A sodium-dependent trehalose transporter contributes to anhydrobiosis in insect cell line, Pv11.

Pv11 is the only animal cell line that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here, we identified a transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the solute carrier 5 (SLC5)-type secondary active transporters cotransport Na+ and carbohydrates including glucose. The heterologous expression systems showed that the transporter belonging to the SLC5 family, whose expression increases upon rehydration, exhibits Na+-dependent trehalose transport activity. Therefore, we named it STRT1 (sodium-ion trehalose transporter 1). We report an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parental cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration.

In vivo direct cell-penetrating peptide mediated protein transduction system in Acyrthosiphon pisum.

OBJECTIVE: The principal delivery method for CRISPR-based genome editing in insects is now based on microinjection into single cells or embryos. The direct protein transduction systems cannot be employed in aphids because oogenesis occurs without apparent vitellogenesis. Given the limited timing of injection into the embryonic stage in oviparous aphids, a protein delivery system from the hemolymph to the germline and embryos would be a useful tool for genome editing. This study reports a newly developed direct protein delivery system for aphids using cell-penetrating peptides (CPPs). CPPs are short peptides that translocate across the plasma membrane when bound to cargo proteins. RESULTS: Penetratin (PEN), a widely conserved CPP among insects, was identified in this study. We used mVenus, a recombinant fluorescent protein, as a visual marker for CPP availability assessments, and fused it with PEN by bacterial protein expression. The mVenus-PEN recombinant proteins were introduced into the hemolymph of adult unwinged Acyrthosiphon pisum females using a nanoinjector. Fluorescence emitted by mVenus-PEN was observed in various tissues, such as the gut, trachea, bacteriocytes, and their progeny. This study shows that PEN can deliver exogenously expressed proteins into tissues in vivo, indicating that CPPs are powerful tools for protein transduction.

Proton gradient mediates hemolymph trehalose influx into aphid bacteriocytes.

Aphids harbor proteobacterial endosymbionts such as Buchnera aphidicola housed in specialized bacteriocytes derived from host cells. The endosymbiont Buchnera supplies essential amino acids such as arginine to the host cells and, in turn, obtains sugars needed for its survival from the hemolymph. The mechanism of sugar supply in aphid bacteriocytes has been rarely studied. It also remains unclear how Buchnera acquires its carbon source. The hemolymph sugars in Acyrthosiphon pisum are composed of the disaccharide trehalose containing two glucose molecules. Here, we report for the first time that trehalose is transported and used as a potential carbon source by Buchnera across the bacteriocyte plasma membrane via trehalose transporters. The current study characterized the bacteriocyte trehalose transporter Ap_ST11 (LOC100159441) using the Xenopus oocyte expression system. The Ap_ST11 transporter was found to be proton-dependent with a Km value ≥700 mM. We re-examined the hemolymph trehalose at 217.8 mM using a fluorescent trehalose sensor. The bacteriocytes did not obtain trehalose by facilitated diffusion along the gradient across cellular membranes. These findings suggest that trehalose influx into the bacteriocytes depends on the extracellular proton-driven secondary electrochemical transporter.

In vitro larval rearing method of eusocial bumblebee Bombus terrestris for toxicity test.

Bumblebees are important pollinators of wild and agricultural plants but recently have been declining due to various stressors, such as pesticides and diseases. Because of the haplo-diploid sex determination system in hymenopterans, experiments using micro-colonies (small sub colonies without a queen) to identify risks to bumblebee health are limited as they are only able to produce males. Therefore, an experimental protocol for rearing bumblebee larvae in vitro is needed to better understand effects on worker larvae. Here, we aimed to establish a rearing method for larvae of Bombus terrestris for use in risk assessment assays. To confirm the validity of our rearing method, we tested two insecticides used for tomato cultivation, chlorfenapyr and dinotefuran. Bombus terrestris larvae fed with a high nutrient quantity and quality diet increased growth per day. All chlorfenapyr-exposed individuals died within 10 days at 2000-fold dilution, an application dose used for tomatoes. There were significant differences in adult emergence rate among almost all chlorfenapyr treatments. On the other hand, sublethal dinotefuran-exposure did not affect rates of pupation and adult emergence, growth, or larval and pupal periods. Although larvae were smaller than in the natural colony, this rearing method for B. terrestris larvae proved to be effective at evaluating realistic sub-colonies to pesticide exposures.

The Cytotoxic Effect of Genistein, a Soybean Isoflavone, against Cultured Tribolium Cells.

The red flour beetle Tribolium castaneum is a known pest of various grains and stored-products such as wheat flours; however, T. castaneum feeds on and infests soybean and soy products. For more than 60 years, soy flour has been suggested to be unstable food for Tribolium spp. because it causes larval development failure. However, it remains unknown whether soy flour affects adult beetles. The objective of the present study was to examine the effects of soy flour and its related isoflavones against T. castaneum using an artificial dietary intake assay. Beetles were fed gypsum (a non-digestible compound) mixed with either water (control) or soy flour. Significantly fewer beetles survived after being fed the soy flour treatment. Although the soy isoflavone genistein, a defensive agent and secondary metabolite, decreased the T. castaneum adult survival, it required a long time to have a lethal effect. Therefore, the cytotoxic effects of soy flour, i.e., the rapid biological responses following isoflavone addition, were also examined using a cultured cell line derived from T. castaneum. Both genistin and genistein significantly affected the survival of the cultured cells, although genistein had a stronger lethal effect. This study demonstrated the toxicity of genistein found in soybean against T. castaneum cultured cells within 24 h period. Genistein may be used as an oral toxin biopesticide against T. castaneum.

Response of Tribolium castaneum to dietary mannitol, with remarks on its possible nutritive effects.

Mannitol, one of the sugar alcohols, is often used as a low-calorific carbohydrate by animals. In some insects, mannitol acts as a cryoprotectant to endure coldness, but also become a poisonous agent. Adults of the red flour beetle Tribolium castaneum were shown to recognize mannitol as a factor stimulating their feeding behavior, but it remains unclear whether T. castaneum can utilize mannitol as a source of nutrition, because the enzymes needed to metabolize mannitol are unknown in this species. This study shows that T. castaneum utilizes mannitol as a nutrient in a dietary assay based on a sole carbon source added to artificial gypsum diet. The amount of mannitol excreted was less than that ingested, suggesting that it is absorbed in the insect body. The hemolymph of T. castaneum contained no mannitol but contained trehalose, a known blood sugar in insects, even after being fed mannitol. This study also revealed that dietary mannitol was metabolized to triglyceride, the main component of the fat body, forming lipid droplets. It was found that metabolites of a mannitol-supplemented diet extend the lifespan of T. castaneum, compared with those obtained by metabolizing a mannitol-free diet. Given that the insects presented transcriptional changes upon being fed carbohydrates, it might be possible to identify specific genes related to mannitol-specific metabolism by their upregulation upon mannitol intake in T. castaneum. The present study investigated mannitol-responsive gene expression using RNA-Seq. Twenty-eight genes, including those encoding trehalose-6-phosphate synthase and fatty acid synthase, were differentially expressed between beetles that were fed or not fed mannitol. The identification of upregulated genes provides us with important insights into the molecular events following mannitol intake.

Molecular and functional characterization of glucose transporter genes of the fox tapeworm Echinococcus multilocularis.

Alveolar echinococcosis (AE) is a zoonotic parasitosis caused by larvae of the fox tapeworm, Echinococcus multilocularis. E. multilocularis is distributed widely in the Northern hemisphere, causing serious health problems in various animals and humans. E. multilocularis, like other cestodes, lacks a digestive tract and absorbs essential nutrients, including glucose, across the syncytial tegument on its external surface. Therefore, it is hypothesized that E. multilocularis uses glucose transporters on its surface similar to a closely-related species, Taenia solium. Based on this hypothesis, we cloned and characterized glucose transporter homologues from E. multilocularis. As a result, we obtained full-length sequences of 2 putative glucose transporter genes (EmGLUT1 and EmGLUT2) from E. multilocularis. In silico analysis predicted that these were classified in the solute carrier family 2 group. Functional expression analysis using Xenopus oocytes demonstrated clear uptake of 2-deoxy-D-glucose (2-DG) by EmGLUT1, but not by EmGLUT2 in this experimental system. EmGLUT1 was shown to have relatively high glucose transport activity. Further analyses using the Xenopus oocyte system revealed that 2-DG uptake of EmGLUT1 did not depend on the presence or concentration of Na+ nor H+, respectively. Immunoblot analyses using cultured metacestode, ex vivo protoscolex, and adult worm samples demonstrated that both EmGLUTs were stably expressed during each developmental stage of the parasite. Based on the above-mentioned findings, we conclude that EmGLUT1 is a simple facilitated glucose transporter and possibly plays an important role in glucose uptake by E. multilocularis throughout its life cycle.

Extracellular loop structures in silkworm ABCC transporters determine their specificities for Bacillus thuringiensis Cry toxins.

Bacillus thuringiensis Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm (Bombyx mori) ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant B. mori strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.

The intracellular region of silkworm cadherin-like protein is not necessary to mediate the toxicity of Bacillus thuringiensis Cry1Aa and Cry1Ab toxins.

The cadherin-like protein in lepidopteran insects, known as a receptor for Bacillus thuringiensis Cry1A toxins, is a single-pass membrane protein that can be divided into extracellular and intracellular regions. The extracellular region is important for toxin binding and oligomerization, whereas the role of the intracellular region during Cry1A intoxication is unclear. In the present study, we generated a deletion mutant of Bombyx mori cadherin-like protein (BtR175) that lacked the intracellular region to investigate its role in mediating Cry1A toxicity. Like wild-type BtR175, the mutant protein conferred susceptibility to Cry1Aa and Cry1Ab toxins in Sf9 cells, suggesting that the intracellular region is not required to mediate intoxication. The deletion mutant maintained another role of cadherin-like proteins; that it, synergistic activity with B. mori ABC transporter C2 (ABCC2) when mediating Cry1Aa and Cry1Ab toxicity. In addition, we evaluated the effects of reagents that have been reported to inhibit Cry1A toxicity (e.g., protein kinase A inhibitors, EDTA, and sucrose) on Cry1A toxicity in BtR175-expressing cells. Our results suggest that Cry1Aa-induced cell death in BtR175-expressing cells was not caused by signal transduction but by osmotic lysis. Overall, our data indicate that BtR175 mediates the toxicity of Cry1Aa and Cry1Ab toxins entirely via its extracellular region. They also indicate that the synergism between cadherin-like protein and ABCC2 occurs outside of cells or in the cell membrane.

Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role.

Honey bee (Apis mellifera) workers contribute to the maintenance of colonies in various ways. The primary functions of workers are divided into two types depending on age: young workers (nurses) primarily engage in such behaviors as cleaning and food handling within the hive, whereas older workers (foragers) acquire floral nutrients beyond the colony. Concomitant with this age-dependent change in activity, physiological changes occur in the tissues and organs of workers. Nurses supply younger larvae with honey containing high levels of glucose and supply older larvae with honey containing high levels of fructose. Given that nurses must determine both the concentration and type of sugar used in honey, gustatory receptors (Gr) expressed in the chemosensory organs likely play a role in distinguishing between sugars. Glucose is recognized by Gr1 in honey bees (AmGr1); however, it remains unclear which Gr are responsible for fructose recognition. This study aimed to identify fructose receptors in honey bees and reported that AmGr3, when transiently expressed in Xenopus oocytes, responded only to fructose, and to no other sugars. We analyzed expression levels of AmGr3 to identify which tissues and organs of workers are involved in fructose recognition and determined that expression of AmGr3 was particularly high in the antennae and legs of nurses. Our results suggest that nurses use their antennae and legs to recognize fructose, and that AmGr3 functions as an accurate nutrient sensor used to maintain food quality in honey bee hives.

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin.

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that 770DYWL773 of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around 770DYWL773 of ECL 4 in the ABCC2.

A mannitol/sorbitol receptor stimulates dietary intake in Tribolium castaneum.

In insects, perception of chemical stimuli is involved in the acceptance or rejection of food. Gustatory receptors (Grs) that regulate external signals in chemosensory organs have been found in many insects. Tribolium castaneum, a major pest of stored products, possesses over 200 Gr genes. An expanded repertoire of Gr genes appears to be required for diet recognition in species that are generalist feeders; however, it remains unclear whether T. castaneum recognizes a suite of chemicals common to many products or whether its feeding is activated by specific chemicals, and whether its Grs are involved in feeding behavior. It is difficult to determine the food preferences of T. castaneum based on dietary intake due to a lack of appropriate methodology. This study established a novel dietary intake estimation method using gypsum, designated the TribUTE (Tribolium Urges To Eat) assay. For this assay, T. castaneum adults were fed a gypsum block without added organic compounds. Sweet preference was determined by adding sweeteners and measuring the amount of gypsum in the excreta. Mannitol was the strongest activator of T. castaneum dietary intake. In a Xenopus oocyte expression, TcGr20 was found to be responsible for mannitol and sorbitol responses, but not for responses to other tested non-volatile compounds. The EC50 values of TcGr20 for mannitol and sorbitol were 72.6 mM and 90.6 mM, respectively, suggesting that TcGr20 is a feasible receptor for the recognition of mannitol at lower concentrations. We used RNAi and the TribUTE assay to examine whether TcGr20 expression was involved in mannitol recognition. The amounts of excreta in TcGr20 dsRNA-injected adults decreased significantly, despite the presence of mannitol, compared to control adults. Taken together, our results indicate that T. castaneum adults recognized mannitol/sorbitol using the TcGr20 receptor, thereby facilitating the dietary intake of these compounds.

Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells.

Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic midge embryo endure complete desiccation in an ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells could preserve an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase (Luc), preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including preincubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pv11-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pv11-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation tolerance-related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

Cry toxin specificities of insect ABCC transporters closely related to lepidopteran ABCC2 transporters.

In this study, we examined insect and human ABCC transporters closely related to the lepidopteran ABC transporter C2 (ABCC2), a powerful receptor for the Bacillus thuringiensis Cry toxin, for their responses to various Cry toxins. ABCC2 and the lepidopteran ABC transporter C3 (ABCC3) conferred cultured cells with susceptibility to a lepidopteran-specific Cry1Aa toxin but not to lepidopteran-specific Cry1Ca and Cry1Da. One coleopteran ABCC transporter specifically responded to a coleopteran-specific Cry8Ca toxin. ABCC transporters from a dipteran insect and humans did not respond to any of the tested Cry toxins that are active to lepidopteran and coleopteran insects. These results yield important information for our understanding of insect specificity of Cry toxins and provide the first demonstration of a coleopteran ABCC transporter that serves as a Cry toxin receptor.

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.

The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor.

Information about the receptor-interaction region of Cry toxins, insecticidal proteins produced by Bacillus thuringiensis, is needed to elucidate the mode of action of Cry toxins and improve their toxicity through protein engineering. We analyzed the interaction sites on Cry1Aa with ABC transporter C2 (ABCC2), one of the most important Cry1A toxin receptors. A competitive binding assay revealed that the Bombyx mori ABCC2 (BmABCC2) Cry1A binding site was the same as the BtR175 binding site, suggesting that the loop region of Cry1Aa domain II is a binding site. Next, we constructed several domain II loop mutant toxins and tested their binding affinity in an SPR analysis, and also performed a cell swelling assay to evaluate receptor-mediated cytotoxicity. Our results indicate that the loop regions required for BtR175 and BmABCC2 binding and the regions important for cytotoxicity partially overlap. Our results also suggest that receptor binding is necessary but not sufficient for cytotoxicity. This is the first report showing the region of interaction between ABCC2 and Cry1Aa and the cytotoxicity-relevant properties of the Cry1Aa domain II loop region.

Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line, Pv11.

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5-34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

Functional characterization of Bacillus thuringiensis Cry toxin receptors explains resistance in insects.

Bacillus thuringiensis produces Cry toxins, which are used as insecticides in sprays and in transgenic crops. However, little is known about the function of Cry toxin receptors and the mechanisms that determine their binding specificity and activity. In this study, the cRNAs of Bombyx mori ABC transporter C2 (BmABCC2), the toxin-binding region of cadherin-like receptor (BtR175-TBR), or aminopeptidase N1 (BmAPN1) were injected into Xenopus oocytes, and the Cry1Aa-dependent cation-selective pore formation activities of these receptors were analyzed using a two-electrode voltage clamp. Cation current passing through the pores was detected within 25 s, and increased in a linear fashion in BmABCC2-expressing oocytes treated with 88 nm Cry1Aa. This result suggested that Cry1Aa continuously made stable pores with the help of BmABCC2. In contrast, no cation current was observed until 60 min after incubation with 500 nm Cry1Aa in BtR175TBR-expressing oocytes even though oligomerization of Cry1Aa progressed. This result indicated that in the presence of BtR175-TBR most of the oligomerized toxin could not enter the cell membrane. However, oocytes that simultaneously expressed both receptors demonstrated that BtR175-TBR exerted a synergistic effect with BmABCC2 on pore formation in the presence of 22 nm Cry1Aa. These results confirm that the main reason for moderate-level resistance in insects lacking the cadherin-like receptor but expressing ABCC2 is the absence of a similar synergistic promotion of toxin oligomerization. Similar to results from our previous report evaluating ectopic expression in the Sf9/Baculovirus system, BmAPN1 could not by itself cause Cry1A-related pore formation, despite the fact that BmAPN1 gathered toxin on the oocytes as well as BmABCC2 did.

Expression of the fructose receptor BmGr9 and its involvement in the promotion of feeding, suggested by its co-expression with neuropeptide F1 in Bombyx mori.

Insect gustatory receptors (Grs) are members of a large family of proteins with seven transmembrane domains that provide insects with the ability to detect chemical signals critical for feeding, mating, and oviposition. To date, 69 Bombyx mori Grs (BmGrs) genes have been identified via genome studies. BmGr9 has been shown to respond specifically to fructose and to function as a ligand-gated ion channel selectively activated by fructose. However, the sites where this Gr are expressed remain unclear. We demonstrated using reverse transcription (RT)-PCR that BmGr9 is widely expressed in the central nervous system (CNS), as well as oral sensory organs. Additionally, immunohistochemistry was performed using anti-BmGr9 antiserum to show that BmGr9 is expressed in cells of the oral sensory organs, including the maxillary galea, maxillary palps, labrum, and labium, as well as in putative neurosecretory cells of the CNS. Furthermore, double immunohistochemical analysis showed that most BmGr9-expressing cells co-localized with putative neuropeptide F1-expressing cells in the brain, suggesting that BmGr9 is involved in the promotion of feeding behaviors. In addition, a portion of BmGr9-expressing cells in the brain co-localized with cells expressing BmGr6, a molecule of the sugar receptor clade, suggesting that sugars other than fructose are involved in the regulation of feeding behaviors in B. mori larvae.

Characterization of a ligand-gated cation channel based on an inositol receptor in the silkworm, Bombyx mori.

Insect herbivores recognize non-volatile compounds in plants to direct their feeding behavior. Gustatory receptors (Gr) appear to be required for nutrient recognition by gustatory organs in the mouthparts of insects. Gr10 is expressed in Bombyx mori (BmGr10) mouthparts such as maxillary galea, maxillary palp, and labrum. BmGr10 is predicted to function in sugar recognition; however, the precise biochemical function remains obscure. Larvae of B. mori are monophagous feeders able to find and feed on mulberry leaves. Soluble mulberry leaf extract contains sucrose, glucose, fructose, and myo-inositol. In this study, we identified BmGr10 as an inositol receptor using electrophysiological analysis with the Xenopus oocyte expression system and Ca(2+) imaging techniques using mammalian cells. These results demonstrated that Xenopus oocytes or HEK293T cells expressing BmGr10 specifically respond to myo-inositol and epi-inositol but do not respond to any mono-, di-, or tri-saccharides or to some sugar alcohols. These inositols caused Ca(2+) and Na(+) influxes into the cytoplasm independently of a G protein-mediated signaling cascade, indicating that BmGr10 is a ligand-gated cation channel. Overall, BmGr10 plays an important role in the myo-inositol recognition required for B. mori larval feeding behavior.