Effects of exposure to diethyl phthalate, 4-(tert)-octylphenol,and 2,4,5-trichlorobiphenyl on activity of chitobiase in the epidermis and hepatopancreas of the fiddler crab,Uca pugilator

Seven-day exposure of fiddler crabs, Uca pugilator, to diethyl phthalate at 50.0 mg l⁻¹ significantly inhibited the activity of chitobiase (also known as N-acetyl-β-glucosaminidase) in the epidermis and hepatopancreas. Epidermal chitobiase activity of crabs exposed to 10.0 mg l⁻¹ 4-(tert)-octylphenol for 7 days significantly decreased. PCB29 at 0.5 and 2.0 mg l⁻¹ significantly inhibited chitobiase activity in the epidermis and hepatopancreas of crabs exposed for 3 days. The inhibitory effects rendered by diethyl phthalate and PCB29 can at least partly account for the delayed molting they cause because chitobiase is needed to break down the old exoskeleton of crustaceans prior to ecdysis. Since chitinolytic enzymes are apparently the products of ecdysteroid regulated genes in arthropods, the decline in chitobiase activity after exposure to diethyl phthalate, 4-(tert)-octylphenol, and PCB29 along with the delayed molting they cause strongly suggests that these xenobiotics disturb the Y-organ–ecdysteroid receptor axis. Such disturbance may involve an interaction between ecdysteroid receptors and steroid mimics where the steroid mimics act as antagonists of endogenous steroid molting hormones, and/or arise from the interference with synthesis and excretion of ecdysteroids by these compounds.     

Interaction Between Ecdysteroid, Eclosion Hormone, and Bursicon Titers inManduca sexta

SYNOPSIS. The end of the molting process in the tobacco hornwormincludes the rapid digestion of the old cuticle, molting fluidresorption, ecdysis of the old cuticle, and expansion and hardeningof the new cuticle. The coordination of these processes is accomplishedby three hormones. Each ecdysis during the life of Manduca appearsto be triggered by eclosion hormone. Depending on developmentalstage, the hormone comes either from the brain-corpora cardiacacomplex or from the chain of ventral ganglia. The neural programstriggered by eclosion hormone include a neuroendocrine event,the release of the tanning hormone, bursicon, thereby ensuringthat tanning of the new cuticle must follow ecdysis. Ecdysis,itself, appears to be controlled by the ecdysteroid levels sinceecdysteroid injections delay ecdysis at physiological concentrationsand in a dose dependent fashion. This delay is due to inhibitionof eclosion hormone secretion and to the retardation of theterminal phases of the molt including the digestion of the oldcuticle and the onset of sensitivity to eclosion hormone. Thus,eclosion hormone secretion and the ecdysis it triggers are coordinatedwith the end of development because both are influenced by thesame endocrine signal—the decline in the ecdysteroid titer.     

Non-target effects of the insecticide methoprene on molting in the estuarine crustacean Neomysis integer (Crustacea: Mysidacea)

Ecdysteroids, the molting hormones in crustaceans and other arthropods, play a crucial role in the control of growth, reproduction and embryogenesis of these organisms. Insecticides are often designed to target specific endocrine-regulated functions such as molting and larval development such as methoprene, a juvenile hormone analogue.The aim of this study was to examine the effects of methoprene on molting in a non-target species, the estuarine mysid Neomysis integer (Crustacea: Mysidacea). Mysids have been proposed as standard test organisms for evaluating the endocrine disruptive effect of chemicals. Juveniles (< 24 h) were exposed for 3 weeks to the nominal concentrations 0.01, 1 and 100 μg methoprene/l. Daily, present molts were checked and stored in 4% formaldehyde for subsequent growth measurements. Methoprene significantly delayed molting at 100 μg/l by decreasing the growth rate and increasing the intermolt period. This resulted in a decreased wet weight of the organism. The anti-ecdysteroidal properties of methoprene on mysid molting were also evaluated by determining the ability of exogenously administered 20-hydroxyecdysone, the active ecdysteroid in crustaceans, to protect against the observed methoprene effects. Co-exposure to 20-hydroxyecdysone did not mitigate methoprene effects on mysid molting. This study demonstrates the need for incorporating invertebrate-specific hormone-regulated endpoints in regulatory screening and testing programs for the detection of endocrine disruption caused by man-made chemicals.     

Conserved role of cyclic nucleotides in the regulation of ecdysteroidogenesis by the crustacean molting gland

Molting processes in crustaceans are regulated by ecdysteroids produced in the molting gland (Y-organ), and molting is indirectly controlled by circulating factors that inhibit the production of these polyhydroxylated steroids. Two of these regulatory factors are the neuropeptides molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). CHH appears to inhibit ecdysteroidogenesis in the Y-organ through the activation of a receptor guanylyl cyclase. The signaling pathway activated by MIH, however, remains a subject of controversy. It is clear that neuropeptides inhibit ecdysteroidogenesis by simultaneously suppressing ecdysteroid biosynthetic processes, protein synthesis, and uptake of high density lipoproteins. Data demonstrate that cAMP is the primary regulator of critical catabolic, anabolic, and transport processes, which ultimately support the capacity for ecdysteroid production by the Y-organ. While cAMP also regulates acute ecdysteroidogenesis to some extent, data indicate that cGMP is the primary signaling molecule responsible for acute inhibition by neuropeptides. It is clear that the regulatory roles filled by cAMP and cGMP are conserved among decapod crustaceans. It is unknown if these complementary second messengers are linked in a single signaling pathway or are components of independent pathways activated by different factors present in extracts of eyestalk ganglia.     

Stress reduces hemolymph ecdysteroid levels in the crab: mediation by the eyestalks

In decapod crustaceans, molt hormone (ecdysone) production by Y-organs is suppressed by an eyestalk neurosecretory product, molt-inhibiting hormone (MIH). Environmental stressors are known to delay or prevent molting in crabs. The present study assesses the function of the MIH-Y-organ neuroendocrine system in the crab Cancer antennarius under conditions of daily handling stress. After three days, stressed crabs showed significant suppression of hemolymph ecdysteroid levels, which continued to fall to 20% of controls by day 14. Ecdysteroid titers of stressed crabs returned to prestress levels seven days after stress termination. Ecdysteroid levels in de-eyestalked (DES) crabs rose 160% within 48 hr post-DES. Stressing DES crabs over 16 subsequent days did not significantly alter ecdysteroid levels compared with unstressed DES controls. Handling stress thus depresses hemolymph ecdysteroid levels in the crab, a response that is mediated by eyestalks and appears to result from stress-induced MIH release.     

Setogenesis and molting in planktonic crustaceans

The formation of new setae, termed setogenesis, is describedfor two taxa of planktonic crustaceans: euphausiids, Euphausiapacifica and Thysanoessa spinifera, and a calanoid copepod,Calanus marshallae. Characteristics of setal formation and eversionat ecdysis are described from two time-series of animals preservedat known intervals in their molt cycle. Results from these laboratoryreference series indicate that previous interpretations of setogenesisin the literature can by synthesized to describe a dynamic processof setal formation which is common to all crustacean taxa. The morphological characteristics of developing setae are usedto designate three specific phases in the molt cycle of planktoniccrustaceans (premolt, postmolt, and intermolt). This stagingtechnique may be used to study field-oriented problems relatedto molting in small planktonic crustaceans.     

Hormonal Control of Molting and Reproduction in Ticks

SYNOPSIS. Among ticks there are two developmental and threereproductive patterns that correlate with taxonomic groupings(Argasidae, prostriate and metastriate Ixodidae). Feeding isa prerequisite for molting; feeding and mating are necessaryfor reproduction in all except a few parthenogenetic species.Growth and development in ticks and other chelicerates appearto be controlled by molting hormones (ecdysteroids), as theyare in insects and crustaceans. Ecdysone and 20-hydroxyecdysoneappear to be present in most or all of the major cheliceratetaxa. Epidermis is the site of ecdysone production and fat bodythe site of 20-hydroxylation in the argasid Ornithodoros parkeri,as is probably the case in all ticks. Ecdysteroids influenceearly stages of spermatogenesis by stimulation of DNA synthesisin spermatocytes, but controls for later stages of meiosis areunknown. A polypeptide (12,000 daltons) from male genital accessoryglands stimulates capacitation (maturation) of spermatids intosperm at the time of spermatid transfer to females. Knowledgeof control of egg development and oviposition is incomplete.Stimuli from the synganglion are necessary for completion ofoogenesis and two synganglial factors have been proposed. AnEgg Development Stimulation Factor (EDSF) in O. parkeri is synthesizedand/or released three to six days after feeding. VitellogenesisInducing Factor (VIF) in O. moubata is synthesized and/ or releasedwithin one hour after feeding. The VIF is hypothesized to impactan unidentified tissue which in turn produces a Fat Body StimulationFactor (FSF) that stimulates fat body to synthesize vitellogenin(Vg). Roles of ecdysteroids and juvenile hormones during eggdevelopment and oviposition are unclear.     

Sterols and steroids in a freshwater crustacean (Proasellus meridianus): hormonal response to nutritional input

For crustaceans that eat shredded plant material in freshwater habitats, the amount and the composition of food greatly vary over time because of the seasonal succession of plant fragments and algal biomass. The acquisition of elements necessary for growth, immune defense, and reproduction depends largely on this variation in food type and availability. In particular, sterols that are required as cellular membrane components and as precursors of ecdysteroids (molting hormones) must be acquired through food because crustaceans do not synthesize the steroid nucleus de novo. The present study examined the possible link between nutrition, sterols, and ecdysteroids in an isopod, Proasellus meridianus. In a first step, quantitative and qualitative analyses of sterols of P. meridianus were performed by gas‐chromatography/mass spectrometry. The results suggested that members of P. meridianus are able to convert dietary plant sterols into cholesterol required for growth and reproduction. In a second step, by manipulating food availability and using an enzyme immuno‐assay, we showed that ecdysteroid content in males and females (ovigerous or not) of P. meridianus decreased significantly after a starvation period. A nutritional input following this starvation period triggered an increase in the ecdysteroid contents of these animals. The comparable ecdysteroid responses to food pulses in males and females suggested that a nutritional control on steroid hormones regulated growth or gametogenesis rather than egg maturation. Thus, it appears that P. meridianus possesses an efficient stop‐and‐go endocrine system that may have been selectively favored in response to seasonal pulses of food.     

Inhibition of ecdysteroid production in nymphs of Rhodnius prolixus treated with ethoxyprecocene II

Rhodnius prolixus nymphs fed 7-ethoxy-6-methoxy-2,2-dimethylchromene (ethoxyprecocene II, EPII) show a variety of responses, including precocious molting to diminutive adults, severe retardation of molting, or a condition of permanent ecdysial stasis. The latter two conditions are reversible by subsequent treatment with 20-hydroxyecdysone. Ecdysteroid titers in the hemolymph of individual insects, determined by radioimmunoassay (RIA), show that the ecdysteroid cycle in nymphs undergoing precocious metamorphosis is similar to that of untreated fifth stage nymphs during normal imaginal molting. Nymphs in ecdysial stasis, following EPII treatment, were found to have very low ecdysteroid titers. Analysis of ecdysteroid synthesis by the prothoracic glands (PG), cultured in vitro, showed that: 1) only traces of ecdysteroid were detectable in PG from nymphs treated in vivo with EPII; 2) the PG from untreated nymphs incubated in culture medium with EPII possessed significantly lower ecdysteroid synthesis compared with controls. These studies sought to determine if the inhibition of ecdysteroid biosynthesis observed in Rhodnius, following exposure to EPII in vivo and in vitro, is due to a direct action on the PG or result as an indirect effect perhaps mediated by the neuroendocrine system.     

Hormonal Control of Molting in Decapod Crustacea

The involvement of the molting hormone, 20-hydroxyecdysone,in the mediation of molting in decapod crustaceans is brieflyreviewed. Aspects of the secretion and metabolism of its precursor,ecdysone, are discussed. Experiments are described that demonstratethe presence of a molt-inhibiting hormone (MIH) in the sinusglands of juvenile lobsters (Homarus americanus). Assays forMIH include measurement of the molt interval and radioimmunoassayof circulating titers of ecdysteroids in eyestalk-ablated lobsters.This latter assay indicates that sinus gland extracts significantlydecrease the concentration of circulating ecdysteroids 24 hrafter injection. Data are also presented on the circulatingtiters of ecdysteroids during multiple molt cycles of lobstersfollowing eyestalk ablation. These data indicate that theremust be another factor that ultimately regulates the circulatinglevels of the molting hormone.     

Hormones in the Lives of Crustaceans: An Overview

We present an overview of the isolation and characterizationof three hormones (or hormone families) important for the growthand development of decapod crustaceans. These hormones includethe ecdysteroids (steroid molting hormones), the hyperglycemichormone neuropeptide family, and the terpenoid methyl farnesoate.Using examples primarily from our laboratory, we describe workon these hormones using various life stages of the lobster (Homarusamericanus) as the principal model.     

Antipeptide antibodies for detecting crab (Callinectes sapidus) molt-inhibiting hormone

In crustaceans, the synthesis of ecdysteroid molting hormones is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by an eyestalk neuroendocrine system, the X-organ/sinus gland complex. Using sequence analysis software, two regions of the blue crab (Callinectes sapidus) MIH peptide were selected for antibody production. Two 14-mer peptides were commercially synthesized and used to generate polyclonal antisera. Western blot analysis revealed that each antiserum bound to proteins of the predicted size in extracts of C. sapidus sinus glands, and lysates of insect cells containing recombinant MIH. Thin section immunocytochemistry using either antiserum showed specific immunoreactivity in X-organ neurosecretory cell bodies, their associated axons and collaterals, and their axon terminals in the sinus gland.     

三疣梭子蟹蜕皮周期中MIH基因mRNA水平与蜕皮激素浓度变化

甲壳动物的蜕皮过程被认为是由位于眼柄的X器-窦腺复合体(XO-SG)分泌蜕皮抑制激素(MIH)通过调节Y器(YO)合成蜕皮激素而调控的。通过实时荧光定量PCR(qRT-PCR)发现MIH基因在三疣梭子蟹眼柄X器-窦腺复合体中表达最强。采用qRT-PCR分析了MIH基因在三疣梭子蟹蜕皮周期中的表达变化, 结果表明; A期为(0.42±0.08)倍, B期为(1.09±0.09)倍, C期为(1.35±0.16)倍, D₀亚期为(1.00±0.10)倍, D₁亚期(0.78±0.07)倍, D₂亚期为(0.27±0.08)倍, D_3/4亚期为(0.20±0.04)倍。采用高效液相色谱-电喷雾串联质谱(LC-MS/MS)法完成了三疣梭子蟹蜕皮周期中蜕皮激素(20E)浓度变化的测定。A/B期蜕皮激素的浓度较低, 低于仪器检测限0.33 pg, C期为(1.666±0.762) ng/mL, D₀亚期为(4.047±1.5133) ng/mL, D₁亚期为(6.756±4.928) ng/mL, D₂亚期为(8.609±3.827) ng/mL, D₃亚期为(19.534±4.799) ng/mL, D₄亚期为11.616 ng/mL。在三疣梭子蟹蜕皮周期中, MIH基因表达量与血淋巴中蜕皮激素浓度呈现一定拮抗性, 揭示MIH抑制Y器合成蜕皮激素而调控着三疣梭子蟹蜕皮的发生和进行。     

Hemocyanins and the immune response: defense against the dark arts

The innate immune response is a conserved trait shared by invertebratesand vertebrates. In crustaceans, circulating hemocytes playsignificant roles in the immune response, including the releaseof prophenoloxidases. Activated phenoloxidase (tyrosinase) participatesin encapsulation and melanization of foreign organisms as wellas sclerotization of the new exoskeleton after wound-repairor molting. Hemocyanin functions as a phenoloxidase under certainconditions and thus also participates in the immune responseand molting. The relative contributions of hemocyte phenoloxidaseand hemocyanin in the physiological ratio at which they occurin hemolymph have been investigated in the crab Cancer magister.Differences in activity, substrate affinity, and catalytic abilitybetween the two enzymes indicate that hemocytes are the predominantsource of phenoloxidase activity in crabs. In contrast, hemocyaninis the primary source of phenoloxidase activity in isopods andchelicerates whose hemocytes show no phenoloxidase activity.Quantitative PCR studies on the distribution of prophenoloxidasemRNA in the tissues of Carcinus maenas showed little effectrelative to salinity stress. Phylogenetic analysis of hemocyanin,phenoloxidase, and other members of this arthropod gene familyare consistent with the possibility that a common ancestralmolecule had both phenoloxidase and oxygen-binding capabilities.     

Regulation of Crustacean Molting: A Multi-Hormonal System

SYNOPSIS. In order to increase in size, arthropods must firstmolt (shed) their confining exoskeleton. This molting processis under the immediate control of the steroid molting hormone20-hydroxyecdysone (20-HE). Both the initial rise in circulatinghormone concentration and a coordinated decline are necessaryfor successful molting. Synthesis and/or release of ecdysone,the precursor to 20-HE, is regulated by the neuropeptide molt-inhibitinghormone (MIH). We have determined the primary amino acid sequenceof MIH in the lobster, Homarus americanus. This peptide hasa high degree of identity with the lobster hyperglycemic hormone.Another endocrine factor that appears to be involved in moltingis the juvenile hormone-like terpenoid methyl farnesoate (MF).We have characterized hemolymph MF binding proteins during themolt cycle. In addition, recent data indicate that MF may stimulatethe secretion of 20-HE in vivo and in vitro.