Nujira Tatun

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

Abstract 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.

Expression of Heat Shock Protein Genes in Different Developmental Stages and After Temperature Stress in the Maize Weevil (Coleoptera: Curculionidae)

ABSTRACT The maize weevil, Sitophilus zeamais Motschulsky, is a major pest of rice and other postharvest grain stocks in tropical countries. Heating and cooling treatments have been adopted to control this pest. Because heat shock protein (hsp) genes respond to temperature stress, we examined the association of hsp genes with development and thermal stress in S. zeamais. The temperature response of the insect to heat and cold treatments was assessed at four developmental stages: egg, larva, pupa, and adult. LT50 values at high temperatures were similar among the four developmental stages, while adults were the most tolerant to low temperatures, and eggs, larvae, and pupae exhibited similar LT50 values. Expression levels of three hsps—Szhsp70, Szhsc70, and Szhsp90—fluctuated substantially throughout the four stages at a rearing temperature of 28°C. Heat shock and cold shock increased the expression of all three hsps, and the highest upregulation was observed at 40°C, although the intensity of upregulation varied among the three genes: strongly in Szhsp70, moderately in Szhsp90, and slightly in Szhsc70. Basal expression of the three hsps at 28°C and gene responses to heat and cold shock also varied significantly at the tissue level.

Trehalase Activity in Fungus-Growing Termite, Odontotermes feae (Isoptera: Termitideae) and Inhibitory Effect of Validamycin

ABSTRACT Trehalase is the hydrolytic enzyme that catalyzed the hydrolysis of trehalose to glucose. In this study, trehalase activity in the fungus-growing termite, Odontotermes feae Wasmann had been examined. Trehalase activity in digestive tract and carcass of O. feae was higher than that in woodfeeding termite, Coptotermes gestroi Wasmann. The intestinal tract of worker caste of O. feae was the main source of trehalase compared with that in salivary, fat body, and carcass. In particular, the highest activity was found in the midgut and hindgut parts. More specifically, the contents of midgut and hindgut had higher enzyme activity compared with that trehalase prepared from their epithelial tissue. The enzyme activity of gut trehalase in three different termite castes, worker, soldier, and reproductive, had been determined. The result showed that female alate had the highest activity, followed by worker, male alate, and soldier castes. Trehalose concentration in the reproductive caste was at lowest level, while soldier and worker contained the high trehalose concentration. This study indicates that high trehalase activity locates in the midgut and hindgut contents and change in trehalase activity in fungus-growing termite is caste-specific. Validamycin inhibited trehalase activity of O. feae in vivo and caused high mortality, indicating that this trehalase inhibitor is valuable tools for termite control.

Developmental and Lethal Effects of Trehalase Inhibitor (Validamycin) on the Tribolium castaneum (Coleoptera: Tenebrionidae)

Abstract The trehalose analogue compound validamycin is a very strong competitive inhibitor of trehalase enzymatic activity in various insects, and it has been studied as a potential material for the development of new insecticides. The inhibitory effect of validamycin on trehalase activity in the red flour beetle, Tribolium castaneum Herbst, was investigated in the present study. We measured changes in glucose concentrations and assessed morphological abnormalities in the wings of T. castaneum after trehalase activity was inhibited by validamycin treatment. We found that the application of validamycin during the larval stage, via both feeding and injection methods, resulted in a significant decrease in trehalase activity during the larval, pupal, and adult stages. Injection of validamycin into pupae caused a reduction in trehalase enzyme activity and caused the glucose concentration to fall by 21% compared with the control. The dose-dependent lethal effect of validamycin treatment was recorded, with the highest mortality rate of ∼78%. This study is the first to demonstrate the dosedependent effect of validamycin on the wing morphology of T. castaneum adults, in that the injected insects exhibited wrinkled elytra and improperly folded hindwings. Reduction of elytral length and width was also recorded. The trehalase inhibitor validamycin is valuable for investigating the physiological function of trehalase in insects and may be useful as a pest control tool for T. castaneum.