Temporal,spatial and induced expression of chitinase in the spruce budworm,Choristoneura fumiferana

Temporal, spatial and induced expression of Choristoneura fumiferana chitinase (CfChitinase) was studied using immunohistochemistry and Western blots. CfChitinase was detected in the integument, the midgut peritrophic membrane, the cuticular lining of the trachea, the spiracle, and salivary glands. The enzyme was expressed as larvae were preparing to molt from one instar to the next. The spatial and temporal expression patterns are consistent with its function in degrading chitin during the molting process. The 20-hydroxyecdysone agonist, tebufenozide (RH5992), induced the expression of the CfChitinase gene in the early stage of the sixth-instar larvae and the enzyme was detected in the epidermis and molting fluid 24 h post treatment.     

Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana

A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.     

Cloning, expression, and localization of a molt-related beta-N-acetylglucosaminidase in the spruce budworm, Choristoneura fumiferana

A beta-N-acetylglucosaminidase cDNA (CfGlcNAcase) was cloned from the spruce budworm, Choristoneura fumiferana. Western blotting analysis of developmental CfGlcNAcase expression revealed high levels of expression of the gene on the last day of the 5th instar larvae and the first day in the 6th instar larvae, followed by a decrease to background levels during the intermolt of the 6th instar. CfGlcNAcase was detected again from the last day of the 6th instar to day 2 of pupal stage. CfGlcNAcase expression was induced by tebufenozide at 24 h post treatment and remained at high levels until 72 h. Immunohistochemical localization analysis of CfGlcNAcase indicated that CfGlcNAcase was present in the molting fluid, epidermis, trachea, and hemolymph in prepupae during the transformation from larva to pupa. CfGlcNAcase cDNA was expressed into a recombinant protein in bacterial and baculovirus systems and the protein expressed in the baculovirus system had a higher chitinolytic activity than in the bacterial system and appeared to be secreted.     

Developmental profiles of the mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta

When fat body mRNA from the tobacco hornworm larva, Manduca sexta, was translated in a rabbit reticulocyte lysate system, three major polypeptides were found, each having a different developmental profile. One mRNA coded for a 74 kilodalton (K) polypeptide doublet precipitated by an antibody to the arylphorin (manducin). This mRNA was present only during the intermolt feeding phase of the penultimate and the final larval instars. Its appearance 16–24 hr after larval ecdysis was dependent upon the incoming nutrient supply and independent of the juvenile hormone (JH) level. Immunoblots of proteins of the fat body, epidermis, and cuticle revealed the presence of arylphorin in all three tissues. Additionally, several small polypeptides that cross-reacted with the arylphorin antibody were found in the fat body during and up to 24 hr after the last larval molt and in the tanning pupal cuticle. The larval epidermis was also found to contain a small amount of arylphorin mRNA. At the time of the JH decline prior to the onset of metamorphosis, a female-specific mRNA coding for a 79 K translation product appeared. In allatectomized larvae this mRNA was detectable earlier, and its appearance in intact larvae was prevented by application of methoprene, indicating that JH regulates its appearance. At wandering a new mRNA that also codes for a 79 K polypeptide appeared in both sexes and was the major messenger present during the prepupal stage. Neither it nor the female-specific mRNA were translatable after pupal ecdysis.     

Developmental expression of Ultraspiracle proteins in the epidermis of the tobacco hornworm, Manduca sexta, during larval life and the onset of metamorphosis

 During the final two larval instars, a changing pattern of three Ultraspiracle (Usp) proteins (50.5, 52.5, and 57 kDa) was detected in immunoblots of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, by a monoclonal antibody against Drosophila Usp that was shown to detect MsUsp. The 57- and 52.5-kDa bands were present during the intermolt periods and the 50.5- and 52.5-kDa bands during the molting phases. The antibody detected a nuclear antigen present in epidermis, muscle, fat body, and the central nervous system from the time of hatching. In the epidermis Usp was present in all cell nuclei but was especially prominent in the tormogen and trichogen cells immediately after ecdysis in both the penultimate and final instars. This latter immunoreactivity disappeared within 12 h whereas the remainder of the epidermis retained high levels throughout the feeding period. During the molt immunostaining reappeared in the hair cell nuclei. During the wandering stage at the onset of metamorphosis and just before pupal ecdysis, immunoblots showed high levels of Usp, but nuclei showed little or no staining. This discrepancy is likely due to the loss of one Usp isoform from the nucleus and its dispersal in the cytoplasm in preparation for the appearance of the second isoform. Received: 10 June 1997 / Accepted: 22 August 1997     

Molecular characterization of an inhibitor of apoptosis in the Egyptian armyworm, Spodoptera littoralis, and midgut cell death during metamorphosis

The cDNA corresponding to an inhibitor of apoptosis (IAP) from the Egyptian armyworm, Spodoptera littoralis, was cloned by RT-PCR. Sequence analysis showed that the IAP of S. littoralis (SlIAP) contains two baculoviral IAP repeat (BIR) motifs, followed by a RING finger, an organization which is very similar to that of other lepidopteran IAPs. SlIAP mRNA was detected in ovary, testis, salivary gland, fat body, epidermis, brain and midgut of S. littoralis. During the last larval instar, prepupal and pupal stages, brain mRNA levels remained approximately constant, whereas those of midgut showed a large peak centred in the prepupal stage. Midgut morphology changed during metamorphosis from a semi-transparent, cylindrical structure in last instar larvae to a brownish globular mass in pupae. TUNEL assays, LysoTracker staining and caspase-3 immunohistochemistry, indicated that programmed cell death in midgut starts actively at the onset of pupation process, coinciding with the dramatic decrease of SlIAP mRNA levels observed at the same time.     

The role of juvenile hormone in endocrine control of pigmentation in Manduca sexta

Spatial and temporal distribution of insecticyanin was studied in the fourth and fifth larval instars of Manduca sexta. The protein was distributed between the epidermis (62%), the hemolymph (37%) and the pericardial cells (0.5%). Hemolymph insecticyanin (HINS) was highest (0.6 mg/ml) in the very early fourth instar, gradually declining to 0.3 mg/ml. Levels in the fifth instar decreased after ecdysis (0.15 mg/ml), began to rise at wandering, and nearly doubled by the time of pupation. Titers of epidermal insecticyanin (EINS) followed the general growth patterns during the fourth and early fifth instar. At 76 hr after fifth instar ecdysis, titers of EINS dropped precipitously and then rose again to peak just after the wandering stage. Levels of EINS again rapidly declined and could not be detected after 180 hr. Ecdysteroids appear to shut off synthesis of EINS but this response is quantitatively modified in the presence of JH. Endocrine manipulation of the last larval-larval molt indicated that juvenile hormone (JH) acts quantitatively on EINS to induce a dose-dependent increase. The JH-induced increase can be as much as 4-fold, depending upon the body region.     

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.     

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Ecdysone oxidase and 3-oxoecdysteroid reductases in Manduca sexta: Developmental changes and tissue distribution

The activities of ecdysone oxidase (EO), 3-oxoecdysteroid 3α-reductase (3α-R), and 3-oxoecdysteroid 3β-reductase (3β-R) were determined for epidermis, hemolymph, and fat body of wandering fifth instar Manduca sexta larvae and for midguts of various developmental stages between 3 days after the last larval and 14 days after the pupal ecdysis. The larval midgut was the only organ showing substantial specific activities of EO and 3α-R, and both increased up to the seventh day after ecdysis. Hemolymph and fat body had only moderate to high 3β-R and low EO activites, and the epidermis did not contain significant activity of any of the enzymes. On the ninth day after the last larval ecdysis the larval midgut epithelium was replaced by a new pupal midgut epithelium. After this event only 3β-R was restored to high activities, whereas EO and 3α-R showed only low to marginal activities. It is concluded that only the larval midgut has a role in the inactivation of ecdysteroids by 3-epimerization. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Host/parasitoid interactions: critical timing of parasitoid-derived products

Short-term in vitro incubations were used to examine the ability of endoparasitoid larvae to produce and release both ecdysteroids and proteins into their environment. Second instar larvae of both Chelonus near curvimaculatus and Ascogaster quadridentata were observed by SDS-PAGE to release temporally-similar polypeptides in the 20-30kD M(r) range. Peak occurrence of these polypeptides coincided with shedding of the anal vesicle, immediately prior to ecdysis to the third instar. Ecdysis also coincided with the switch from endoparasitic to ectoparasitic development in vivo. Polyclonal antibodies were generated against a particular 27kD polypeptide of Chelonus, which was found to be species-specific and localized primarily within the anal vesicle during the latter part of the second stadium and whole body homogenates of third instars. In vitro incorporation studies using (35)S-methionine indicated rapid changes in the synthetic abilities of second instar larvae shortly before ecdysis. The production and release of ecdysteroids, as measured by RIA, was found to precede the peak occurrence of the 27kD polypeptide and ecdysteroid presence was undetectable following the molt. In contrast, the polypeptides were observed to gradually increase prior to the molt and slowly decrease after the molt. The Chelonus polypeptide was not detected in host tissues until after parasitoid egression.     

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.     

Brain-independent development in the moth Sesamia nonagrioides

The caterpillars of Sesamia nonagrioides developing under long-day (LD) photoperiod pupate in the 5th or 6th instar whereas under short day (SD) conditions they enter diapause and undergo several extra larval molts. The diapause is terminated within 1-3 instars upon transfer of SD larvae to the LD conditions. Brain removal from the 6th instar larvae promotes pupation followed by imaginal development; however, one third of the SD larvae and 12% of the LD larvae debrained at the start of the instar first undergo 1-2 larval molts. The incidence of larval molts is enhanced by the brain implants. Exclusively pupal molts occur in the LD larvae debrained late in the 6th instar. Decapitation elicits pupation in both LD and SD larvae, except for some of the 4th and 5th and rarely 6th instar that are induced to a fast larval molt. The pupation of decapitated larvae is reverted to a larval molt by application of a juvenile hormone (JH) agonist. No molts occur in abdomens isolated from the head and thorax prior to the wandering stage. Abdomens isolated later undergo a larval (SD insects) or a pupal (LD insects) molt. Taken together the data reveal that in S. nonagrioides (1) several larval molts followed by a pupal and imaginal molt can occur without brain; (2) an unknown head factor outside the brain is needed for the pupal-adult molt; (3) brain exerts both stimulatory and inhibitory effect on the corpora allata (CA); (4) larval molts induced in CA absence suggest considerable JH persistence.     

Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.