In JoVE (1)

Other Publications (35)

Articles by J. Joe Hull in JoVE

Other articles by J. Joe Hull on PubMed

Identification of a Potent Antidiuretic Factor Acting on Beetle Malpighian Tubules

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2002  |  Pubmed ID: 11756661

Beetles, like other insects, depend on diuretic and antidiuretic hormones to control water balance. We have isolated, using head extracts from the beetle Tenebrio molitor, a peptide that strongly inhibits fluid secretion by the Malpighian tubules of this insect. This antidiuretic factor (ADF) appears to elicit its effect via cGMP as a second messenger but does not stimulate NO production. It has primary structure: Val-Val-Asn-Thr-Pro-Gly-His-Ala-Val-Ser-Tyr-His-Val-Tyr-OH. The ADF inhibits tubule secretion with high potency: the EC(50) is around 10 fM. It bears no significant resemblance to other biologically active neuropeptides. To our knowledge this is the only endogenous insect ADF acting on Malpighian tubules to be sequenced, and the first coleopteran (beetle) antidiuretic factor fully characterized to date.

Isolation, Identification and Localization of a Second Beetle Antidiuretic Peptide

Peptides. Jan, 2003  |  Pubmed ID: 12576082

We isolated from head extracts of Tenebrio molitor a peptide that inhibits fluid secretion by the Malpighian tubules of this insect. This second antidiuretic factor, ADFb, like the previously published ADFa, works through cyclic GMP as a second messenger. It has primary structure Tyr-Asp-Asp-Gly-Ser-Tyr-Lys-Pro-His-Ile-Tyr-Gly-Phe-OH with an EC(50) of approximately 240 pM in a fluid secretion assay. This peptide is now the second sequenced endogenous insect ADF which inhibits Malpighian tubule fluid secretion. Immunohistochemical techniques show that the peptide is localized in the brain; it appears to be produced mainly in two pairs of bilaterally symmetrical cells in the protocerebrum.

Involvement of a Bifunctional Fatty-acyl Desaturase in the Biosynthesis of the Silkmoth, Bombyx Mori, Sex Pheromone

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2004  |  Pubmed ID: 15173596

The straight-chain C(10) to C(18) unsaturated aliphatic compounds containing an oxygenated functional group (aldehyde, alcohol, or acetate ester) derived from saturated C(16) or C(18) fatty acids are a major class of sex pheromone components produced by female moths. In the biosynthesis of these pheromone components, various combinations of limited chain-shortening and regio- and stereospecific desaturation reactions significantly contribute to the production of a vast number of the species-specific pheromone components in Lepidoptera. Biosynthesis of the silkmoth sex pheromone bombykol, (E,Z)-10,12-hexadecadien-1-ol, involves two consecutive desaturation steps, the second of which is unique in that it generates a conjugated diene system from the Delta11-monoene C(16) intermediate. In experiments designed to characterize the acyl-CoA desaturases responsible for bombykol biosynthesis, we have cloned three cDNAs encoding desaturase family members from the pheromone gland of the inbred strain of the silkmoth, Bombyx mori. Transcript analyses by RT-PCR and subsequent functional assays using a Bac-to-Bac baculovirus expression system revealed that desat1 is the only desaturase gene prominently expressed during pheromonogenesis and that its gene product, B. mori Desat1, possesses both Z11 desaturation and Delta10,12-desaturation activities. Consequently, we have concluded that B. mori Desat1 is not only a bifunctional desaturase involved in bombykol biosynthesis but that it is also the enzyme responsible for both desaturation steps.

Cloning and Characterization of the Pheromone Biosynthesis Activating Neuropeptide Receptor from the Silkmoth, Bombyx Mori. Significance of the Carboxyl Terminus in Receptor Internalization

The Journal of Biological Chemistry. Dec, 2004  |  Pubmed ID: 15358772

In most Lepidoptera, pheromone biosynthesis is regulated by a neuropeptide termed pheromone biosynthesis activating neuropeptide (PBAN). Although much is known about the cellular targets of PBAN, identification and functional characterization of the PBAN receptor (PBANR) has proven to be elusive. Given the sequence similarity between the active C-terminal regions of PBAN and neuromedin U, it was hypothesized that their respective receptors might also be similar in structure (Park, Y., Kim, Y. J., and Adams, M. E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11423-11428). Consequently, utilizing primers constructed from the conserved regions of insect neuromedin U receptor homologues, a full-length 2780-nucleotide clone encoding a 46-kDa G protein-coupled receptor was amplified from a Bombyx mori pheromone gland cDNA library. Tissue distribution analyses revealed that the receptor transcript is specific to the pheromone gland where it undergoes significant up-regulation in the day preceding eclosion. When transiently expressed in Sf9 cells, the B. mori PBANR responds to PBAN by mobilizing extracellular calcium in a dose-dependent manner. Confocal microscopic studies demonstrated the specificity of enhanced green fluorescent protein-tagged B. mori PBANR for PBAN and showed that PBAN induces internalization of the PBANR.PBAN complex. The rapid onset of internalization is mediated by a 67-amino acid C-terminal extension absent in the cloned Helicoverpa zea PBANR, which suggests that receptor internalization in that species likely utilizes a different mechanism. From these results, we have concluded that the cloned receptor gene encodes the B. mori PBANR and that it is both structurally and functionally distinct from the H. zea PBANR.

Regulatory Mechanisms Underlying Pheromone Biosynthesis Activating Neuropeptide (PBAN)-induced Internalization of the Bombyx Mori PBAN Receptor

Biochemical and Biophysical Research Communications. Aug, 2005  |  Pubmed ID: 15992769

Internalization of the Bombyx mori pheromone biosynthesis activating neuropeptide receptor (PBANR) has been attributed to the presence of a 67 amino acid C-terminal extension absent in PBANRs from Helicoverpa. To identify the structural motif(s) responsible for internalization, a series of truncation mutants fused with enhanced green fluorescent protein were constructed and transiently expressed in insect Sf9 cells. Confocal microscopy analyses revealed that truncation at Gly357 severely inhibited internalization while truncation at Gln367 did not, indicating that the PBANR internalization motif resides between Gly357-Gln367. Alanine substitution studies suggest that Tyr360 and Leu363 may constitute a YXXL endosomal targeting motif that facilitates endocytosis, however, this motif does not appear to be the primary determinant; an indication that multiple sites are involved. Furthermore, we determined that internalization of the PBANR proceeds via a clathrin-dependent pathway, is dependent on the influx of extracellular calcium, and likely does not involve a G protein-coupled receptor kinase.

Targeted Disruption of Genes in the Bombyx Mori Sex Pheromone Biosynthetic Pathway

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2006  |  Pubmed ID: 16537410

The sex pheromone biosynthetic pathways of lepidopterans require the concerted actions of multiple gene products. A number of pheromone gland (PG)-specific genes have been cloned in recent years and, whereas in vitro characterizations have indicated functions consistent with roles in pheromone production, there have been no clear demonstrations in vivo. Using an RNA interference-mediated loss-of-function approach, we injected newly formed Bombyx mori pupae with dsRNAs corresponding to genes of interest [i.e., PG fatty acyl reductase (pgFAR), B. mori PG Z11/Delta10,12 desaturase (Bmpgdesat1), PG acyl-CoA-binding protein (pgACBP), midgut ACBP, and pheromone biosynthesis activating neuropeptide receptor (PBANR)] to assess their specific roles during pheromonogenesis. In all cases, the introduced dsRNAs induced a dose-dependent reduction in sex pheromone production with the corresponding decrease in transcript levels. No effects on pupal development or adult emergence were observed. Disrupting the PBANR gene resulted in a loss of the lipase activity that liberates pheromone precursors, whereas knockout of the pgACBP gene prevented the daily accumulation and fluctuation of the triacylglycerols that function as the cellular deposits for the pheromone precursors. Taken together, our results provide unequivocal evidence that the pgACBP, Bmpgdesat1, pgFAR, and PBANR gene products are essential during pheromonogenesis and demonstrate the power of this methodology for dissecting the molecular interactions that comprise biosynthetic pathways.

Molecular Mechanisms Underlying Sex Pheromone Production in the Silkmoth, Bombyx Mori: Characterization of the Molecular Components Involved in Bombykol Biosynthesis

Journal of Insect Physiology. Aug, 2007  |  Pubmed ID: 17448494

Many species of female moths produce sex pheromones to attract conspecific males. To date, sex pheromones from more than 570 moth species have been chemically identified. Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester and usually with several double bonds. In contrast, some moth species use unsaturated hydrocarbons or hydrocarbon epoxides, classified as Type II lepidopteran pheromones, as sex pheromones. Studies over the past three decades have demonstrated that female moths usually produce sex pheromones as multi-component blends where the ratio of the individual components is precisely controlled, thus making it possible to generate species-specific pheromone blends. As for the biosynthesis of Type I pheromones, it is well established that they are de novo synthesized in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways. However, as many of the molecular components within the PG cells (i.e., enzymes, proteins, and small regulatory molecules) have not been functionally characterized, the molecular mechanisms underlying sex pheromone production in PG cells remain poorly understood. To address this, we have recently characterized some of the molecules involved in the biosynthesis of the sex pheromone bombykol in the silkmoth, Bombyx mori. Characterization of these, and other, key molecules will facilitate our understanding of the precise mechanisms underlying lepidopteran sex pheromone production.

The Bombyx Mori Sex Pheromone Biosynthetic Pathway is Not Mediated by CAMP

Journal of Insect Physiology. Aug, 2007  |  Pubmed ID: 17449058

In most moths, sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). How the extracellular PBAN signal is turned into a biological response has been the focus of numerous studies. In the classical scheme of signal transduction, activated G proteins relay the extracellular signal to downstream effector molecules such as calcium channels and adenylyl cyclase. The role of calcium in PBAN signaling has been clearly demonstrated, but the possible involvement of cAMP is not as straightforward. While cAMP has been shown to be necessary for PBAN signaling in most heliothine species, there has been no definitive demonstration of its role in Bombyx mori. To address this question, we used degenerate RT-PCR to clone two Gs subunits, designated P50Gs1 and P50Gs2, from B. mori pheromone gland (PG) cDNAs. The two Gs proteins were expressed in all tissues examined and were not up-regulated in accordance with adult eclosion. Even though two bands corresponding to the approximate molecular weights of P50Gs1 and P50Gs2 were detected in PG homogenates, the Gs antagonist, NF449, had no effect on sex pheromone production. Furthermore, no changes in the intracellular cAMP levels were detected following PBAN stimulation.

FXPRL-amide Peptides Induce Ecdysteroidogenesis Through a G-protein Coupled Receptor Expressed in the Prothoracic Gland of Bombyx Mori

Molecular and Cellular Endocrinology. Jul, 2007  |  Pubmed ID: 17590269

The FXPRL-amide peptide family (pyrokinin/PBAN family) consists of insect peptides that function broadly in insect life processes and are characterized by a conserved C-terminal motif. In the silkworm, Bombyx mori, sex pheromone biosynthesis and induction of embryonic diapause are regulated by peptides from this family. To elucidate other functions of Bombyx FXPRL-amide peptides, we analyzed the tissue expression patterns of two known Bombyx G-protein coupled receptors for these peptides. We found that the Bombyx diapause hormone receptor (BmDHR), is expressed in the prothoracic gland (PG), the organ which synthesizes and releases the insect molting hormones, ecdysteroids. Furthermore, diapause hormone (DH), a member of the Bombyx FXPRL-amide peptides, increases both intracellular Ca(2+) and cAMP concentrations and induces ecdysteroidogenesis in late fifth instar PGs coincident with BmDHR expression in the PGs. DH also has the highest prothoracicotropic activity among the FXPRL-amide peptides, which corresponds well to the ligand specificity of heterologously expressed BmDHR. These results demonstrate that FXPRL-amide peptides can function as prothoracicotropic factors through the activation of BmDHR and may play an important role in controlling molting and metamorphosis.

Sex Pheromone Production in the Silkworm, Bombyx Mori, is Mediated by Store-operated Ca2+ Channels

Bioscience, Biotechnology, and Biochemistry. Aug, 2007  |  Pubmed ID: 17690463

In most female moths, pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production by stimulating an influx of extracellular Ca(2+). Little is known about the plasma membrane channel or how the PBAN stimulus is communicated to the channel. Fluorescent Ca(2+) imaging techniques confirmed PBAN-induced Ca(2+) influx in the silkworm, Bombyx mori, and showed that the PBAN response is reduced with repeated stimulation. Compounds known to impact Ca(2+) signaling were examined for their effects on sex pheromone production. These experiments demonstrated that the PBAN signal is likely mediated by a store-operated channel (SOC). SOC blockers, SKF-96365 and 2-aminoethoxydiphenyl borate, abolished sex pheromone production, as did flufenamic acid, a blocker of transient receptor potential (TRP) channels. Thapsigargin mimicked the pheromonotropic effects of PBAN. Similar results were seen when PBAN-induced lipase activity was assayed. Conversely, 1-oleoyl-2-acetyl-sn-glycerol and arachidonic acid, activators of diacylglycerol-dependent Ca(2+) channels, had no effect on bombykol production.

[Mating Behavior and Pheromone Production in Moths]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Oct, 2007  |  Pubmed ID: 18051399

Molecular Mechanisms Underlying PBAN Signaling in the Silkmoth Bombyx Mori

Annals of the New York Academy of Sciences. Apr, 2009  |  Pubmed ID: 19456388

Pheromone biosynthesis in the silkmoth Bombyx mori is under the control of the neurohormone pheromone biosynthesis activating neuropeptide (PBAN) and is triggered upon PBAN binding to its cognate PBAN receptor on the pheromone gland cells of female moths. Using fluorescent Ca(2+) imaging techniques with isolated pheromone glands, we have successfully demonstrated that PBAN specifically evokes an influx of extracellular Ca(2+). Furthermore, results from experiments designed to elucidate the molecular mechanisms underlying PBAN signaling indicate that B. mori utilizes the canonical store-operated channel-activation pathway and that the influx of extracellular Ca(2+) accelerates both lipolysis and fatty acyl reduction through a phosphorylation/dephosphorylation cascade for bombykol production.

De Novo Molecular Modeling and Biophysical Characterization of Manduca Sexta Eclosion Hormone

Biochemistry. Sep, 2009  |  Pubmed ID: 19670911

Eclosion hormone (EH) is an integral component in the cascade regulating the behaviors culminating in emergence of an insect from its old exoskeleton. Little is known regarding the EH solution structure; consequently, we utilized a computational approach to generate a hypothetical structure for Manduca sexta EH. The de novo algorithm exploited the restricted conformational space of disulfide bonds (Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49) and predicted secondary structure elements to generate a thermodynamically stable structure characterized by 55% helical content, an unstructured N-terminus, a helical C-terminus, and a solvent-exposed loop containing Trp28 and Phe29. Both the strain and pseudo energies of the predicted peptide compare favorably with those of known structures. The 62-amino acid peptide was synthesized, folded, assayed for activity, and structurally characterized to confirm the validity of the model. The helical content is supported by circular dichroism and hydrogen-deuterium exchange mass spectrometry. Fluorescence emission spectra and acrylamide quenching are consistent with the solvent exposure predicted for Trp28, which is shielded by Phe29. Furthermore, thermodynamically stable conformations that deviated only slightly from the predicted Manduca EH structure were generated in silico for the Bombyx mori and Drosophila melanogaster EHs, indicating that the conformation is not species-dependent. In addition, the biological activities of known mutants and deletion peptides were rationalized with the predicted Manduca EH structure, and we found that, on the basis of sequence conservation, functionally important residues map to two conserved hydrophobic clusters incorporating the C-terminus and the first loop.

Bombyx Mori Homologs of STIM1 and Orai1 Are Essential Components of the Signal Transduction Cascade That Regulates Sex Pheromone Production

The Journal of Biological Chemistry. Nov, 2009  |  Pubmed ID: 19740753

Sex pheromone production in the pheromone gland (PG) of the silkmoth, Bombyx mori, is mediated by store-operated channels (SOCs) acting downstream of pheromone biosynthesis activating neuropeptide (PBAN) binding. Although recent studies have implicated STIM1 and Orai1 as essential components of SOCs, little is known about the molecular nature of the SOCs involved in sex pheromone production. In this study we cloned silkmoth homologs of STIM1 and Orai1 and sought to determine whether they comprise the PG SOC pathway. BmSTIM1 is expressed in multiple tissues and, in the PG, is encoded by two transcripts of differing size. BmOrai1A and BmOrai1B, which are identical except for a 37-residue N-terminal truncation in BmOrai1B, arise from alternative splicing of the bmorai1 locus and are expressed as independent transcripts in various tissues. In the PG, only BmOrai1B is actively transcribed. Fluorescent chimeras demonstrated that BmSTIM1 expression is restricted to the endoplasmic reticulum, whereas both BmOrai1A and BmOrai1B localize to the cell surface. In Ca(2+)-free medium, thapsigargin-mediated depletion of endoplasmic reticulum Ca(2+) stores resulted in redistribution of BmSTIM1 to the plasma membrane, but only when the BmOrai1 homologs were also overexpressed. Translocation was dependent on the BmSTIM1 C terminus "CRAC activation domain." Ala mutation of Lys(380), Lys(383), Lys(384), Arg(382), and Arg(385) suggests that translocation involves electrostatic interactions. Translocation was also seen following PBAN stimulation in cells co-expressing BmSTIM1, BmOrai1B, and the PBAN receptor. In vivo RNA interference-mediated knockdown of BmSTIM1 and BmOrai1 significantly reduced sex pheromone production without affecting cell viability.

Functional Role of STIM1 and Orai1 in Silkmoth (Bombyx Mori) Sex Pheromone Production

Communicative & Integrative Biology. May, 2010  |  Pubmed ID: 20714403

Store-operated Ca(2+) influx has recently been shown to require the activation of two proteins, stromal interaction molecule 1 (STIM1) and Orai1. In mammals the putative channel ion selectivity filter is thought to comprise conserved charged residues in the first and third transmembrane domains of Orai1 in addition to three residues in the first extracellular loop. The latter residues, however, are not conserved in either of the Bombyx mori Orai1 variants or in most insects, suggesting that selectivity is a relatively recent evolutionary event. In B. mori, thapsigargin-mediated STIM1 redistribution is dependent on a cluster of highly conserved basic residues (amino acids 380-385) in the C terminus that likely interact with acidic residues in the Orai1 C terminus. BmSTIM1 redistribution in vitro also occurs downstream of pheromone biosynthesis activating neuropeptide receptor activation. Activation of in vivo RNA interference mechanisms confirmed the physiological role of BmSTIM1 and Orai1 in sex pheromone production.

Unraveling the Pheromone Biosynthesis Activating Neuropeptide (PBAN) Signal Transduction Cascade That Regulates Sex Pheromone Production in Moths

Vitamins and Hormones. 2010  |  Pubmed ID: 20831957

Studies over the past three decades have demonstrated that female moths usually produce sex pheromones as multicomponent blends in which the ratios of the individual components are precisely controlled, making it possible to generate species-specific pheromone blends. Most moth pheromone components are de novo synthesized from acetyl-CoA in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways. Pheromone biosynthesis activating neuropeptide (PBAN), a neurohormone produced by a cephalic organ (subesophageal ganglion) stimulates sex pheromone biosynthesis in the PG via an influx of extracellular Ca(2+). In recent years, we have expanded our knowledge of the precise mechanisms underlying silkmoth (Bombyx mori) sex pheromone production by characterizing a number of key molecules. In this review, we want to highlight our efforts in elucidating these mechanisms in B. mori and to understand how they relate more broadly to lepidopteran sex pheromone production in general.

RNA Interference in Lepidoptera: an Overview of Successful and Unsuccessful Studies and Implications for Experimental Design

Journal of Insect Physiology. Feb, 2011  |  Pubmed ID: 21078327

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

Hormone Signaling Linked to Silkmoth Sex Pheromone Biosynthesis Involves Ca2+/calmodulin-dependent Protein Kinase II-mediated Phosphorylation of the Insect PAT Family Protein Bombyx Mori Lipid Storage Droplet Protein-1 (BmLsd1)

The Journal of Biological Chemistry. Jul, 2011  |  Pubmed ID: 21572162

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via the fatty acid biosynthetic pathway. This pathway is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets, which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells. PBAN stimulates lipolysis of the stored lipid droplet triacylglycerols (TAGs) and releases the precursor for final modification. PBAN exerts its physiological function via the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family. The PBAN receptor-mediated signal is transmitted via a canonical store-operated channel activation pathway utilizing Gq-mediated phospholipase C activation (Hull, J. J., Kajigaya, R., Imai, K., and Matsumoto, S. (2007) Biosci. Biotechnol. Biochem. 71, 1993-2001; Hull, J. J., Lee, J. M., Kajigaya, R., and Matsumoto, S. (2009) J. Biol. Chem. 284, 31200-31213; Hull, J. J., Lee, J. M., and Matsumoto, S. (2010) Insect Mol. Biol. 19, 553-566). Little, however, is known about the molecular components regulating TAG lipolysis in PG cells. In the current study we found that PBAN signaling involves phosphorylation of an insect PAT family protein named B. mori lipid storage droplet protein-1 (BmLsd1) and that BmLsd1 plays an essential role in the TAG lipolysis associated with bombykol production. Unlike mammalian PAT family perilipins, however, BmLsd1 activation is dependent on phosphorylation by B. mori Ca(2+)/calmodulin-dependent protein kinase II rather than protein kinase A.

Silkworms Transformed with Chimeric Silkworm/spider Silk Genes Spin Composite Silk Fibers with Improved Mechanical Properties

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2012  |  Pubmed ID: 22215590

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

Re-Evaluation of the PBAN Receptor Molecule: Characterization of PBANR Variants Expressed in the Pheromone Glands of Moths

Frontiers in Endocrinology. 2012  |  Pubmed ID: 22654850

Sex pheromone production in most moths is initiated following pheromone biosynthesis activating neuropeptide receptor (PBANR) activation. PBANR was initially cloned from pheromone glands (PGs) of Helicoverpa zea and Bombyx mori. The B. mori PBANR is characterized by a relatively long C-terminus that is essential for ligand-induced internalization, whereas the H. zea PBANR has a shorter C-terminus that lacks features present in the B. mori PBANR critical for internalization. Multiple PBANRs have been reported to be concurrently expressed in the larval CNS of Heliothis virescens. In the current study, we sought to examine the prevalence of multiple PBANRs in the PGs of three moths and to ascertain their potential functional relevance. Multiple PBANR variants (As, A, B, and C) were cloned from the PGs of all species examined with PBANR-C the most highly expressed. Alternative splicing of the C-terminal coding sequence of the PBAN gene gives rise to the variants, which are distinguishable only by the length and composition of their respective C-terminal tails. Transient expression of fluorescent PBANR chimeras in insect cells revealed that PBANR-B and PBANR-C localized exclusively to the cell surface while PBANR-As and PBANR-A exhibited varying degrees of cytosolic localization. Similarly, only the PBANR-B and PBANR-C variants underwent ligand-induced internalization. Taken together, our results suggest that PBANR-C is the principal receptor molecule involved in PBAN signaling regardless of moth species. The high GC content of the C-terminal coding sequence in the B and C variants, which makes amplification using conventional polymerases difficult, likely accounts for previous "preferential" amplification of PBANR-A like receptors from other species.

Establishment of Sf9 Transformants Constitutively Expressing PBAN Receptor Variants: Application to Functional Evaluation

Frontiers in Endocrinology. 2012  |  Pubmed ID: 22654874

To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines constitutively expressing green fluorescent protein chimeras of the recently identified Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants. Fluorescent chimeras included the BommoPBANR-A, -B, and -C variants and the PsesePBANR-B and -C variants. Cell lines expressing non-chimeric BommoPBANR-B and -C variants were also generated. Functional evaluation of these transformed cell lines using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBAN(R2K)) specifically co-localized with all of the respective PBANR variants at the plasma membrane. Near complete internalization of the fluorescent RR-C10PBAN(R2K) ligand 30 min after binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca(2+) imaging further showed that the BommoPBANR-A cell line exhibited drastically different Ca(2+) mobilization kinetics at a number of RR-C10PBAN(R2K) concentrations including 10 μM. These observations demonstrate a clear functional difference between the BommoPBANR-A variant and the BommoPBANR-B and -C variants in terms of receptor regulation and activation of downstream effector molecules. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca(2+).

IDENTIFICATION OF THE WESTERN TARNISHED PLANT BUG (Lygus Hesperus) OLFACTORY CO-RECEPTOR ORCO: EXPRESSION PROFILE AND CONFIRMATION OF ATYPICAL MEMBRANE TOPOLOGY

Archives of Insect Biochemistry and Physiology. Jul, 2012  |  Pubmed ID: 22836832

Lygus hesperus (western tarnished plant bug) is an agronomically important pest species of numerous cropping systems. Similar to other insects, a critical component underlying behaviors is the perception and discrimination of olfactory cues. Consequently, the molecular basis of olfaction in this species is of interest. To begin to address this issue, we utilized homology-based PCR as a commonly accepted abbreviation but if necessary it is polymerase chain reaction methods to identify the L. hesperus olfactory receptor co-receptor (Orco) ortholog, a receptor that has been shown to be essential for olfaction. The L. hesperus Orco (LhOrco) shares significant sequence homology with known Orco proteins in other insects. Parallel experiments using the sympatric sister species, Lygus lineolaris (tarnished plant bug), revealed that the Lygus Orco gene was completely conserved. Surprisingly, a majority of the membrane topology prediction algorithms used in the study predicted LhOrco to have both the N and C terminus intracellular. In vitro immunofluorescent microscopy experiments designed to probe the membrane topology of transiently expressed LhOrco, however, refuted those predictions and confirmed that the protein adopts the inverted topology (intracellular N terminus and an extracellular C terminus) characteristic of Orco proteins. RT-PCR analyses indicated that LhOrco transcripts are predominantly expressed in adult antennae and to a lesser degree in traditionally nonolfactory chemosensory tissues of the proboscis and legs. Expression is not developmentally regulated because transcripts were detected in all nymphal stages as well as eggs. Taken together, the results suggest that LhOrco likely plays a critical role in mediating L. hesperus odorant perception and discrimination.

Sequencing and De Novo Assembly of the Western Tarnished Plant Bug (Lygus Hesperus) Transcriptome

PloS One. 2013  |  Pubmed ID: 23357950

Mirid plant bugs (Hemiptera: Miridae) are economically important insect pests of many crops worldwide. The western tarnished plant bug Lygus hesperus Knight is a pest of cotton, alfalfa, fruit and vegetable crops, and potentially of several emerging biofuel and natural product feedstocks in the western US. However, little is known about the underlying molecular genetics, biochemistry, or physiology of L. hesperus, including their ability to survive extreme environmental conditions.

Molecular Mechanisms Underlying Insect Behaviors: Receptors, Peptides, and Biosynthetic Pathways

Frontiers in Endocrinology. 2013  |  Pubmed ID: 24062724

Characterization of Male-derived Factors Inhibiting Female Sexual Receptivity in Lygus Hesperus

Journal of Insect Physiology. Jan, 2014  |  Pubmed ID: 24333151

Newly mated females of the plant bug, Lygus hesperus Knight, enter a refractory period during which their sexual receptivity to courting males is greatly reduced for several days. This behavioral change appears to be induced by male-derived factors delivered in the spermatophore during copulation. To better understand the source of the factor(s) responsible for the inhibition, the homogenates of spermatophores, or of the individual organs that provide the constituents of the spermatophore, were injected directly into the abdomen of virgin females. The contents of the lateral and medial accessory glands both appear to produce inhibitory effects, but those of the seminal vesicle had no effect. Treatment of the homogenate also indicated that the active factor(s) is heat labile and water soluble. Several unique proteins were found in the water soluble fraction of the spermatophore, one of which is similar in size to the Drosophila melanogaster sex peptide, a male derived compound known to inhibit receptivity in female flies. In addition, spermatophores contained a substantial quantity of juvenile hormone, a key endocrine regulator of reproductive behavior and physiology in most insects. The results support the hypothesized role of males in manipulating the post-mating behavior of females, and suggest this is achieved through multiple components that act in concert to induce both short- and long-term effects.

Molecular and Functional Characterization of Multiple Aquaporin Water Channel Proteins from the Western Tarnished Plant Bug, Lygus Hesperus

Insect Biochemistry and Molecular Biology. Feb, 2014  |  Pubmed ID: 24333473

Aquaporins (AQPs) are integral membrane channel proteins that facilitate the bidirectional transfer of water or other small solutes across biological membranes involved in numerous essential physiological processes. In arthropods, AQPs belong to several subfamilies, which contribute to osmoregulation, respiration, cryoprotection, anhydrobiosis, and excretion. We cloned and characterized five novel AQPs from the western tarnished plant bug, Lygus hesperus, a polyphagous insect pest of food and fiber crops throughout western North America. The L. hesperus AQPs (LhAQP1-5) belong to different phylogenetic subfamilies, have unique transcription profiles and cellular localizations, and all transport water (but not glycerol) when heterologously expressed in Xenopus laevis oocytes. Our results demonstrate that multiple AQPs with possible compensatory functions are produced in L. hesperus that likely play important roles in maintaining water homeostasis in this important insect pest.

Identification of Functionally Important Residues of the Silkmoth Pheromone Biosynthesis-activating Neuropeptide Receptor, an Insect Ortholog of the Vertebrate Neuromedin U Receptor

The Journal of Biological Chemistry. Jul, 2014  |  Pubmed ID: 24847080

The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBAN(R2K)) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca(2+) mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca(2+) mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide.

Transcriptome-based Identification of ABC Transporters in the Western Tarnished Plant Bug Lygus Hesperus

PloS One. 2014  |  Pubmed ID: 25401762

ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate diverse physiological functions by coupling ATP hydrolysis with substrate transport across lipid membranes. In insects, these proteins play roles in metabolism, development, eye pigmentation, and xenobiotic clearance. While ABC transporters have been extensively studied in vertebrates, less is known concerning this superfamily in insects, particularly hemipteran pests. We used RNA-Seq transcriptome sequencing to identify 65 putative ABC transporter sequences (including 36 full-length sequences) from the eight ABC subfamilies in the western tarnished plant bug (Lygus hesperus), a polyphagous agricultural pest. Phylogenetic analyses revealed clear orthologous relationships with ABC transporters linked to insecticide/xenobiotic clearance and indicated lineage specific expansion of the L. hesperus ABCG and ABCH subfamilies. The transcriptional profile of 13 LhABCs representative of the ABCA, ABCB, ABCC, ABCG, and ABCH subfamilies was examined across L. hesperus development and within sex-specific adult tissues. All of the transcripts were amplified from both reproductively immature and mature adults and all but LhABCA8 were expressed to some degree in eggs. Expression of LhABCA8 was spatially localized to the testis and temporally timed with male reproductive development, suggesting a potential role in sexual maturation and/or spermatozoa protection. Elevated expression of LhABCC5 in Malpighian tubules suggests a possible role in xenobiotic clearance. Our results provide the first transcriptome-wide analysis of ABC transporters in an agriculturally important hemipteran pest and, because ABC transporters are known to be important mediators of insecticidal resistance, will provide the basis for future biochemical and toxicological studies on the role of this protein family in insecticide resistance in Lygus species.

Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus Hesperus

Insects. Dec, 2014  |  Pubmed ID: 26463065

The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits.

De Novo Construction of an Expanded Transcriptome Assembly for the Western Tarnished Plant Bug, Lygus Hesperus

GigaScience. 2016  |  Pubmed ID: 26823975

The plant bug Lygus hesperus Knight is a polyphagous pest of many economically important crops. Despite its pest status, little is known about the molecular mechanisms responsible for much of the biology of this species. Earlier Lygus transcriptome assemblies were limited by low read depth, or because they focused on specific conditions. To generate a more comprehensive transcriptome, we supplemented previous datasets with new reads corresponding to specific tissues (heads, antennae, and male reproductive tissues). This transcriptome augments current Lygus molecular resources and provides the foundational knowledge critical for future comparative studies.

Transcriptome Analysis Reveals a Comprehensive Insect Resistance Response Mechanism in Cotton to Infestation by the Phloem Feeding Insect Bemisia Tabaci (whitefly)

Plant Biotechnology Journal. Oct, 2016  |  Pubmed ID: 26923339

The whitefly (Bemisia tabaci) causes tremendous damage to cotton production worldwide. However, very limited information is available about how plants perceive and defend themselves from this destructive pest. In this study, the transcriptomic differences between two cotton cultivars that exhibit either strong resistance (HR) or sensitivity (ZS) to whitefly were compared at different time points (0, 12, 24 and 48 h after infection) using RNA-Seq. Approximately one billion paired-end reads were obtained by Illumina sequencing technology. Gene ontology and KEGG pathway analysis indicated that the cotton transcriptional response to whitefly infestation involves genes encoding protein kinases, transcription factors, metabolite synthesis, and phytohormone signalling. Furthermore, a weighted gene co-expression network constructed from RNA-Seq datasets showed that WRKY40 and copper transport protein are hub genes that may regulate cotton defenses to whitefly infestation. Silencing GhMPK3 by virus-induced gene silencing (VIGS) resulted in suppression of the MPK-WRKY-JA and ET pathways and lead to enhanced whitefly susceptibility, suggesting that the candidate insect resistant genes identified in this RNA-Seq analysis are credible and offer significant utility. Taken together, this study provides comprehensive insights into the cotton defense system to whitefly infestation and has identified several candidate genes for control of phloem-feeding pests.

ECDYSTEROID AND CHITINASE FLUCTUATIONS IN THE WESTERN TARNISHED PLANT BUG (Lygus Hesperus) PRIOR TO MOLT INDICATE ROLES IN DEVELOPMENT

Archives of Insect Biochemistry and Physiology. Jun, 2016  |  Pubmed ID: 27192063

Vital physiological processes that drive the insect molt represent areas of interest for the development of alternative control strategies. The western tarnished plant bug (Lygus hesperus Knight) is a pest of numerous agronomic and horticultural crops but the development of novel control approaches is impeded by limited knowledge of the mechanisms regulating its molt. To address this deficiency, we examined the fundamental relationship underlying the hormonal and molecular components of ecdysis. At 27°C L. hesperus exhibits a temporally controlled nymph-adult molt that occurs about 4 days after the final nymph-nymph molt with ecdysteroid levels peaking 2 days prior to the final molt. Application of exogenous ecdysteroids when endogenous levels had decreased disrupted the nymphal-adult molt, with treated animals exhibiting an inability to escape the old exoskeleton and resulting in mortality compared to controls. Using accessible transcriptomic data, we identified 10 chitinase-like sequences (LhCht), eight of which had protein motifs consistent with chitinases. Phylogenetic analyses revealed orthologous relationships to chitinases critical to molting in other insects. RT-PCR based transcript profiling revealed that expression changes to four of the LhChts was coordinated with the molt period and ecdysteroid levels. Collectively, our results support a role for ecdysteroid regulation of the L. hesperus molt and suggest that cuticle clearance is mediated by LhCht orthologs of chitinases that are essential to the molt process. These results provide the initial hormonal and molecular basis for future studies to investigate the specific roles of these components in molting.

A Class-A GPCR Solubilized Under High Hydrostatic Pressure Retains Its Ligand Binding Ability

Biochimica Et Biophysica Acta. Sep, 2016  |  Pubmed ID: 27342372

The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200MPa) at room temperature for 1-16h in the presence of 0-2.0% (w/v) n-dodecyl-β-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~1.5-fold after 1h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.

Molecular and Functional Characterization of Bemisia Tabaci Aquaporins Reveals the Water Channel Diversity of Hemipteran Insects

Insect Biochemistry and Molecular Biology. Oct, 2016  |  Pubmed ID: 27491441

The Middle East-Asia Minor 1 (MEAM1) whitefly, Bemisia tabaci (Gennadius) is an economically important pest of food, fiber, and ornamental crops. This pest has evolved a number of adaptations to overcome physiological challenges, including 1) the ability to regulate osmotic stress between gut lumen and hemolymph after imbibing large quantities of a low nitrogen, sugar-rich liquid diet; 2) the ability to avoid or prevent dehydration and desiccation, particularly during egg hatching and molting; and 3) to be adapted for survival at elevated temperatures. One superfamily of proteins involved in the maintenance of fluid homeostasis in many organisms includes the aquaporins, which are integral membrane channel proteins that aid in the rapid flux of water and other small solutes across biological membranes. Here, we show that B. tabaci has eight aquaporins (BtAqps), of which seven belong to the classical aquaporin 4-related grade of channels, including Bib, Drip, Prip, and Eglps and one that belongs to the unorthodox grade of aquaporin 12-like channels. B. tabaci has further expanded its repertoire of water channels through the expression of three BtDrip2 amino-terminal splice variants, while other hemipteran species express amino- or carboxyl-terminal isoforms of Drip, Prip, and Eglps. Each BtAqp has unique transcript expression profiles, cellular localization, and/or substrate preference. Our phylogenetic and functional data reveal that hemipteran insects lost the classical glp genes, but have compensated for this by duplicating the eglp genes early in their evolution to comprise at least three separate clades of glycerol transporters.

Prediction of a Peptidome for the Western Tarnished Plant Bug Lygus Hesperus

General and Comparative Endocrinology. Mar, 2017  |  Pubmed ID: 27789347

Many strategies for controlling insect pests require an understanding of their hormonal signaling agents, peptides being the largest and most diverse single class of these molecules. Lygus hesperus is a pest species of particular concern, as it is responsible for significant damage to a wide variety of commercially important plant crops. At present, little is known about the peptide hormones of L. hesperus. Here, transcriptomic data were used to predict a peptidome for L. hesperus. Fifty-three L. hesperus transcripts encoding peptide precursors were identified, with a subset amplified by PCR for sequence verification. The proteins deduced from these transcripts allowed for the prediction of a 119-sequence peptidome for L. hesperus. The predicted peptides include isoforms of allatostatin A, allatostatin B (AST-B), allatostatin C, allatotropin, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, GSEFLamide, insulin-like peptide, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pyrokinin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide. Of note were several isoforms of AST-B that possess -WX7Wamide carboxyl-termini rather than the stereotypical -WX6Wamide (e.g., KWQDMQNPGWamide), an allatotropin ending in -SARGFamide rather than -TARGFamide (GLKNGPLNSARGFamide), a GSEFLamide ending in -GTEFLamide (TVGTEFLamide), several orcokinins with PMDEIDR- rather than NFDEIDR- amino-termini (e.g., PMDEIDRAGFTHFV), and an eight rather than 12 amino acid long isoform of SIFamide (PPFNGSIFamide). Collectively, the L. hesperus peptidome predicted here provides a resource for initiating physiological investigations of peptidergic signaling in this species, including studies directed at the biological control of this agricultural pest.

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