Larval diapause termination in the bamboo borer, Omphisa fuscidentalis.

In insects, juvenile hormone (JH) and 20-hydroxyecdysone (20E) regulate larval growth and molting. However, little is known about how this cooperative control is terminating larval diapause especially in the bamboo borer, Omphisa fuscidentalis. In both in vivo and in vitro experiments, we here measured the expression levels of genes which were affected by juvenile hormone analogue (JHA: S-methoprene) and 20-hydroxyecdysone (20E) in diapausing O. fuscidentalis larvae. Corresponding mRNA expression changes in the subesophageal ganglion (SG) and prothoracic gland (PG) were evaluated using qRT-PCR. The data showed similar response patterns of JH receptor gene (OfMet), diapause hormone gene (OfDH-PBAN), ecdysone receptor genes (OfEcR-A and OfEcR-B1) and ecdysone inducible genes (OfBr-C, OfE75A, OfE75B, OfE75C and OfHR3). JHA induced the expressions of OfMet and OfDH-PBAN in both SG and PG, whereas ecdysone receptor genes and ecdysone inducible genes were induced by JHA only in PG. For 20E treatment group, expressions of ecdysone receptor genes and ecdysone inducible genes in both SG and PG were increased by 20E injection. In addition, the in vitro experiments showed that OfMet and OfDH-PBAN were up-regulated by JHA alone, but ecdysone receptor genes and ecdysone inducible genes were up-regulated by JHA and 20E. However, OfMet and OfDH-PBAN in the SG was expressed faster than OfMet and OfDH-PBAN in the PG and the expression of ecdysone receptor genes and ecdysone inducible genes induced by JHA was much later than observed for 20E. These results indicate that JHA might stimulate the PG indirectly via factors (OfMet and OfDH-PBAN) in the SG, which might be a regulatory mechanism for larval diapause termination in O. fuscidentalis.

Three new species and a new record of the spider genus Mallinella Strand, 1906 from Brunei (Araneae, Zodariidae).

This article is the first formal record of the genus Mallinella Strand, 1906 in Brunei Darussalam. Four Mallinella species are documented. Mallinella merimbunenis sp. nov., M. myrmecophaga sp. nov., and M. platyrhyncha sp. nov. are newly described. The females of M. bigemina Dankittipakul, Jocqué & Singtripop, 2012 are here recorded from Brunei for the first time. The latter species was previously known only from an evergreen hill forest at 2,000 m elevation at Mt. Kinabalu of Kinabalu Park in Sabah.

Molecular characterization and gene expression of juvenile hormone binding protein in the bamboo borer, Omphisa fuscidentalis.

Juvenile hormone (JH) plays an important role in many physiological processes in insect development, diapause and reproduction. An appropriate JH titer in hemolymph is essential for normal development in insects. Information concerning its carrier partner protein, juvenile hormone binding protein (JHBP), provides an alternative approach to understanding how JH regulates metamorphosis. In this study, we cloned and sequenced the Omphisa juvenile hormone binding protein (OfJHBP). The full-length OfJHBP cDNA sequence is comprised of 849 nucleotides with an open reading frame of 726bp encoding 242 amino acids. The molecular mass of the protein was estimated to be 26.94kDa. The deduced protein sequence of OfJHBP showed moderate homology with the lepidopteran, Heliothis virescens JHBP (52% amino acid identity) and lower homology with the Bombyx mori JHBP (45%) and the Manduca sexta JHBP (44%). The OfJHBP was expressed mainly in the fat body. OfJHBP transcripts in the fat body was moderately high during 3rd, 4th and 5th instars, then rapidly increased, reaching a peak during early diapause. The expression remained high in mid-diapause, then decreased in late-diapause until the pupal stage. Both juvenile hormone analog (JHA), methoprene, 20-hydroxyecdysone (20E) exhibited a similar stimulatory pattern in OfJHBP expression of diapausing larvae. OfJHBP mRNA levels gradually increased and showed a peak of gene expression on the penultimate, then declined to low levels in the pupal stage. For in vitro gene expression, both of JHA and 20E induced OfJHBP mRNA expression in fat body. Fat body maintenance in vitro in the presence of 0.1µg/50µl JHA induced OfJHBP mRNA expression to high levels within the first 30min whereas 0.1µg/50µl 20E induced gene expression at 120min. To study the synergistic effect of these two hormones, fat body was incubated in vitro with 0.1µg/50µl JHA or 0.1µg/50µl 20E or a combination of both hormone for 30min. Induction of OfJHBP expression by JHA and 20E was significantly greater than that of either hormone alone. These results should contribute to our understanding of how JHBP and JH regulate the termination of larval diapause in the bamboo borer.

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 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.

Characteristic expression of three heat shock-responsive genes during larval diapause in the bamboo borer Omphisa fuscidentalis.

Three heat shock-responsive genes, Ofhsp70, Ofhsc70, and Ofhsp90, were identified in Omphisa fuscidentalis. These genes encode proteins with molecular weights of 70, 72, and 82 kDa, respectively, and were upregulated during heat shock. Ofhsp70 was expressed actively in the pre-diapause period, but its expression was low during diapause. While Ofhsc70 expression was low during pre-diapause and the first half of diapause, its expression increased greatly in the second half before diapause termination. In contrast, Ofhsp90 expression decreased by half during the transition to diapause and conspicuously decreased during late diapause. The results suggest that Ofhsp90 is associated with the maintenance of diapause and Ofhsc70 with the termination of diapause, whereas the expression of Ofhsp70 is unrelated to diapause. The insect molting hormone 20-hydroxyecdysone (20E) and juvenile hormone analog (JHA) caused increases in the mRNA expression of Ofhsc70, but not Ofhsp70 or Ofhsp90, in diapausing larvae. These transcriptional responses to 20E are consistent with the changes in ecdysteroid titer in the hemolymph relative to gene expression during diapause.

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.

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.

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.

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.

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.

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.