Water influx via aquaporin directly determines necrotic cell death induced by the Bacillus thuringiensis Cry toxin.

The Bacillus thuringiensis Cry toxin causes swelling and necrosis in insect cells, but the route(s) and significance of the water influx involved in its cytotoxicity are unclear. Here, we assessed the role of aquaporins (AQPs), known as water channels, in Cry toxin intoxication. An AQP inhibitor did not interfere with any known process to form the toxin pore, but it diminished the cell swelling and loss of membrane integrity induced by the Cry toxin. Overexpression of AQPs facilitated water influx and cytotoxicity. Our results demonstrate that water influx via aquaporin directly determines necrotic cell death induced by the Cry toxin.

Insect water-specific aquaporins in developing ovarian follicles of the silk moth Bombyx mori: role in hydration during egg maturation.

Egg formation in terrestrial insects is an absorptive process, accommodated not only by packing proteins and lipids into yolk but also by filling chorions with water. An osmotic swelling of ovarian follicles takes place during oocyte maturation. This study investigated the role of the aquaporin (AQP) water channel in the osmotic uptake of water during oogenesis in the silk moth Bombyx mori Linnaeus, 1758. Using the antibodies that specifically recognize previously characterized AQPs, two water-specific subtypes-AQP-Bom1 and AQP-Bom3-belonging to the Drosophila integral protein (DRIP) and Pyrocoelia rufa integral protein (PRIP) subfamilies of the insect AQP clade, respectively, were identified in the developing ovaries of B. mori. During oocyte growth, Bombyx PRIP was distributed at the oocyte plasma membrane, where it likely plays a role in water uptake and oocyte swelling, and may be responsible for oocyte hydration during fluid absorption by ovarian follicles. During the transition from vitellogenesis to choriogenesis during oocyte maturation, Bombyx DRIP expression became abundant in peripheral yolk granules underlying the oocyte plasma membrane. The restricted DRIP localization was not observed in non-diapause-destined follicles, where DRIP was evenly distributed in medullary yolk granules. There was no difference in PRIP distribution between diapause- and non-diapause-destined follicles. The diapause-destined oocytes encase DRIP protein in the peripheral yolk granules, where DRIP might be inert. This would be reflected in the metabolic arrest associated with diapause after fertilization and egg oviposition.

Differential distribution of glutamate- and GABA-gated chloride channels in the housefly Musca domestica.

l-Glutamic acid (glutamate) mediates fast inhibitory neurotransmission by affecting glutamate-gated chloride channels (GluCls) in invertebrates. The molecular function and pharmacological properties of GluCls have been well studied, but not much is known about their physiological role and localization in the insect body. The distribution of GluCls in the housefly (Musca domestica L.) was thus compared with the distribution of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Quantitative PCR and ligand-binding experiments indicate that the GluCl and GABACl transcripts and proteins are predominantly expressed in the adult head. Intense GluCl immunostaining was detected in the lamina, leg motor neurons, and legs of adult houseflies. The GABACl (Rdl) immunostaining was more widely distributed, and was found in the medulla, lobula, lobula plate, mushroom body, antennal lobe, and ellipsoid body. The present findings suggest that GluCls have physiological roles in different tissues than GABACls.

Identification and characterisation of a functional aquaporin water channel (Anomala cuprea DRIP) in a coleopteran insect.

Water transport across the plasma membrane depends on the presence of the water channel aquaporin (AQP), which mediates the bulk movement of water through osmotic and pressure gradients. In terrestrial insects, which are solid and/or plant feeders, the entrance and exit of water is primarily executed along the alimentary tract, where the hindgut, particularly the rectum, is the major site of water conservation. A cDNA encoding the homologue of the water-specific Drosophila AQP [Drosophila integral protein (DRIP)] was identified through the RT-PCR of RNA isolated from the rectum of the cupreous chafer larvae, Anomala cuprea, a humus and plant root feeder. This gene (Anocu AQP1) has a predicted molecular mass of 26.471 kDa, similar to the DRIP clade of insect AQPs characterised from caterpillars, flies and several liquid-feeding insects. When expressed in Xenopus laevis oocytes, Anocu AQP1 showed the hallmarks of aquaporin-mediated water transport but no glycerol or urea permeability, and the reversible inhibition of elevated water transport through 1 mmol l(-1) HgCl2. This is the first experimental demonstration of the presence of a water-specific AQP, namely DRIP, in the Coleoptera. The genome of the model beetle Tribolium castaneum contains six putative AQP sequences, one of which (Trica-1a, XP_972862) showed the highest similarity to Anocu AQP1 (~60% amino acid identity). Anocu AQP1 is predominantly expressed in the rectum. Using a specific antibody raised against DRIP in the silkworm Bombyx mori (AQP-Bom1), Anocu AQP1 was localised to the apical plasma membrane of rectal epithelial cells, and lacking in the midgut and gastric caecal epithelia. Based on the BeetleBase prediction, there are three putative AQPs (Trica-3a, 3b, 3c: XP_970728, 970912, 970791) that are homologous to B. mori aquaglyceroporin [AQP-Bom2 (GLP)]. The immunocytochemical studies using the specific anti-peptide antibody against AQP-Bom2 revealed the presence of the GLP homologue at the apical plasma membrane of enterocytes in the midgut and gastric caeca. Thus, DRIP (Anocu AQP1) and the putative GLP share epithelial fluid-transporting roles along the alimentary tract in cupreous chafer larvae.

A water-specific aquaporin is expressed in the olfactory organs of the blowfly, Phormia regina.

The high sensitivity and selectivity of perireceptor events in insect olfactory organs requires the concerted action of odorant-binding proteins (OBPs), odorant receptors (ORs), and odorant-degrading enzymes (ODEs). Sensillum lymph in the sensillum cavity is a physiological saline that not only mediates the olfactory signaling pathway described above, but also protects the olfactory neurons against desiccation. The molecular mechanism of how water balance is maintained in the sensillum cavity still remains to be elucidated. Here, we characterize an aquaporin from the blowfly, Phormia regina (PregAQP1). PregAQP1 possesses six predicted transmembrane domains and two asparagine-proline-alanine (NPA) motifs, and belongs to the Drosophila melanogaster integral protein (DRIP) subfamily. Transcript levels were high in the maxillary palp and moderate in the antenna. PregAQP1 accumulated in accessory cells located underneath a long-grooved hair in the maxillary palp and also in a receptor neuron in a thick-walled sensillum in the antenna. Expression of PregAQP1 in Xenopus oocytes showed water permeability in a mercury-sensitive manner. These results suggest that PregAQP1 plays a role in the maintenance of the aqueous environment of olfactory organs.

Two water-specific aquaporins at the apical and basal plasma membranes of insect epithelia: molecular basis for water recycling through the cryptonephric rectal complex of lepidopteran larvae.

Larval lepidopteran and coleopteran insects have evolved a specialised cryptonephric system in the hindgut in which water is constantly and rapidly taken up before defecation. In the silkworm, Bombyx mori, the movement of water through the epithelia within the cryptonephric rectal complex is likely facilitated by the two aquaporins, AQP-Bom1 and AQP-Bom3. Both are functionally water-specific and are predominantly expressed in the hindgut (colon and rectum). Phylogenetically, AQP-Bom1 and AQP-Bom3 belong to the DRIP (Drosophila integral protein) and PRIP (Pyrocoelia rufa integral protein) subfamilies, respectively, of the insect AQP clade. In immunoblot analyses using antipeptide antibodies for each Bombyx AQP, the predicted molecular mass for the respective AQPs were around 25 kDa, and further indicated that both tended to be oligomerised as a homotetramer (∼110 kDa). AQP-Bom1 [DRIP] was exclusively expressed at the apical plasma membrane of colonic and rectal epithelial cells, whereas AQP-Bom3 [PRIP] was expressed at the basal plasma membrane of these cells. This polarised localisation of DRIP/PRIP was also observed in the outer cryptonephric Malpighian tubules (outer cMT) and in the six tubules just outside the cryptonephric rectal complex (rectal lead MT). In the rectal epithelia, water is transported from the rectal lumen to the perinephric space and then deposited into the lumen of the outer cMT; the water then goes through the tubular lumen to exit the complex and is finally transported across the rectal lead MT. We conclude that rectal water retrieval into the haemocoele occurs at the very limited region of the water-permeable sites in MT epithelia after passing the rectal and cMT epithelia and that the high osmotic permeability is due to the presence of two distinct water-specific AQPs (DRIP and PRIP) in the epithelial cells of lepidopteran hindgut.

Changes in the expression of soluble and integral-membrane trehalases in the midgut during metamorphosis in Bombyx mori.

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.

Mating attraction by Stenotus rubrovittatus (Heteroptera: Miridae) females and its relationship to ovarian development.

Stenotus rubrovittatus (Matsumura) (Heteroptera: Miridae) causes brown or black marks on rice, Oryza sativa L., grains (pecky rice), and it is becoming of increasing importance in Japan. Attractiveness of adult females or males of S. rubrovittatus to conspecific individuals was examined in the field in 2003 and 2004. Unmated female-baited traps captured significantly more males than did the unmated male-baited traps. However, the numbers of females captured by female- or male-baited traps were low, and they were not significantly different from the numbers caught by the control traps. No nymphs were captured by any traps. In 2004, we examined the effects of age and mating experience on female attraction ability with the goal of understanding the role of reproductive development in the observed behavior. The daily number of males captured by young unmated female (3-d-old)-baited traps increased from the first day until the fourth day of experiments, and then capture started to decrease. The peak in the number of captured males corresponded to the preoviposition period. When we observed ovarian development of S. rubrovittatus females under 25 degrees C and a photoperiod of 16:8 (L:D) h, we found that vitellogenesis had already started in approximately 30-50% of 1-d-old individuals. By the fifth day after emergence, 50-70% of individuals had mature eggs. These results indicated that the attractiveness of females is the strongest when egg laying becomes possible. Therefore, S. rubrovittatus females attract males selectively for mating, and it is probable that females use a sex pheromone for the attraction.

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori.

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.