Hormonal mechanisms underlying termination of larval diapause by juvenile hormone in the bamboo borer, Omphisa fuscidentalis

Topical application of methoprene, a juvenile hormone analogue (JHA), induces pupation by activating the prothoracic glands (PGs) in diapausing larvae of the bamboo borer, Omphisa fuscidentalis. To determine the minimum stimulation period for PG activation, we transplanted PGs of JHA-treated larvae (donors) into non-treated larvae (recipients) on successive days after JHA treatment and observed the recipients for pupation. JHA stimulation for 1 day was sufficient to induce pupation. In recipient larvae, the hemolymph ecdysteroid titer increased transiently on day 18 after transplantation and significantly on days 24-28, prior to pupation. Secretory activity of recipient PGs increased transiently on day 16 and days 22-28. Because the recipient PG activity was too low to account for an increased ecdysteroid titer, the JHA-stimulated donor PGs must produce the major part of hemolymph ecdysteroids. In addition, the ecdysteroid produced by the donor PGs might have stimulated the recipient PGs. We examined the possible involvement of two ecdysone receptor (EcR) isoforms, OfEcR-A and OfEcR-B1, in PG activation by JHA, and found that although both isoforms were up-regulated, accompanied by an increased ecdysteroid titer in the hemolymph, the isoform mRNA levels were not altered at all before the increase in PG secretory activity. Thus, EcR expression might not be involved in feedback activation of PGs.     

Intensity of larval diapause in the bamboo borer,Omphisa fuscidentalis

Larvae of the bamboo borer, Omphisa fuscidentalis, enter larval diapause in September and pupate in the following June (Singtripop et al., 1999). We examined the changes in the responses of larvae to exogenous 20-hydroxyecdysone (20E) in order to estimate the progress of diapause development. In this respect, we adopted two terms, responsiveness and sensitivity of larvae to 20E. Responsiveness was estimated by the percentage of larvae that pupated, and sensitivity was evaluated by the duration from the day of 20E injection to pupation. The responsiveness of larvae declined gradually from September to November when larvae were least responsive to 20E, and then increased markedly from January to February. This indicates that the intensity of diapause increases from September to November and terminated gradually thereafter. Thus the sequence of events as the larval responses to 20E is characterized by a V-shaped curve. Sensitivity of larvae to 20E was at the same level from September to December, and increased remarkably from December to January. The abrupt increase in the sensitivity occurred one month earlier than the bottom of the V-shaped curve of larval responsiveness, suggesting that the increases in the responsiveness and sensitivity in the latter half of diapause may be brought about by respective mechanisms.     

Identification, characterization, and developmental regulation of two storage proteins in the bamboo borer Omphisa fuscidentalis

Two insect storage proteins, OfSP1 (75 kDa) and OfSP2 (72 kDa), were purified using three different chromatographies from the hemolymph of Omphisa fuscidentalis larvae during diapause, and their genes were cloned. OfSP1 and OfSP2 concentrations in the hemolymph were high during diapause. During pupation, OfSP1 levels decreased in the male hemolymph and disappeared from the female hemolymph. OfSP1 and OfSP2 mRNA levels in the fat bodies were low during the third instar, but increased greatly during the fourth and fifth larval instars. During diapause, mRNA expression continued at a lower level than during the feeding period. The injection of 20-hydroxyecdysone (20E) into diapausing larvae caused an increase in OfSP1 and OfSP2 mRNA levels 2-3 days post-injection, followed by a decrease in expression until pupation, which occurred 2-4 days thereafter. When larvae were treated with juvenile-hormone analog (JHA), OfSP1 and OfSP2 mRNA levels gradually decreased until the onset of pupation. In Omphisa, OfSP1 and OfSP2 proteins are produced and released by the larval fat bodies in the fourth and fifth-instar larvae, and the proteins accumulate in the hemolymph until the insects enter diapause. OfSP1 may be reabsorbed by the fat bodies at the end of diapause for subsequent re-use during pupation.     

Juvenile hormone-mediated termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

Larvae of the bamboo borer, Omphisa fuscidentalis are in diapause for more than nine months (Singtripop, T., Wanichaneewa, S., Tsuzuki, S., Sakurai, S. 1999. Larval growth and diapause in a tropical moth, Omphisa fuscidentalis Hampson. Zool. Sci. 16, 725-733). To examine the endocrine mechanisms underlying this larval diapause, we assayed the responsiveness of the diapausing larvae to 20-hydroxyecdysone (20E) and a juvenile hormone analogue (JHA: S-methoprene). 20E injection caused the larvae to halt movement, followed by deposition of a pupal cuticle. Topical application of JHA induced pupation in a dose-dependent manner. JHA also induced pupation of the larvae whose brains were removed before JHA application. In those larvae, the prothoracic glands became active and competent to respond to brain extracts within seven days after JHA treatment, and the hemolymph ecdysteroid concentration began to increase 12 days after JHA application. These results indicate that JHA stimulates the prothoracic glands of diapausing Omphisa larvae, terminating larval diapause, in contrast with previous findings that JH inhibits the brain-prothoracic gland axis and thus maintains the larval diapause. Current results therefore suggest a novel regulatory mechanism for larval diapause in this species.     

Molecular cloning,developmental expression and tissue distribution of diapause hormone and pheromone biosynthesis activating neuropeptide in the bamboo borer Omphisa fuscidentalis

Diapause, an arrested period of post‐embryonic development in insects, is under the control of hormonal interactions. In the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae), larvae remain in diapause for as long as 9 months during the dry season, from September to the following June, although the factors that regulate larval diapause are poorly understood. The present study describes the cloning and expression analysis of the diapause hormone and pheromone biosynthesis activating neuropeptide (DH‐PBAN) precursor of O. fuscidentalis (Ompfu‐DH‐PBAN cDNA), aiming to reveal how it may be involved regulating larval diapause in this species in combination with environmental factors. The open reading frame (ORF) of the cDNA encodes a 199‐amino acid precursor protein that contains DH, PBAN and three other neuropeptides, all of which share a conservative C‐terminal pentapeptide motif FXPR/KL (X = G, T or S). The Ompfu‐DH‐PBAN is highly similar (74%) to the DH‐PBAN of the legume pod borer (Maruca vitrata). A quantitative real‐time polymerase chain reaction reveals that Ompfu‐DH‐PBAN mRNA is expressed only in neural tissues and that expression is highest in the suboesophageal ganglion. In addition, the expression level of Ompfu‐DH‐PBAN mRNA in the suboesophageal ganglion is consistently high during the fifth larval instar, increasing moderately in early diapause before reaching a peak during late diapause. After pupation, expression of the Ompfu‐DH‐PBAN precursor decreases to a low level. In addition to endocrine factors, the results demonstrate that photoperiod increases the expression level of Ompfu‐DH‐PBAN mRNA in larval diapause. These results also suggest that the expression of the Ompfu‐DH‐PBAN gene correlates with larval diapause development and may be activated by photoperiod in O. fuscidentalis.     

Programmed cell death of larval tissues induced by juvenile hormone in the bamboo borer, Omphisa fuscidentalis

Programmed cell death (PCD) plays a critical role during animal development through the destruction of unneeded cells and tissues. In some insects, the prothoracic glands (PGs) and anterior silk glands (ASGs) are larval-specific tissues that are normally eliminated by PCD after pupation. Previous studies report that juvenile hormone analog (JHA) terminates the larval diapause of Omphisa fuscidentalis by increasing the hemolymph ecdysteroids that trigger PCD. Because JHA may indirectly induce the PCD of the PGs and ASGs of Omphisa diapausing larvae, the effects of JHA on the induction of PCD were determined. The application of 1μg JHA induced PCD in the PGs and ASGs of larvae identified as stage G0 (prior to pupation). The injection of 1μg 20E triggered the PCD of the ASGs when the larvae expressed a G0-G1 morphology, whereas PCD occurred in the PGs on day 1 post-injection. Histological studies revealed similar patterns of morphological changes during the PG and ASG PCD in the JHA- and 20E-treated larvae. Furthermore, to confirm that PCD was induced by a high ecdysteroid level that increases after JHA application, the expression profiles of EcR-A and EcR-B1 in the PGs and ASGs from the JHA-treated larvae were examined, and the results showed that the expression levels of EcR-A and EcR-B1 mRNA increased during the G0 stage. These results suggest that JHA may be involved in PCD by increasing the ecdysteroid titer, leading to termination of the larval diapause period in Omphisa fuscidentalis.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson

During larval diapause lasting 9 months from September to May, trehalase activity in the midgut of the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae) was low from December to April, followed by a fourfold increase in May that remained high during the pupal stage in July. An application of juvenile hormone analog (JHA) produced increases in the ecdysteroid titer, while trehalase activity was increased by both JHA and 20-hydroxyecdysone (20E) injection. The trehalase activity in the midgut of diapausing larvae was doubled by incubating the midgut with 20E for 48h. During diapause as well as after JHA application, expression of two ecdysone receptor isoform genes (EcR-A and EcR-B1) in the midgut increased simultaneously with the increase in hemolymph ecdysteroid titer, followed by an increase in trehalase activity. The hemolymph of diapausing larvae contained a trehalase inhibitor and inhibitory activity was high during diapause. After 20E injection, trehalase inhibition decreased as midgut trehalase activity increased. Taken together, at least two factors may participate in the change in midgut trehalase activity: increase in trehalase activity and decrease in trehalase inhibitor activity, both of which may be induced by 20E.     

Juvenile hormone esterases in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

Haemolymph levels of juvenile hormone esterase, 1-naphthyl acetate esterase, and juvenile hormone were measured in synchronously staged diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis. Juvenile hormone esterase levels were monitored using juvenile hormone I as a substrate while juvenile hormone titres were measured with the Galleria bioassay. Haemolymph of nondiapause larvae showed two peaks of juvenile hormone hydrolytic activity: one near the end of the feeding phase and a smaller one just prior to pupal ecdysis. These peaks of enzyme activity correlated well with the low levels of haemolymph juvenile hormone. Juvenile hormone titres were high early in the stadium then showed a second peak during the prepupal stage coinciding with low esterase activity. Diapause haemolymph had peak juvenile hormone esterase activity nearly 4 times the nondiapause level, reaching a peak near the end of the feeding phase. Diapause-destined larvae retained high juvenile hormone titres even during the rise of the high esterase levels. 1-naphthyl acetate esterase levels did not correlate with the juvenile hormone esterase levels in either the diapause or nondiapause haemolymph. High levels of 1-naphthyl acetate esterase activity were associated with moulting periods.     

The role of juvenile hormone in the larval diapause of the European corn borer,Ostrinia nubilalis

The possible role of juvenile hormone (JH) in the induction and termination of larval diapause in the European corn borer, Ostrinia nubilalis, was investigated using topical applications of both JH I and a JH mimic as well as by monitoring JH titers with the Galleria bioassay. Neither JH nor the JH mimic ZR515 was capable of influencing diapause termination when administered topically. The Galleria bioassay revealed little or no JH in the hemolymph of mid diapause (>30 days) insects, indicating no demonstrable role for JH in diapause maintenance. When ZR515 was administered to nondiapause, newly ecdysed fifth instar larvae the pupal molting cycle was delayed. By use of photoperiodic regimes we were able to show that the molting delay was not equivalent to diapause induction. The Galleria bioassay showed differences in JH titer profiles between diapause and nondiapause animals during the final larval stadium. The nondiapause insects showed titers that decline rapidly to trace amounts following the molt to fifth instar then rose prior to pupation. The diapause insects had generally higher titers and exhibited a more gradual decline after the molt. No evidence was obtained to support the hypothesis that JH plays a key role in the induction, maintenance, or termination of larval diapause.     

Sensitivities to juvenile hormone and ecdysteroid in the diapause larvae of Omphisa fuscidentalis based on the hemolymph trehalose dynamics index

The final instar larva of the bamboo borer, Omphisa fuscidentalis, is in diapause for 9 months from September to the following June. Trehalose and ecdysteroid concentrations in hemolymph were measured through the larval diapause period and in the pupal stage. The ecdysteroid concentration remained low until November, followed by a gradual increase to about 30 ng/ml in May. The trehalose concentration remained at levels ranging between 40-50 mM until May, and decreased to an almost undetectable level after pupation. Since a juvenile hormone analogue (JHA), methoprene, is capable of terminating diapause by stimulating larval prothoracic glands, we examined its effects on ecdysteroid and trehalose concentrations in larvae in December and February. The hemolymph ecdysteroid increased more quickly in February than in December, indicating that the sensitivity of the prothoracic glands to JHA increased towards the end of diapause termination. Similarly, hemolymph trehalose in February decreased within a few days after JHA application, while in December the decrease occurred in the third week. Exogenous 20-hydroxyecdysone (20E) caused a decrease in trehalose concentration in a dose-dependent manner. The effective dose of 20E, however, did not change from January until April, implying that the sensitivity of tissue(s) to 20E may not change until the end of diapause. Taken together, our results suggest that the sensitivities of tissues to JH and 20E do not increase simultaneously with the progress of diapause development and that termination of larval diapause is not associated simply with the restoration of hormone deficiencies.     

Diapause of the southwestern corn borer, Diatraea grandiosella: Further evidence showing juvenile hormone to be the regulator

Further evidence is presented to demonstrate the involvement of juvenile hormone (JH) in regulating diapause in the final larval stage of the southwestern corn borer. Diatraea grandiosella. JH titres in the haemolymph were measured throughout the entire diapause period. Additional results showed that actively secreting corpora allata are necessary to maintain diapause because allatectomized larvae terminated diapause prematurely. A topical application of JH mimic 2 days after the allatectomy prevented this premature termination of diapause. Intact nervous connections between the brain and the corpora allata were necessary for the maintenance of JH secretion. Other surgical work showed that the brains of nondiapausing larvae exhibited a higher ecdysiotropic activity than those of pre-, early-or mid-diapausing larvae.A single application of a JH mimic was more effective in maintaining a diapause-like state in nondiapausing larvae than were repeated topical applications of C₁₈-JH or an implantation of active corpora allata, suggesting that JH was more rapidly metabolized than was the JH mimic. The oxygen consumption of diapausing larvae which had received repeated topical applications of JH mimic was not significantly elevated over that of the controls indicating that treated larvae maintained a low metabolic rate even though they reverted to the spotted morph. A single application of 0.03 μg JH mimic/larva was sufficient to prolong diapause, thereby confirming that JH is necessary for diapause maintenance.     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.     

Morphological study of the reproductive system and ovarian growth of the univoltine bamboo borer,Omphisa fuscidentalis (Hampson, 1896) (Lepidoptera: Crambidae)

ABSTRACT

The female reproductive system and protein deposition in the ovaries during development have never been examined in the bamboo borer Omphisa fuscidentalis Hampson. The aim of this study was thus to study the morphology of the female reproductive system of each stage of development. The female reproductive system of the borer consists of a pair of ovary, oviduct and accessory glands. Each ovary is composed of four polytrophic ovarioles that connect to lateral oviducts, fused with a common oviduct. The size of the ovary in diapausing larvae for 9 months was determined. The length and width of the ovaries were the smallest in September larvae (0.343 ± 0.03 and 0.071 ± 0.01 mm, respectively). The ovaries were the largest during ovarian development in May (0.752 ± 0.08 mm long and 0.084 ± 0.01 wide). Additionally, ovarian size was significantly larger in adults than in pupae. The ovarian protein concentration of larvae in May was 0.59 ± 0.06 mg/ml and increased to 16.61 ± 7.5 and 37.42 ± 5.5 mg/ml in pupae (June) and adults (July), respectively. The results showed ovarian development in all life stages of this holometabolous insect, which has a longer life cycle than other lepidopterans.

Abbreviation: TEM: transmission electron microscope     

Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.     

Juvenile hormone and regulation of dichotomous spermatogenesis during the larval diapause of the codling moth

Spermatogenesis ceases during diapause and resumes after diapause. A possible causal relationship between the regulation of these phenomena and the fluctuations of the juvenile hormone titre connected with the diapause was examined in the codling moth. Spermatocyte lysis and spermatogenesis arrest are directly and reversibly related to these fluctuations. They are induced in the last-larval instar by sole application of the juvenoid Altosid to the penultimate-instar larvae predetermined not to enter diapause. Conversely, allatectomy of early last-instar larvae predetermined to diapause induces renewal of spermatogenesis and disruption of diapause. However, the continuation of the arrest of the spermatogenesis during diapause is not directly related to the presence of a high titre of juvenile hormone. The arrest extends throughout diapause which may continue for months, although no juvenile hormone could be detected in the species after 35 days of larval diapause.     

Switchover in the sensitivity of the prothoracic glands to juvenile hormone in the cotton leafworm, Spodoptera littoralis

Switchover in the sensitivity of the prothoracic glands to juvenile hormone analogue during the last-larval instar of Spodoptera littoralis occurs in the middle of the third scotophase i.e. at the end of phagoperiod when the body weight is maximal and the ecdysteroid is increasing in the haemolymph. Application of the analogue to larvae neck-ligated before the switchover completely inhibits or delays metamorphosis due to an inhibitory effect on the prothoracic gland cells and is not mediated by the nervous system. This inhibition by the analogue is dose-dependent, and when complete inhibition of metamorphosis occurs, the prothoracic glands cells degenerate. Treatment of neck-ligated larvae with the analogue after the switchover stimulates metamorphosis by accelerating the appearance of an ecdysteroids peak in the haemolymph. The stimulatory effect of the analogue to the prothoracic glands in neck-ligated larvae is not direct one, and some unknown factors seem to play a role therein.

The probale role of prothoracicotropic hormone as a synchronizing factor in the switchover in the sensitivity of the prothoracic glands to juvenile hormone is discussed.     

Regulation of soluble and membrane-bound trehalase activity and expression of the enzyme in the larval midgut of the bamboo borer Omphisa fuscidentalis

Diapausing larvae of Omphisa fuscidentalis contain soluble and membrane-bound trehalase in the midgut. Soluble trehalase activity accounts for three-fourths of the total trehalase activity in midgut homogenates. The exposure of diapausing larvae to juvenile hormone analog (JHA) induced pupation, accompanied by an increase in soluble trehalase activity at the beginning of the prepupal period. Injection of 20-hydroxyecdysone (20E) increased the level of soluble trehalase activity 5 days postinjection in a dose-dependent manner. In contrast, no increase in membrane-bound trehalase activity was observed under the same conditions. We cloned the cDNAs that encode the soluble and membrane-bound forms of trehalase in O. fuscidentalis trehalase-1 (OfTreh-1) and trehalase-2 (OfTreh-2), respectively. Treh-1 encodes a 581-aa protein while Treh-2 encodes a 648-aa protein with one putative transmembrane domain near the C-terminus. The mRNA expression level of Treh-1 was 27-fold higher than that of Treh-2 in diapausing larval midgut. Following the exposure of diapausing larvae to JHA, Treh-1 mRNA expression increased gradually until the prepupal period whereupon it increased dramatically; in contrast, the mRNA expression of Treh-2 remained at its initial level. Similarly, 20E upregulated Treh-1 expression but had no effect on Treh-2 expression. Taken together, these results suggest that an increase in the soluble trehalase activity at pupation is caused by upregulation of Treh-1 gene. Moreover, membrane-bound trehalase does not appear to be involved in the dynamic changes in the hemolymph trehalose concentration that occur during the larval-pupal transformation.