The main blood sugar in insects, trehalose, differs from glucose in mammals. To incorporate trehalose into cells and utilize it, tissue cells possess the enzyme trehalase (EC3.2.1.28), which catalyses trehalose into glucose, in the organellar membrane or in the cytoplasm. Soluble and membrane-bound trehalase proteins have been isolated from insects. To date, however, only genes encoding the soluble trehalase have been reported in insects. Soluble trehalase is therefore believed to become localized on the cell surface via modification. In contrast, cDNAs encoding trehalase localized on the apical cell surface via the glycosylphosphatidylinositol-anchor have been isolated from mammalian small intestines. The amino acid sequence contains a specific hydrophobic region and an upstream omega site, which is cleaved for glycosylphosphatidylinositol-attachment, at the C-terminus. Here, we describe a cDNA from the silkworm Bombyx mori that encodes a novel trehalase (type-2) with one transmembrane domain and lacking the omega site. Immunoblotting and immunohistochemical analyses demonstrated that in the midgut tissue of Bombyx larvae, soluble trehalase-1 is present mainly in goblet cell cavities, but membrane-bound trehalase-2 is predominantly seen on the visceral muscle surrounding the midgut. To our knowledge, this is the first report of a cDNA encoding trehalase that penetrates the cell membrane in insects and its cellular localization.

Two cDNAs similar to aquaporins (AQPs) from other insect species were identified and characterized from the silkworm larva, Bombyx mori. The first cDNA (AQP-Bom1) cloned from the anterior silk gland encodes a 25 900 Da protein similar to insect AQPs isolated from several liquid-feeding insects. The second cDNA (AQP-Bom2) cloned from the posterior midgut encodes a 27 694 Da protein. Northern blot analysis has revealed that the AQP-Bom1 mRNA (2.3 kb) is expressed predominantly in the hindgut (colon and rectum), and moderately or minimally in the silk gland, midgut and Malpighian tubules, while the AQP-Bom2 mRNA (1.3 kb) is mainly expressed in the posterior midgut and Malpighian tubules. Functional analysis in Xenopus oocytes microinjected with the cRNA of these AQPs revealed that the AQP-Bom1 mRNA encodes a water-specific aquaporin, likely involved in the water retrieval function of the hindgut, while the AQP-Bom2 mRNA encodes an aquaglyceroporin, increasing glycerol and urea uptake.

The stoichiometry of K+/H + antiport was measured fiuorometrically by the static head method in highly purified ~esicles from goblet cell apical membranes of larval lepidopteran midgut. The measured stoichiometry of 1 K+I2 H ÷ explains how the antiport results in electrophoretic exchange of extracellular tl + for intracellular K +, driven by the voltage component of the proton-motive force of an H + translocating V-ATPase that is located in the same membrane. In turn, the exchange of K ÷ for It + helps to explain how the midgut contents are alkalinized to :i pH of 11. ffey words': K+/H + antiport; V-ATPase; K + transport; \lkalinization; Manduca sexta • IntroductionA remarkable property of lepidopteran insect midgut is the 0roduction of a luminal fluid that is the most alkaline in a i~iological system and can exceed pH 12 in some species [1]. The tdaptive role of the high luminal pH is not known, although t may represent an evolutionary response to tannins that tend o reduce the digestibility of plant foods [2] by cross-linking ~roteins [3]. The mechanism by which the gut lumen is alkalinzed is also unknown. It is clear that the overall mechanism of dkalinization has to involve the exchange of H ÷ for cations ,uch as K + or Na +, or the exchange of anions such as CI-for mions such as OH-or CO 2-. Obviously, the mechanisms that ;enerate weakly alkaline fluids in vertebrates can not account "or this strong alkalinization since they are based on HCO 3 ransport and thus could produce pH values no higher than 9. Insect gastrointestinal and sensory epithelial possess a mique alkali metal ion transport system that resides in the tpical plasma membrane and actively pumps ions out of cells 4,5]. In the larval midgut of the tobacco hornworm, Manduca ¢exta, it is situated in the apical membrane of goblet cells [4]. It transports K + electrogenically into the gut lumen, thereby .,reating a transmembrane voltage in excess of 250 mV, lumen 13ositive [6]. One of the most appealing hypotheses of insect gut tlkalinization was proposed by Dow [1], who suggested that "Corresponding

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A prominent 16-kDa protein copurifies with the V-ATPase isolated from both posterior midgut and Malpighian tubules of Manduca sexta larvae and thus was believed to represent a V-ATPase subunit. [14C]N,N'-dicyclohexylcarbodiimide labeling and its position on SDS-electrophoresis gels revealed that this protein was different from the 17-kDa proteolipid. A cDNA clone encoding a highly hydrophilic protein with a calculated molecular mass of 13,692 Da was obtained by immunoscreening. Monospecific antibodies, affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16-kDa protein of the purified V-ATPase, confirming that a cDNA encoding this protein had been cloned. In vitro translation of the cRNA showed that the cloned 13-kDa subunit behaved like a 16-kDa protein on SDS-electrophoresis gels. The cloned protein showed 37% amino acid sequence identity to the 13-kDa V-ATPase subunit Vma10p recently cloned from yeast and some similarity to subunit b of bacterial F-ATPases. In contrast to the Vma10p protein, which behaved like a V0 subunit, the M. sexta 13-kDa protein behaved like a V1 subunit, since it could be stripped from the membrane by treatment with the chaotropic salt KI and by cold inactivation. When KI dissociated V-ATPase subunits were reassociated by dialysis that removed the KI, a soluble, 450-kDa complex of the M. sexta V-ATPase could be purified by gel chromatography. This V1 complex consisted of subunits A, B, E, and the 13-kDa subunit, confirming that the cloned protein is a new V-ATPase subunit and a member of the peripheral V1 complex of the V-ATPase. We designate this new V1 component subunit G.

To elucidate the relationship between soluble trehalase (Treh1) and integral-membrane trehalase (Treh2) in the Bombyx mori midgut, expression profiles for both proteins and mRNAs were examined during metamorphosis by using Western-blotting and quantitative real-time PCR analyses. Two bands of Treh2 (about 74 kDa) were detected in the midgut of 0-day-old 5th (last) instar larvae. Levels of Treh2 decreased as the developing larvae approached spinning (8 days old). In contrast, towards the onset of the spinning stage, Treh1 (68 kDa) was clearly observed, and levels increased until the middle of the pupal stage. Treh2 mRNA expression relative to Bmrp49 mRNA expression was almost constant, although fluctuations were detected. Treh1 mRNA expression relative to Bmrp49 mRNA increased sharply just after spinning. To further examine the expression mechanism of the Treh1 gene in midgut, actively feeding larvae (4 days old) were starved or ligated between the 4th and 5th segments. Injection of a molting hormone into the larval-isolated abdomen led to activation of Treh1, demonstrating that molting hormone acts on the midgut and activates this gene.

A cDNA of aquaporin (AQP, water channel) belonging to the major intrinsic protein (MIP) family was obtained from the digestive tract of the Formosan subterranean termite, Coptotermes formosanus. The cDNA encoded an AQP homolog designated as CfAQP with 249 amino acids. RT-PCR analysis revealed the expression of CfAQP in the digestive tract and other tissues, suggesting that CfAQP may play important roles not only in water recycling but also other essential water transport processes in the termite. Phylogenetic analysis of CfAQP with 71 insect MIP sequences from 17 species indicated that insect MIPs could be classified into 4 groups, designated as insect MIP Group 1 to Group 4, and that CfAQP is a member of Group 1, a sister group of vertebrate AQPs.

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