchronic Disruption: Eyestalk CHH activity remains unchanged in exposed crabs. Exogenous CHH injection ($32\,\text{pmol/crab}$ purified from Cancer pagurus) fully restores normal blood glucose in cadmium-exposed crabs (confirming cadmium selectively inhibits CHH secretion). In copper-exposed crabs, CHH injection yields only partial recovery, indicating copper inhibits CHH secretion AND impairs CHH signal transduction at target tissues (hepatopancreas).

• Chromatic and Ocular Disruption:

  • Chromatophore Pigment Migration: Exposure of Uca pugilator to Aroclor 1242, naphthalene, or cadmium inhibits black pigment dispersion in integumentary chromatophores by blocking black pigment-dispersing hormone (BPDH) release from the sinus gland.
  • Retinal Adaptation: Cadmium suppresses light-adapting distal retinal pigment migration within ommatidia.

Ecotoxicological Relevance and Sentinel Species

• Field Observations of Endocrine Disruption:

  • Intersexuality in Copepods: Up to $93\%$ of harpacticoid copepods near Edinburgh sewage outfalls display intersexuality.
  • Demasculinization in Amphipods: Marine amphipods in polluted Scottish estuaries show high rates of intersexuality and gnathopod size reduction in phenotypically normal males, signaling androgenic gland impairment.
  • Estuarine Estrogen Exposure: UK estuarine fish exhibit feminization and vitellogenin production, whereas sympatric decapod crustaceans (crabs and shrimps) do not synthesize vitellin upon estrogen exposure.

• Biomarker Criteria and Sentinel Selection:

  • Sensitivity Requirements: Useful biomarkers must show endocrine sensitivity at contaminant concentrations well below acute lethal thresholds ($\text{LC}_{50}$).
  • Sentinel Organism Criteria: Sentinel species must possess wide geographical distribution, well-characterized seasonal biology, high ecological abundance, insensitivity to general non-EDC toxicities, ease of laboratory culture, clear sexual dimorphism, rapid generation times, and thoroughly understood endocrinology.
  • Species Trade-offs:
  • Cladocerans (Daphnia magna): Fast generation time and simple culture, but high sensitivity to non-specific toxic stress and poorly understood endocrine control.
  • Mysid Shrimps (Neomysis integer): Cosmopolitan distribution across fresh, estuarine, and marine waters, with quantitative vitellin ELISA bioassays available, though non-specific toxicity sensitivity remains high.
  • Decapod Crustaceans: Highly resistant to general chemical toxicity and possess well-characterized endocrine systems, but long life cycles and complex culture requirements limit rapid multi-generational screening.