EFFECTS OF DIURETIC HORMONE 31, DROSOKININ,AND ALLATOSTATIN A ON TRANSEPITHELIAL K+ TRANSPORT AND CONTRACTION FREQUENCY IN THE MIDGUT AND HINDGUT OF LARVAL Drosophila melanogaster

Recent studies have identified paracrine and endocrine cells in the midgut of larval Drosophila melanogaster as well as midgut and hindgut receptors for multiple neuropeptides implicated in the control of fluid and ion balance. Although the effects of diuretic factors on fluid secretion by isolated Malpighian tubules of D. melanogaster have been examined extensively, relatively little is known about the effects of such factors on gut peristalsis or ion transport across the gut. We have measured the effects of diuretic hormone 31 (DH31), drosokinin and allatostatin A (AST‐A) on both K⁺ transport and muscle contraction frequency in the isolated gut of larval D. melanogaster. K⁺ absorption across the gut was measured using K⁺‐selective microelectrodes and the scanning ion‐selective electrode technique. Allatostatin A (AST‐A; 1 μM) increased K⁺ absorption across the anterior midgut but reduced K⁺ absorption across the copper cells and large flat cells of the middle midgut. AST‐A strongly inhibited gut contractions in the anterior midgut but had no effect on contractions of the pyloric sphincter induced by proctolin. DH31 (1 μM) increased the contraction frequency in the anterior midgut, but had no effect on K⁺ flux across the anterior, middle, or posterior midgut or across the ileum. Drosokinin (1 μM) did not affect either contraction frequency or K⁺ flux across any of the gut regions examined. Possible functions of AST‐A, DH31, and drosokinin in regulating midgut physiology are discussed.     

Diapause hormone directly stimulates the prothoracic glands of diapause larvae under juvenile hormone regulation in the bamboo borer,Omphisa fuscidentalis Hampson

Larval diapause in many lepidopteran insects is induced and maintained by high juvenile hormone (JH). In the case of the bamboo borer, Omphisa fuscidentalis, the effect of JH is the opposite: The application of juvenile hormone analog (JHA: S‐methoprene) terminates larval diapause, unlike in other insect species. Here, we analyzed the expression of JH‐receptor Met, DH‐PBAN, and Kr‐h1 in the subesophageal ganglion (SG) from October to April using semi‐quantitative polymerase chain reaction (PCR). The results show that OfMet and OfDH‐PBAN messenger RNA in the SG are mainly expressed during the larval diapause stage, while OfKr‐h1 increases during the pupal stage. Using tissue culture techniques and an enzyme‐linked immunosorbent assay (ELISA), diapause hormone (DH) was found to induce ecdysteroidogenesis in the culture medium of the prothoracic gland (PG) after incubation for 30 min with 25 ng and 50 ng of DH. Thus, DH is a novel stimulator for the PG. We identified a DHR homolog in the bamboo borer and confirmed that it is expressed in the PG. In addition, for in vitro experiments, DH increased the expression levels of OfDHR, OfEcR‐A, and ecdysone‐inducible genes in the PG. These results demonstrate that DH can function as a prothoracicotropic factor, and this function of DH might be through of DHR expressed on PG cells. Consequently, DH is one of the key factors in larval diapause break which is triggered by JH in the bamboo borer, O. fuscidentalis.     

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism

The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography‐tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α‐amylase) or other reactions (β‐1,3‐glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C‐Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase‐2 (McCHS‐2), chitinase (McCHI), and β‐N‐acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS‐2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS‐2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.     

Regulatory peptides in fruit fly midgut

Regulatory peptides were immunolocalized in the midgut of the fruit fly Drosophila melanogaster. Endocrine cells were found to produce six different peptides: allatostatins A, B and C, neuropeptide F, diuretic hormone 31, and the tachykinins. Small neuropeptide-F (sNPF) was found in neurons in the hypocerebral ganglion innervating the anterior midgut, whereas pigment-dispersing factor was found in nerves on the most posterior part of the posterior midgut. Neuropeptide-F (NPF)-producing endocrine cells were located in the anterior and middle midgut and in the very first part of the posterior midgut. All NPF endocrine cells also produced tachykinins. Endocrine cells containing diuretic hormone 31 were found in the caudal half of the posterior midgut; these cells also produced tachykinins. Other endocrine cells produced exclusively tachykinins in the anterior and posterior extemities of the midgut. Allatostatin-immunoreactive endocrine cells were present throughout the midgut. Those in the caudal half of the posterior midgut produced allatostatins A, whereas those in the anterior, middle, and first half of the posterior midgut produced allatostatin C. In the middle of the posterior midgut, some endocrine cells produced both allatostatins A and C. Allatostatin-C-immunoreactive endocrine cells were particularly prominent in the first half of the posterior midgut. Allatostatin B/MIP-immunoreactive cells were not consistently found and, when present, were only weakly immunoreactive, forming a subgroup of the allatostatin-C-immunoreactive cells in the posterior midgut. Previous work on Drosophila and other insect species suggested that (FM)RFamide-immunoreactive endocrine cells in the insect midgut could produce NPF, sNPF, myosuppressin, and/or sulfakinins. Using a combination of specific antisera to these peptides and transgenic fly models, we showed that the endocrine cells in the adult Drosophila midgut produced exclusively NPF. Although the Drosophila insulin gene Ilp3 was abundantly expressed in the midgut, Ilp3 was not expressed in endocrine cells, but in midgut muscle.     

Gene cloning and expression of cadherin in midgut of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> and its Cry1A binding region

Cadherins belong to one of the families of animal glycoproteins responsible for calcium-dependent cell-cell adhesion. Recent literatures showed that the cadherin-like in midgut of several insects served as the receptor of Bt toxin Cry1A and the variation of cadherin-like is related to insect’s resistance to Cry1A. The full-length cDNA encoding cadherin-like of Helicoverpa armigera is cloned by degenerate PCR and RACE techniques and the gene was designated as BtR-harm, which is 5581 bp in full-length, encoding 1730 amino acid residues (BtR-harm was deposited in GenBank and the accession number is AF519180). Its predicted molecular weight and isoelectric point were 195.39 kDa and 4.23, respectively. The inferred amino acid sequence includes a signal sequence, 11 cadherin repeats, a membrane-proximal region, a transmembrane region and a cytoplasmic region. Sequence analysis indicated that the deduced protein sequence was most similar to the cadherin-like from Heliothis virescens with 84.2% identity and highly similar to three other lepidopteran cadherin from Bombyx mori, Manduca sexta and Pectinophora gossypiella, with the sequence identities of 60.3.6%, 57.5% and 51.0%, respectively. The cDNA encoding cadherin gene was expressed successfully in E. coli and the recombinant proteins can bind with Cry1Ac. Truncation analysis and binding experiment of BtR-harm revealed that the Cry1A binding region was a contiguous 244-amino acid sequence, which located between amino acid 1217 and 1461. Semi-quantitative RT-PCR analysis showed that BtR-harm was highly expressed in midgut of H. armigera, very low expressed in foregut and hindgut and was not expressed in other tissues. After H. armigera producing resistance to Cry1Ac, the expression quantity of BtR-harm significantly decreased in midgut of H. armigera. It is the first confirmation that BtR-harm can function as receptor of Cry1Ac in H. armigera and the binding region was located on a contiguous 244 amino acid sequence, suggesting that the decrease of expression quantity of BtR-harm is one of the main reasons for H. armigera resistance to Cry1Ac.     

Expression analysis of chlorophyllid a binding protein a secretory,red fluorescence protein in the midgut of silkworm,Bombyx mori

Chlorophyllid a binding protein （ chbp） was recently characterized by its ability to bind the prosthetic group of chlorophylls and little information is known regarding its expression. In the present study, we found that chpb was expressed highly and exclusively in the midgut of silkworm, Bombyx mori. The expression level of chbp was very high in the newly molted fifth instar larvae followed by gradual decline in the same instar. Our results demonstrated that CHBP was a secretory protein and located mainly in the apical of midgut epithelial cells. Real-time polymerase chain reaction analysis results showed that chpb highly expressed in the anterior midgut, threefold and sixfold higher compared with that of the middle midgut and posterior midgut, respectively, and chpb expression declined in darkness. In addition, the expression of chbp was affected by high-dose virus or bacterium infection.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

<Emphasis Type="Italic">Prays oleae</Emphasis> Midgut Putative Receptor of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Vegetative Insecticidal Protein Vip3LB Differs from that of Cry1Ac Toxin

Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins.     

Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos

Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2‐dimensional electrophoresis coupled with matrix‐assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide‐induced responses in the mosquito.     

Characterization of midgut stem cell‐ and enteroblast‐specific Gal4 lines in drosophila

The homeostasis of Drosophila midgut is maintained by multipotent intestinal stem cells (ISCs), each of which gives rise to a new ISC and an immature daughter cell, enteroblast (EB), after one asymmetric cell division. In Drosophila, the Gal4‐UAS system is widely used to manipulate gene expression in a tissue‐ or cell‐specific manner, but in Drosophila midgut, there are no ISC‐ or EB‐specific Gal4 lines available. Here we report the generation and characterization of Dl‐Gal4 and Su(H)GBE‐Gal4 lines, which are expressed specifically in the ISCs and EBs separately. Additionally, we demonstrate that Dl‐Gal4 and Su(H)GBE‐Gal4 are expressed in adult midgut progenitors (AMPs) and niche peripheral cells (PCs) separately in larval midgut. These two Gal4 lines will serve as invaluable tools for navigating ISC behaviors. genesis 48:607–611, 2010. Published 2010 Wiley‐Liss, Inc.     

Proteolysis, histopathological effects, and immunohistopathological localization of delta-endotoxins of Bacillus thuringiensis subsp. kurstaki in the midgut of lepidopteran olive tree pathogenic insect Prays oleae

Considering the fact that Prays oleae is one of the most pathogenic insects to the olive tree in the Mediterranean particularly in Tunisia, the mode of action of Cry insecticidal toxins of Bacillus thuringiensis kurstaki in Prays oleae midgut was investigated. The proteolysis of Bacillus thuringiensis δ-endotoxins in the midgut was a key step in determining their potency against Prays oleae. The latter's proteases activated the δ-endotoxins early, yielding stable toxins. The in vitro and in vivo binding of these toxins to Prays oleae larvae midgut was studied immunohistochemically, evidencing a midgut columnar cell vacuolization, microvilli damage, and then a pass of epithelium cell content into the larvae midgut. Moreover, Bacillus thuringiensis toxins were shown to bind to the apical microvilli of the midgut epithelial cells. The in vitro study of the interaction of Prays oleae midgut proteins with biotinylated Bacillus thuringiensis toxins allowed the prediction of four suitable receptor proteins in Prays oleae.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

Molecular Cloning and Chromosomal Localization of the MouseGpr37Gene Encoding an Orphan G-Protein-Coupled Peptide Receptor Expressed in Brain and Testis

We report the cloning of the mouse ortholog of the humanGPR37gene, which encodes an orphan G-protein-coupled receptor highly expressed in brain tissues and homologous to neuropeptide-specific receptors ([20],Genomics 45:68–77;[45],Biochem. Biophys. Res. Commun. 233:559–567). The genomic organization of theGPR37gene is conserved in both mouse and human species with a single intron interrupting the receptor-coding sequence within the presumed third transmembrane domain. Comparative genetic mapping of theGPR37gene showed that it maps to a conserved chromosomal segment on proximal mouse chromosome 6 and human chromosome 7q31. The mouseGpr37gene contains an open reading frame coding for a 600-amino-acid protein 83% identical to the humanGPR37gene product. The predicted mouse GPR37 protein contains seven putative hydrophobic transmembrane domains, as well as a long (249 amino acid residues), arginine- and proline-rich amino-terminal extracellular domain, which is also a distinctive feature of the human GPR37 receptor. Northern blot analysis of mouse tissues withGpr37-specific probes revealed a main 3.8-kb mRNA and a much less abundant 8-kb mRNA, both expressed in the brain. A 3-kb mRNA is also expressed in the testis. Both the mouse and the humanGPR37genes may belong to a class of highly conserved mammalian genes encoding a novel type of G-protein-coupled receptor predominantly expressed in the brain.     

Vibrio cholerae laboratory infection of the adult house fly Musca domestica

The present study was designed to test the hypothesis that house flies may be capable of specifically harbouring ingested Vibrio cholerae in their digestive tracts. Flies were continuously fed green fluorescent protein (GFP)‐labelled, non‐O1/non‐O139 environmental strains of V. cholerae. Bacterial burdens were quantitatively measured using plate counts and localization was directly observed using confocal microscopy. Vibrio cholerae were present in the fly alimentary canal after just 4 h, and reached a plateau of ～10⁷ colony‐forming units (CFU)/fly after 5 days in those flies most tolerant of the pathogen. However, individual flies were resistant to the pathogen: one or more flies were found to carry < 180 V. cholerae CFU at each time‐point examined. In flies carrying V. cholerae, the pathogen was predominantly localized to the midgut rather than the rectal space or crop. The proportion of house flies carrying V. cholerae in the midgut was dose‐dependent: the continuous ingestion of a concentrated, freshly prepared dose of V. cholerae increased the likelihood that fluorescent cells would be observed. However, V. cholerae may be a transient inhabitant of the house fly. This work represents the first demonstration that V. cholerae can inhabit the house fly midgut, and provides a platform for future studies of host, pathogen and environmental mediators of the successful colonization of this disease vector.     

Gene cloning,bacterial expression,and purification of calreticulin from Anopheles stephensi (AsCRT)

During the invasion of Plasmodium ookinetes to the mosquito midgut epithelium, several proteins or glycoproteins are involved. Recent study has shown that the calreticulin (CRT) of the midgut from Anopheles albimanus can bind to the protein receptor Pvs25 on surface of Plasmodium vivax ookinetes. Thus, in order to get more insight into the potential roles of Anopheles stephensi calreticulin (AsCRT) in the midgut, we amplified and cloned the full‐length of calreticulin coding sequence from Anopheles stephensi. The AsCRT consists of 1221 bp nucleic acids with one open reading frame (ORF) encoding 406 amino acids and an apparent molecular weight around 46 KDa. Subsequently, the recombinant calreticulin as Glutathione S‐transferase (GST) fusion in pGEX ?6p‐1 expression vector (GST‐AsCRT) was produced in the prokaryotic system under optimum conditions. GST‐AsCRT fusion protein has a molecular weight around 73 KDa. The recombinant protein was detected by Western blotting using a rabbit anti‐GST polyclonal antibody. Here, we report via single protein purification procedure using MagneGST beads, 25 mg of the recombinant protein was obtained per liter of bacterial culture. This is the first report describing the heterologous expression of Anopheles stephensi calreticulin in the prokaryotic system. The production of this recombinant protein will now allow us to further investigate AsCRT molecular protein analyses, characterization of physiochemical properties, as well as interaction between calreticulin and plasmodium protein surface.