Juvenile hormone esterase activity in the pupating and diapausing larvae of Sesamia nonagrioides

The development of the Mediterranean corn borer, Sesamia nonagrioides, under long-day (LD) photoperiod is associated with juvenile hormone (JH) decline and pupation in the 5th or 6th larval instar. The larvae grown under short-day (SD) conditions maintain a moderate JH titer and enter diapause during which they undergo several extra larval molts. Both types of larvae exhibit similar levels of juvenile hormone esterase (JHE) activity that increases in each instar during the period of low ecdysteroid titer and drops when the titer rises to a molt-inducing peak. A suppression of JHE activity within 24h after application of an ecdysteroid agonist suggests that the drop of activity is a rapid and possibly direct response to ecdysteroids or their agonist. Esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP) suppressed more than 98% of the JHE activity without affecting pupation timing and adult development. The data indicate that JHE is not crucial for the switch between larval development, diapause, and metamorphosis in S. nonagrioides.     

Developmental expression and hormonal regulation of different isoforms of the transcription factor E75 in the tobacco hornworm Manduca sexta

E75A and E75B, isoforms of the E75 orphan nuclear receptor, are sequentially up-regulated in the abdominal epidermis of the tobacco hornworm Manduca sexta by 20-hydroxyecdysone (20E) during larval and pupal molts, with E75A also increasing at pupal commitment (Zhou et al., Dev. Biol. 193, 127-138, 1998). We have now cloned E75C and show that little is expressed in the epidermis during larval life with trace amounts seen just before ecdysis. Instead, E75C is found in high amounts during the development of the adult wings as the ecdysteroid titer is rising, and this increase was prevented by juvenile hormone (JH) that prevented adult development. By contrast, E75D is expressed transiently during the larval and pupal molts as the ecdysteroid titer begins to decline and again just before ecdysis, but in the developing adult wings is expressed on the rise of 20E. Removal of the source of JH had little effect on either E75C or E75D mRNA expression during the larval and pupal molts. At the time of pupal commitment, in vitro experiments show that 20E up-regulates E75D and JH prevents this increase. Neither E75A nor E75D mRNA was up-regulated by JH alone. Thus, E75C is primarily involved in adult differentiation whereas E75D has roles both during the molt and pupal commitment.     

Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Verson's gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Verson's gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Verson's glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.     

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.     

家蚕蜕皮与变态的内分泌调控

家蚕的蜕皮与变态是由前胸腺分泌的脱皮素（ｍｏｌｔｉｎｇ ｈｏｒｍｏｎｅ或 ｅｃｄｙｓｔｅｒｏｉｄ简称 ＭＨ）及由咽侧体分泌的保幼激素（ｊｕｖｅｎｉｌｅ ｈｏｒｍｏｎｅ）控制的,而促有前胸腺激素（ｐｒｏｔｈｏｒａｃｉｃｏｔｒｏｐｉｃ ｈｏｒｍｏｎｅ,以下简称ＰＴＴＨ）的功能为刺激前胸腺分泌蜕皮素。笔者近１０年来从家蚕内分泌体系的一系列研究中发现,蜕皮素浓度的变化可以通过控制咽侧体的保幼激素的生物合成来影响幼虫发育,而ＰＴＴＨ的信息传递可通过调控前胸腺的功能,进而影响血淋巴中蜕皮素浓度。     

Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization showed that a 7.5 kb MHR4 mRNA appeared in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappeared about 12 h later, then transiently reappeared shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression was seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA was also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis showed that the >9.5 kb betaFTZ-F1 mRNA appeared in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declined, then it disappeared in the larval and pupal molts before the second peak of MHR4 appeared. betaFTZ-F1 mRNA was also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs were found in the testis during the onset of spermatogenesis in the prepupal period.     

Choristoneura fumiferana entomopoxvirus prevents metamorphosis and modulates juvenile hormone and ecdysteroid titers

Larvae of the spruce budworm, Choristoneura fumiferana, infected with C. fumiferana entomopoxvirus (CfEPV) continue to feed and grow without undergoing metamorphosis and die as moribund larvae. The lethal dose (LD(50)) and lethal time (LT(50)) values for fourth instar larvae are 2.4 spheroids and 25.2 days, respectively. One hundred percent of the control fourth instar larvae, which were fed water instead of virus, pupated by 18 days post feeding (PF). Only 30% of the larvae that were fed the LD(50) dose and none of the larvae that were fed the LD(95) dose pupated by 18 days PF. Of the control larvae, 95% became adults by 24 days PF, whereas in the treated group only 2% of larvae that were fed the LD(50) dose and none of the larvae that were fed the LD(95) dose became adults by 24 days PF. Some of the virus-treated larvae died as either larval/pupal or pupal/adult intermediates. These phenotypic effects were similar to the larval/pupal and pupal/adult intermediates, resulting from treating larvae with juvenile hormone (JH) or its analogs, which suggests that EPV may cause such abnormalities by modulating JH and/or ecdysteroid titers. In untreated sixth instar larvae the JH titer decreased to low levels by 24 h after ecdysis and remained low throughout larval life. EPV-fed sixth instar larvae had 2112 pg/ml on day 0, 477 pg/ml on day 1 and 875 pg/ml on day 8 of the sixth instar. Control larvae contained 860 ng of ecdysteroids per ml hemolymph on day 8 of the sixth instar, whereas EPV-treated larvae of the same age (30 days PF) had only 107 ng of ecdysteroids per ml of hemolymph. Thus, EPV infection results in increased JH titer and decreased ecdysteroid titer. Northern hybridization analysis was performed using RNA isolated from control and EPV-fed larvae and cDNA probes for (i) juvenile hormone esterase (JHE), which is JH inducible, (ii) Choristoneura hormone receptor 3 (CHR3), which is ecdysteroid inducible, and (iii) larval specific diapause associated protein 1 (DAP1), whose expression is larval specific. EPV-treated larvae showed higher levels of JHE and DAP1 mRNA and lower levels of CHR3 mRNA, indicating that they had higher levels of JH and lower levels of ecdysteroids. Thus, our data show that EPV prevents metamorphosis by modulating ecdysteroid and JH levels.     

Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta

 Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10^–5 M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC₅₀=6.3×10^–7 M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 μg/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC₅₀ as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected. Received: 16 July 1998 / Accepted: 5 August 1998     

Granulovirus prevents pupation and retards development of Adoxophyes honmai larvae

Abstract Larvae of Adoxophyes honmai (Lepidoptera: Tortricidae) infected with granulovirus (AdhoGV) do not pupate; instead, they undergo prolonged larval development and die during the final stadium. Non-infected larvae, however, pupate after five larval stadia. Insect metamorphosis is regulated by fluctuations of ecdysteroid and Juvenile Hormone (JH). JH esterase activity and titres of ecdysteroid must be measured to understand fully the interaction of an insect virus and its host. JH esterase activity is consistently low in AdhoGV-infected larvae, which suggests that JH in AdhoGV-infected larvae is not degraded during the final stadium. The ecdysteroid titre in non-infected larvae showed a large peak in the final stadium before pupation, whereas that in AdhoGV-infected larvae increased from day 2 to day 5 in the final stadium, and then remained at a high level until death. Furthermore, an ecdysteroid UDP-glucosyltransferase (EGT) assay showed that this activity occurs in haemolymph from AdhoGV-infected larvae, but not in haemolymph of non-infected larvae. PCR and sequencing analysis revealed that the AdhoGV genome contains an egt gene, which encodes a protein of 445 amino acids, located approximately 1 kbp upstream from the granulin gene. These results suggest that AdhoGV-infected larvae are prevented from pupating because JHE activity is suppressed and EGT expression inactivates ecdysteroid in the haemolymph.