Cellular localization and proposed function of midguttrehalase in the silkworm larva, Bombyx mori

doi:10.1016/0040-8166(85)90030-8

Tissue and Cell

Volume 17, Issue 4, 1985, Pages 539-551

https://doi.org/10.1016/0040-8166(85)90030-8 Get rights and content

Abstract

A sorbitol density gradient analysis with the aid of several marker enzymes demonstrated that midgut trehalase of the silkworm larvae. Bombyx mori, was localized in the microsomal membranes, but not in mitochondria, lysosomes and microvilli at the apical surface. Electron microscopic examination showed that trehalase-enriched membrane fraction consisted of heterogeneous mixtures of membrane vesicles derived from the endoplasmic reticulum and plasma membrane parts other than the microvillus membrane. The enzyme-histochemical stains of trehalase activity on the midgut section could be detected only at the basal surface of the epithelium against haemocoel. Such a specific localization was further confirmed by immunohistochemistry with the peroxidase-conjugated antibody technique. Thus, it is concluded that midgut trehalase of silkworm larvae is situated on the plasma membrane at the basal surface of the epithelium. An intact preparation of midgut incubated in vitro in the medium containing [¹⁴C]trehalose could hydrolyse trehalose into glucose and take it up into the cell, although some glucose was liberated into the medium when incubated for extended periods. These results suggest that midgut trehalase plays a physiological role in utilization of haemolymph trehalose not in nutrient absorption.

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The enzyme trehalase aids the breakdown of trehalose to glucose molecules for energy purpose. The trehalase activity in B. mori has been reported in tissues such as fat body, midgut, ovary, and silk gland (Azuma and Yamashita, 1985; Keshan and Ray, 1998, 2001). In haemolymph, the trehalose level fluctuates between 7 and 21 mM during the feeding phase of the last larval instar in B. mori (Kh. and Keshan, 2019).
Insulin in mammals is known for its effect on carbohydrate metabolism and maintenance of blood sugar levels. In the present study, we explored the effect of exogenous insulin and 20-hydroxyecdysone (20E) on carbohydrate metabolism in Bombyx mori under the fed and food-deprived conditions. The study showed that insulin and 20E regulate the trehalose (major circulating sugar) level in B. mori, and larval feeding status plays a decisive role in influencing the action of these two hormones. At feeding, both insulin and 20E showed its hypertrehalosemic action but at food deprivation, these hormones acted as hypotrehalosemic factors. Although both insulin and 20E showed the same effect on the haemolymph trehalose level either at feeding or food deprivation, the metabolic regulation was different for these two hormones. Insulin treatment to fed larvae increased the haemolymph trehalose level without altering the effectiveness of trehalose utilization but possibly by inducing the activity of glycogen phosphorylase enzyme and releasing glucose-1-P for the increased synthesis of trehalose. The treatment of 20E to fed larvae also increased the trehalose level, but concurrently it also increased both the enzyme activity of trehalase and glycogen phosphorylase. Insulin treatment to food-deprived larvae decreased the circulating trehalose level by increasing the trehalose breakdown as the mRNA expression level of trehalase-2 and enzyme activity of trehalase increased in these larvae. The treatment of 20E to food-deprived larvae decreased the haemolymph trehalose possibly by decreasing its synthesis, as glycogen phosphorylase enzyme activity decreased in these larvae, thus restricting the availability of glucose-1-P for trehalose synthesis. The study, thus suggests that both insulin and 20E regulate carbohydrate metabolism in B. mori.
Effects of the plant volatile trans‑2-hexenal on the dispersal ability, nutrient metabolism and enzymatic activities of Bursaphelenchus xylophilus
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Trehalase activity can be enhanced under environmental stresses, promoting the transformation of trehalose into glucose [46]. In our study, trehalase activity initially increased, which might help in resisting the stress of trans‑2-hexenal by converting trehalose to metabolic energy [51]. However, trehalase activity then decreased at 48 h, which might be due to the metabolic disturbance caused by trans‑2-hexenal and might also explain the increase in carbohydrate content at 48 h.
Bursaphelenchus xylophilus causes pine wilt disease (PWD), which severely damages pine species. The plant volatile trans‑2-hexenal has strong activity against nematodes, although the precise mechanism of this inhibitory action remains unclear. In this paper, the fumigant effects of the LC₁₀ and LC₃₀ of trans‑2-hexenal on B. xylophilus were demonstrated. The trans‑2-hexenal treatments significantly inhibited the dispersal ability of nematodes. The results also indicated that trans‑2-hexenal affects the metabolism of nutrients and the activity of digestive enzymes. Among detoxifying enzymes, after treatment with trans‑2-hexenal, glutathione S-transferase activity increased significantly and general esterase activity decreased significantly. Based on these results, trans‑2-hexenal disturbs the normal physiological and biochemical activities of this nematode. These results provide valuable insight into the nematicidal mechanisms of trans‑2-hexenal.
Regulation of hemolymph trehalose level by an insulin-like peptide through diel feeding rhythm of the beet armyworm, Spodoptera exigua
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Like vertebrate insulins, some insect insulin-like peptides (ILPs) play crucial roles in controlling immature growth, adult lifespan, and hemolymph sugar level. An ILP gene (SeILP1) was predicted from a transcriptome database of Spodoptera exigua. SeILP1 encodes 95 amino acid sequence and shares sequence homologies (33–83%) with other insect ILPs, in which six conserved cysteine residues are found in the predicted B–A chains. SeILP1 was expressed in all developmental stages of S. exigua. However, SeILP1 expression was tissue-specific because the transcript was detected in fat body and epidermis, but not in hemocytes and gut. Its expression increased with feeding activity. Hemolymph trehalose levels of the fifth instar larvae maintained a relatively constant level at 2.31 ± 0.62 mM. However, starvation induced a significant increase of the hemolymph trehalose level by more than twofold in 48 h, at which few SeILP1 was transcribed. RNA interference of SeILP1 using its specific double-stranded RNA induced a significant increase of hemolymph trehalose level. Interestingly, a bovine insulin decreased hemolymph trehalose level in a dose-dependent manner. These results indicate that SeILP1 plays a role in suppressing hemolymph trehalose level in S. exigua.
Molecular cloning and expression analysis of soluble and membrane-bound trehalase genes in the cotton bollworm, Helicoverpa armigera
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Citation Excerpt :
SeTre-2 in S. exigua has an important role in CHSB expression and chitin synthesis in the midgut (Chen et al., 2010). HaTreh-2 strongly expressed in the midgut may be involved in incorporating trehalose from the hemolymph into muscular cells and supporting peristaltic movement of the midgut by providing energy to visceral muscles (Azuma and Yamashita, 1985; Mitsumasu et al., 2005) and the chitin synthesis pathway in the midgut (Tang et al., 2008; Chen et al., 2010). Higher expression of HaTreh-2 in the head may be adapted to the energy requirements of brain activity and chitin synthesis.
Trehalase plays a significant role in various physiological processes in insects. In this study, we cloned and characterized a soluble trehalase gene (HaTreh-1) and a membrane-bound trehalase gene (HaTreh-2) from Helicoverpa armigera, a serious polyphagous pest of crops. HaTreh-1 contained an open reading frame of 1716 bp that encodes a protein of 572 amino acids. HaTreh-2 has an open reading frame of 1938 bp, encoding a protein of 646 amino acids. Sequence alignment and phylogenetic analysis of the two putative proteins revealed that HaTreh-1 and HaTreh-2 belong to soluble and membrane-bound trehalase groups, respectively. Quantitative real-time PCR (qPCR) analyses of the spatiotemporal expression pattern of HaTreh in H. armigera revealed that HaTreh-1 was expressed mainly in the midgut, with lower expression in the integument and head, Malpighian tubules, trachea, and fat body. The expression levels of HaTreh-2 were detected in all 6 tissues, and HaTreh-2 was mainly expressed in the midgut and head. Expression of HaTreh-1 was higher throughout the larval stages, but lower on days 1 and 2 of the pupal stage. Expression of HaTreh-2 was higher during the 4th- and 5th-instar larval stages. Taken together, these results suggest that HaTreh-1 and HaTreh-2 have different functions in various developmental stages and tissues.
Characterization of putative soluble and membrane-bound trehalases in a hemipteran insect, Nilaparvata lugens
2009, Journal of Insect Physiology
Trehalose is the main blood sugar of insects, and the enzyme trehalase is involved in energy metabolism and controlling trehalose levels in cells. Two forms (soluble and membrane-bound) of trehalase and the corresponding genes (NlTre-1 and NlTre-2) were identified from the brown planthopper, Nilaparvata lugens. Both NlTre-1 and NlTre-2 contain trehalase signature motifs, and NlTre-2 contains a putative transmembrane domain. Comparison of trehalase activity and gene mRNA level at different developmental stages, or following application of 20-hydroxyecdysone (20E), suggests that NlTre-1 and NlTre-2 encode a soluble trehalase and a membrane-bound trehalase respectively. Soluble trehalase activity accounted for the majority of total trehalase activity in N. lugens. Only soluble trehalase activity and NlTre-1 mRNA level could be induced by 20E. Additionally, only soluble trehalase activity was significantly higher in macropterous individuals than in brachypterous morphs. These results indicate that only soluble trehalase is differentially expressed between macropterous and brachypterous individuals and is more responsive to hormone stimulus.
Regulation of soluble and membrane-bound trehalase activity and expression of the enzyme in the larval midgut of the bamboo borer Omphisa fuscidentalis
2008, Insect Biochemistry and Molecular Biology
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.

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Cellular localization and proposed function of midguttrehalase in the silkworm larva, Bombyx mori

Abstract

Tissue & Cell

Anal. Biochem.

Insect Biochem.

Biochim. biophys. Acta

Biochim. biophys. Acta

Archs Biochem. Biophys.

J. Insect Physiol.

J. biol. Chem.

Insect Biochem.

Biochim. biophys. Acta

Insect Biochem.

Adv. Insect Physiol.

J. Insect Physiol.

Immunohistochemical and biochemical localization of trehalase in the developing ovaries of the silkworm, Bombyx mori

Insect Biochem.

Histochemical localization of trehalase activity in dorsal flight muscle of the flesh fly Sarcophaga bullata with light and electron microscopy

J. Histochem. Cytochem.