Hemolymph ecdysteroids during the last three molt cycles of the blue crab,Callinectes sapidus: quantitative and qualitative analyses and regulation

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210–330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20‐OH ecdysone (20‐OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt‐inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20‐OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. © 2009 Wiley Periodicals, Inc.     

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。     

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.     

Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris

At 25 degrees C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at approximately 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of approximately 1.1 ng/ml was maintained even 30-60 days into the instar, when they were in diapause. Ecdysteroid titers remained approximately 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers.     

Canalization of development and ecdysteroid timing during the last instar in lubber grasshoppers

Development in many phytophagous, holometabolous insects is flexible at the beginning but inflexible at the end of the last larval instar. A prominent feature of the inflexible period is a peak in hemolymph levels of ecdysteroids. We tested whether this pattern holds true for the final molt of a phytophagous, hemimetabolous insect, Romalea microptera (the Eastern lubber grasshopper). We fed one group of grasshoppers a high quantity diet (H) throughout the 5th (final) instar and a second group a low quantity diet (L) throughout the instar. Three other diet treatments involved starting the instar on the high diet and then abruptly switching to the low diet at 3, 8, or 13 days (H3L, H8L, and H13L respectively) and continuing the low diet until adult molt. Diet treatment did not affect the maximum hemolymph level of ecdysteroids (E(max)); this peak typically reached ~4000 ng/ml. Ecdysteroid levels were elevated for ~4 days in all groups. In contrast, diet significantly affected age at adult molt and age at E(max) such that H = H13L = H8L < H3L = L. We identified estimates of thresholds for weight gain (20% initial weight) and hemolymph ecdysteroids (100 ng/ml), after which diet did not affect the time to the adult molt. The weight gain threshold was less precise than the ecdysteroid threshold. These results suggest that R. microptera has an extended period of inflexible (canalized) development during the final instar that includes a peak of ecdysteroids. We hypothesize this pattern holds for many phytophagous, hemimetabolous insects.     

Changes in the Amounts of the Molt-Inhibiting Hormone in Sinus Glands during the Molt Cycle of the American Crayfish, Procambarus clarkii

The purposes of this study are to determine the molt cycle of the American crayfish, Procambarus clarkii, and to quantify the amounts of the molt-inhibiting hormone (Prc-MIH) in the hemolymph and neurohemal sinus glands during the molt cycle of the American crayfish. The molt cycle was classified into six stages based on the changes in volumes of gastroliths in the stomach and ecdysteroid titers in the hemolymph. A sandwich-type enzyme immunoassay using specific antibodies raised against N-terminal and C-terminal segments of Prc-MIH was developed for the Prc-MIH assay. It is sensitive to as little as 0.5 fmol of Prc-MIH (3.3 x10(-12) M). In the hemolymph, no Prc-MIH could be detected at any of the molt stages tested. However, in the sinus gland, it was demonstrated that the amount of Prc-MIH changes in a molt-stage-specific manner during the molt cycle. It was particularly noteworthy that the initiation of a molting sequence (i.e., entering the early premolt stage) corresponded to the increase in Prc-MIH content in the sinus gland, because the finding is consistent with the hypothesis that crustaceans enter the premolt stage when the MIH secretion from the sinus gland is reduced or ceases.     

Changes in intracellular calcium concentration in crustacean (Callinectes sapidus) Y-organs: relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated (inhibited) by molt-inhibiting hormone (MIH), a neuropeptide produced by neurosecretory cells in eyestalk ganglia. The inhibitory effect of MIH is mediated by one or more cyclic nucleotide second messengers. In addition, available data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular calcium. However, despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(2+) in Y-organs cells has not been previously determined. In studies reported here, eyestalks were ablated from blue crabs (Callinectes sapidus) to remove the endogenous source of MIH and activate Y-organs. At 0, 3, 6, and 9 days after eyestalk ablation (D0, D3, D6, and D9, respectively), the level of Ca(2+) in Y-organ cells was determined using a fluorescent calcium indicator (Fluo-4), and the hemolymphatic ecdysteroid titer was determined by radioimmunoassay. Calcium fluorescence in D6 Y-organs was 3.5-fold higher than that in D0 controls; calcium fluorescence in D9 Y-organs was 3.9-fold higher than in D0 controls (P<0.05). Measurement of fluorescence along a transect drawn through representative cells indicated that the calcium fluorescence was localized to cytoplasm and not to nuclei. Associated with the increase in intracellular Ca(2+) was a significant increase in the hemolymphatic ecdysteroid titer: The level of ecdysteroids in hemolymph rose from 5.5?ng/mL on D0 to 49.6?ng/mL on D6 and 87.2?ng/mL on D9 (P<0.05). The results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(2+).     

Manipulation of fifth-instar host (Manduca sexta) ecdysteroid levels by the parasitoid wasp Cotesia congregata

Although 5th (last) instar parasitized Manduca sexta larvae undergo developmental arrest and do not wander, they exhibit a small hemolymph ecdysteroid peak (300-400pg/&mgr;l) which begins one day prior to the parasitoid's molt to the 3rd (last) instar and concomitant emergence from the host. Ecdysteroids present in this peak were 20-hydroxyecdysone, 20,26-dihydroxyecdysone and one or more very polar ecdysteroids, as well as small amounts of 26-hydroxyecdysone and ecdysone. In parasitized day-1 and -2 5th instars ligated just behind the 1st abdominal proleg, hemolymph ecdysteroid levels increased in both anterior and posterior portions (100-500pg/&mgr;l), while in unparasitized larvae, hormone levels only increased in the anterior portion (100-350pg/&mgr;l). Thus, the ecdysteroid peak observed in host 5th instars was probably produced, at least in part, by the parasitoids. It may serve to promote Cotesia congregata's molt from the second to the third instar and/or to facilitate parasitoid emergence from the host. In parasitized day-1 and -2 5th instars ligated between the last thoracic and 1st abdominal segments, hemolymph ecdysteroid titers reached much higher levels (500-3500pg/&mgr;l) in the anterior portion (no parasitoids present) than in the posterior portion (150-450pg/&mgr;l). Therefore, it appears that the parasitoid's regulation of hemolymph ecdysteroid titers occurs at two levels. First, parasitization neutralizes the host's ability to maintain its normal hemolymph ecdysteroid levels. Second, in a separate action, the parasitoid manipulates the ecdysteroid-producing machinery so that hemolymph levels are maintained at the 200-400pg/&mgr;l characteristic of day 3-4 hosts. This is the first report of a parasitoid's ability to interfere with the normal inhibitory mechanisms which prevent prothoracic gland production of ecdysteroid at inappropriate periods of insect growth and development.     

Expression of a recombinant molt-inhibiting hormone of the kuruma prawn Penaeus japonicus in Escherichia coli.

The crustacean molt-inhibiting hormone (MIH) suppresses ecdysteroid synthesis by the Y-organ. The MIH of the kuruma prawn Penaeus japonicus has recently been isolated and its cDNA cloned. In this study, we expressed the MIH in Escherichia coli to obtain a large quantity of this hormone with biological activity. The MIH cDNA was processed and ligated into an expression plasmid. E. coli was transformed with this plasmid, and then the recombinant MIH (r-MIH) was expressed. The r-MIH was put through the refolding reaction and was purified by reverse-phase HPLC. N-terminal amino acid sequence and time-of-flight mass spectral analyses supported the idea that the r-MIH had the entire sequence. By in vitro bioassay using the Y-organ of the crayfish, the r-MIH was found to be comparable to natural MIH in inhibiting ecdysteroid synthesis.     

In vivo fluctuation of JH,JH acid,and ecdysteroid titer,and JH esterase activity,during development of fifth stadium Manduca sexta

Juvenile hormone (JH), JH acid, and ecdysteroid titer, and JH esterase activity, were measured in hemolymph from synchronous last stadium larvae of Manduca sexta. JH and JH acids were identified and quantified by GC-MS: JH I and II (and the corresponding acids) were the predominant JH homologs detected in males or females. Maximum levels of JHs and JH acids were observed just following ecdysis to the fifth (last) stadium (day 0, 0 hr) and at the prepupal stage (day 6–day 7). JH titer (≥ 1 ng JH I or II/ml) was higher than JH acid titer (∼0.7 ng JH I acid or JH II acid/ml) in very early fifth stadium larvae. However, this was reversed at the prepupal stage when higher titers of JH acids than JH were observed. JH acid titer began to rise prior to JH titer at the prepupal stage. JH esterase activity rose significantly only after JH or JH acid titers had begun to decline; maximum JH esterase activity was observed at day 3 and day 8. Ecdysteroid titer (measured by RIA) decreased during the last larval molt to a low level by day 0 (0 hr) and to undetectable levels at day 0 (12 hr) of the fifth stadium, by which time JH and JH acid levels had also declined substantially. Just prior to wandering, a small ecdysteroid peak was noted and a slightly elevated level of ecdysteroid was maintained for a further 2 days before a surge in ecdysteroid titer occurred at the prepupal stage, in synchrony with JH and JH acid titer maxima. There was no sexual dimorphism in timing or magnitude of JH, JH acid, and ecdysteroid titer or JH esterase activity.     

Stage-specific changes in calcium concentration in crustacean (Callinectes sapidus) Y-organs during a natural molting cycle, and their relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is suppressed by molt-inhibiting hormone (MIH). The suppressive effect of MIH on ecdysteroidogenesis is mediated by one or more cyclic nucleotide second messengers. In addition, existing data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular Ca(++). Despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(++) in Y-organ cells has not been previously measured during a natural molting cycle for any crustacean species. In studies reported here, a fluorescent calcium indicator (Fluo-4) was used to measure Ca(++) levels in Y-organs during a molting cycle of the blue crab, Callinectes sapidus. Mean calcium fluorescence increased 5.8-fold between intermolt (C4) and stage D3 of premolt, and then dropped abruptly, reaching a level in postmolt (A) that was not significantly different from that in intermolt (P>0.05). The level of ecdysteroids in hemolymph of Y-organ donor crabs (measured by radioimmunoassay) showed an overall pattern similar to that observed for calcium fluorescence, rising from 2.9 ng/mL in intermolt to 357.1 ng/mL in D3 (P<0.05), and then dropping to 55.3 ng/mL in D4 (P<0.05). The combined results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(++).     

Corazonin reduces the spinning rate in the silkworm,Bombyx mori

The insect neuropeptide, [Arg⁷]-corazonin was injected into larvae of the silkworm, Bombyx mori to investigate its influence on development and behavior. A single injection of 50 pmol of corazonin into the fourth and fifth instar larvae induced prolongation of the spinning period in all experimental groups except for those injected on day 10 of the fifth instar. The injection also caused a prolongation of the pupal period in some experimental groups, while it had no effect on the timing of larval ecdysis and the length of feeding period of the fifth instar. The spinning period was significantly prolonged even at a low dose of 1 pmol. Both the spinning rate and the rate of increase in hemolymph ecdysteroid level during the spinning stage were reduced by injection of corazonin. However, corazonin injection during days 5-7 of the fifth instar reduced the spinning rate without influencing the ecdysteroid level until the end of day 8, thereafter the rate of increase in hemolymph ecdysteroid level was slower in the corazonin-injected larvae than in the control larvae. Therefore, the suppressed ecdysteroid level observed in the corazonin-injected larvae appears to be a result rather than a cause of the reduced spinning rate. This study is the first published report for the corazonin effect on the behavior in insects.     

THE ROLE OF ECDYSTEROIDS IN THE REPRODUCTIVE DIAPAUSE OF DERMACENTOR NIVEUS NEUMANN

Abstract  The ecdysteroid levels in hemolymph, ovary, synganglion and whole body of diapausing female Dermacentor niveus were detected by HPLC, and compared with the results of nondiapausing female. It is revealed that the ecdysteroid levels in hemolymph and ovary of diapausing female are similar basically to that of nondiapausing female in the first few days after engorgement. From the 10th day after engorgement, the ecdysteroid levels of diapausing female decreased and even became distinctly lower than that of nondiapausing female. The paucity of ecdysteroids in these individuals would influence the normal development of oocytes. In order to explore the effect of ecdyateroids on the diapausing female, we injected 20-hydroxyecdysone with different dosages at different time into the ticks, and found that after just complete engorgement the injection with large dosages (10000 and 1375 ng/tick) caused death of the ticks. From 10th to 20th day after engorgement the ecdysteroid levels of diapausing female are lower than that of nondiapausing one before oviposition, the injection with certain dosages 50, 70 and 100 ng/tick> of 20E can accelerate vitellogenesis and terminate reproductive diapause, but the amount of eggs produced by them is less than that produced by nondiapausing female. The termination of diapause in female of ixcdid tick by exogenous ecdysteroids is reported for the first time.     

The juvenile hormone analog pyriproxyfen affects ecdysteroid-dependent cuticle melanization and shifts the pupal ecdysteroid peak in the honey bee (Apis mellifera)

The control of the pupal melanization in the honey bee by ecdysteroids, and the modulation of these processes by a juvenile hormone analog were investigated by a combination of in vivo and in vitro experiments. Injection of 1-5 microg of 20-hydroxyecdysone (20E) into unpigmented pupae showed a dose- and stage-dependent effect. The higher the dose and the later the injection was performed, the more pronounced was the delay in cuticle pigmentation. This inhibition of cuticular melanization by artificially elevated ecdysteroid titers was corroborated by in vitro experiments, culturing integument from unpigmented, dark-eyed pupae for 1-4 days in the presence of 20E (2 or 5 microg/ml culture medium). Topical application (1 microg) of pyriproxyfen to unpigmented, white-eyed pupae had the opposite effect, leading to precocious and enhanced melanization of the pupal cuticle. In vitro incubation of integuments in the presence of this juvenile hormone analog (1 microg/ml) confirmed these results, showing that pyriproxyfen is apparently capable of triggering melanization. The in vivo mode of action of pyriproxyfen was further investigated by quantifying hemolymph ecdysteroids by radioimmunoassays. Topical application leads to a delay of the pupal ecdysteroid peak by 4 days. The pyriproxyfen-induced low ecdysteroid titers during early pupal development could account for precocious pigmentation by removing an inhibition on prophenoloxidase activation normally imposed by the elevated ecdysteroid titer during this phase.     

Bacterial expression of the shrimp molt-inhibiting hormone (MIH): antibody production, immunocytochemical study and biological assay

Molting in shrimp is controlled by the molt-inhibiting hormone (MIH) and ecdysone. MIH inhibits the synthesis of ecdysone in the Y-organ, resulting in molt suppression; it is a neuropeptide member belonging to the eyestalk CHH/MIH/GIH family. The cloning of MIH (formerly MIH-like) of the shrimp Metapenaeus ensis has been reported in a previous study. To obtain a large quantity of fusion protein for antibody production and biological assay, the cDNA encoding the shrimp MIH was inserted into the pRSET bacterial expression vector. His-tagged fusion protein was produced and purified by an Ni2+-charged affinity column. Polyclonal antibody to rMIH was subsequently obtained by immunizing rabbits with purified recombinant proteins. Results from Western blot analysis indicated that the antibody was specific. Furthermore, results from immunocytochemical analysis showed that specific cells in three different clusters of the X-organ, the sinus gland and the axonal tract of the eyestalk contain MIH. To test for the molt-inhibiting activity of rMIH, shrimp at intermolt stage were injected with rMIH and the molt cycle duration of the injected shrimp was monitored. A significant increase in molt cycle duration was recorded for the shrimp injected with the recombinant protein.     

In vivo effects of allatostatins in crickets,Gryllus bimaculatus (Ensifera: Gryllidae)

Two types of cricket allatostatins, Grb-AST A1 and Grb-AST B1, were injected into adult female crickets three times each day on days 0–3 and once on day 4 after adult emergence to test their activity in vivo. On day 4, body weight, ovary weight, number of eggs per ovary, length of the terminal oocytes, ovarian ecdysteroid biosynthesis, and hemolymph titers of ecdysteroids were lower in the allatostatin-injected animals compared with untreated and Ringer-injected controls. Effects of the injected allatostatins on hemolymph juvenile hormone titers were inhomogeneous, and no differences were found in the capacity of the corpora allata to produce juvenile hormone ex vivo. The hemolymph titers of yolk proteins (vitellogenins) were almost twice as high in the allatostatin-injected animals as in the control animals. The effects of the injected allatostatins and their interactions with the endocrine system of the animal are discussed. Arch. Insect Biochem. Physiol. 38:32– 43, 1998. © 1998 Wiley-Liss, Inc.     

Stimulation of nitric oxide synthesis and protective role of insulin in acute thrombosis in vivo

Administration of physiologic amounts of insulin in mice (200 microunits/g body weight) resulted in 9 fold increase of basal nitric oxide level from 0.51+/-0.1224 nmol/ml (mean+/-SD, n=12) to 4.45+/-0.645 nmol/ml after 30min of the injection of the hormone. Since NO is a potent inhibitor of platelet aggregation both in vitro and in vivo, we tested the possibility whether the administration of the hormone would result in the in vivo inhibition of thrombosis through the increase of NO level in the circulation. It was found that administration of insulin (200 microunits/g body weight) in mice protected >90%(p<0.00001, n=500) of these animals from death due to thrombosis in the coronary arteries induced by ADP injection in the heart. This effect of insulin in vivo was found to be directly related to the hormone induced increase of NO level in the system. The thromboprotective effect of insulin could not be achieved by using either prostacyclin, a well known antithrombotic agent or its stable probe prostaglandin E1 instead of insulin. The efficacy of insulin was neither related to the blood glucose level nor was the consequence of the hypoglycemic effect of the hormone. In contrast, inhibition of insulin induced increase of NO level resulted in the complete loss of the thromboprotective effect of the hormone. These results suggest that insulin besides being a hypoglycemic hormone could also be a potent antithrombotic humoral factor.