JournalTOCs

Insect Biochemistry and Molecular Biology

[4 followers] Follow
Full-text available via subscription

Subscription journal
ISSN (Print) 0965-1748
Published by Elsevier

[2556 journals]

Functional analysis of the RNAi response in ovary-derived silkmoth Bm5 cells
- Abstract: Publication date: Available online 10 May 2013
  Source:Insect Biochemistry and Molecular Biology
  Author(s): Anna Kolliopoulou , Luc Swevers
  Experiments of dsRNA-mediated gene silencing in lepidopteran insects in vivo are characterized by high variability although lepidopteran cell cultures have shown an efficient response to RNAi in transfection experiments. In order to identify the core RNAi factors that regulate the RNAi response of Lepidoptera, we employed the silkmoth ovary-derived Bm5 cells as a test system since this cell line is known to respond potently in silencing after dsRNA transfection. Two parallel approaches were used; involving knock-down of the core RNAi genes or over-expression of the main siRNA pathway factors, in order to study possible inhibition or stimulation of the RNAi silencing response, respectively. Components from all three main small RNA pathways (BmAgo-1 for miRNA, BmAgo-2/BmDcr-2 for siRNA, and BmAgo-3 for piRNA) were found to be involved in the RNAi response that is triggered by dsRNA. Since BmAgo-3, a factor in the piRNA pathway that functions independent of Dicer in Drosophila, was identified as a limiting factor in the RNAi response, sense and antisense ssRNA was also tested to induce gene silencing but proved to be ineffective, suggesting a dsRNA-dependent role for BmAgo-3 in Bombyx mori. After efficient over-expression of the main siRNA factors, immunofluorescence staining revealed a predominant cytoplasmic localization in Bm5 cells. This is the first study in Lepidoptera to provide evidence for possible overlapping of all three known small RNA pathways in the regulation of the dsRNA-mediated silencing response using transfected B. mori-derived Bm5 cells as experimental system.
  Graphical abstract
  
  PubDate: 2013-05-14T06:28:31Z
Editorial Board
- Abstract: Publication date: June 2013
  Source:Insect Biochemistry and Molecular Biology, Volume 43, Issue 6
  
  PubDate: 2013-05-06T06:27:47Z
Abamectin resistance in Drosophila is related to increased expression of P-glycoprotein via the dEGFR and dAkt pathways
- Abstract: Publication date: Available online 3 May 2013
  Source:Insect Biochemistry and Molecular Biology
  Author(s): Liang Luo , Ying-Jian Sun , Yi-Jun Wu
  Many insects have evolved resistance to abamectin but the mechanisms involved in this resistance have not been well characterized. P-glycoprotein (P-gp), an ATP-dependent drug-efflux pump transmembrane protein, may be involved in abamectin resistance. We investigated the role of P-gp in abamectin (ABM) resistance in Drosophila using an ABM-resistant strain developed in the laboratory. A toxicity assay, Western blotting analysis and a vanadate-sensitive ATPase activity assay all demonstrated the existence of a direct relationship between P-gp expression and ABM resistance in these flies. Our observations indicate that P-gp levels in flies’ heads were higher than in their thorax and abdomen, and that both P-gp levels and LC50 values were higher in resistant than in susceptible and P-gp-deficient strains. In addition, P-gp levels in the brain blood barrier (BBB) of resistant flies were higher than in susceptible and P-gp-deficient flies, which is further evidence that a high level of P-gp in the BBB is related to ABM resistance. Furthermore, we found greater expression of Drosophila EGFR (dEGFR) in the resistant strain than in the susceptible strain, and that the level of Drosophila Akt (dAkt) was much higher in resistant than in susceptible flies, whereas that in P-gp-deficient flies was very low. Compared to susceptible flies, P-gp levels in the resistant strain were markedly suppressed by the dEGFR and dAkt inhibitors lapatinib and wortmannin. These results suggest that the increased P-gp in resistant flies was regulated by the dEGFR and dAkt pathways and that increased expression of P-gp is an important component of ABM resistance in insects.
  Graphical abstract
  
  PubDate: 2013-05-06T06:27:47Z
Aldehyde Dehydrogenase 3 Converts Farnesal into Farnesoic Acid in the Corpora Allata of Mosquitoes
- Abstract: Publication date: Available online 29 April 2013
  Source:Insect Biochemistry and Molecular Biology
  Author(s): Crisalejandra Rivera-Perez , Marcela Nouzova , Mark. E. Clifton , Elena Martin Garcia , Elizabeth LeBlanc , Fernando G. Noriega
  The juvenile hormones (JHs) play a central role in insect reproduction, development and behavior. Interrupting JH biosynthesis has long been considered a promising strategy for the development of target-specific insecticides. Using a combination of RNAi, in vivo and in vitro studies we characterized the last unknown biosynthetic enzyme of the JH pathway, a fatty aldehyde dehydrogenase (AaALDH3) that oxidizes farnesal into farnesoic acid (FA) in the corpora allata (CA) of mosquitoes. The AaALDH3 is structurally and functionally a NAD+-dependent class 3 ALDH showing tissue and developmental-stage-specific splice variants. Members of the ALDH3 family play critical roles in the development of cancer and Sjögren-Larsson syndrome in humans, but have not been studies in groups other than mammals. Using a newly developed assay utilizing fluorescent tags, we demonstrated that AaALDH3 activity, as well as the concentrations of farnesol, farnesal and FA were different in CA of sugar and blood-fed females. In CA of blood-fed females the low catalytic activity of AaALDH3 limited the flux of precursors and caused a remarkable increase in the pool of farnesal with a decrease in FA and JH synthesis. The accumulation of the potentially toxic farnesal stimulated the activity of a reductase that converted farnesal back into farnesol, resulting in farnesol leaking out of the CA. Our studies indicated AaALDH3 plays a key role in the regulation of JH synthesis in blood-fed females and mosquitoes seem to have developed a ‘‘trade-off’’ system to balance the key role of farnesal as a JH precursor with its potential toxicity.
  Graphical abstract
  
  PubDate: 2013-05-02T06:34:26Z
Characterization of Tudor-sn-containing granules in the silkworm, Bombyx mori
- Abstract: Publication date: Available online 1 May 2013
  Source:Insect Biochemistry and Molecular Biology
  Author(s): Li Zhu , Tsuneyuki Tatsuke , Hiroaki Mon , Zhiqing Li , Jian Xu , Jae Man Lee , Takahiro Kusakabe
  The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.
  Graphical abstract
  
  PubDate: 2013-05-02T06:34:26Z
Characterization of a spruce budworm chitin deacetylase gene: stage- and tissue- specific expression, and inhibition using RNA interference
- Abstract: Publication date: Available online 27 April 2013
  Source:Insect Biochemistry and Molecular Biology
  Author(s): Guoxing Quan , Tim Ladd , Jun Duan , Fayuan Wen , Daniel Doucet , Michel Cusson , Peter J. Krell
  Chitin deacetylase (CDA) catalyzes the conversion of chitin into chitosan, thereby modifying the physical properties of insect cuticles and peritrophic matrices. A lepidopteran chitin deacetylase gene (CfCDA2) was cloned from the spruce budworm, Choristoneura fumiferana, and found to generate two alternatively spliced transcripts, CfCDA2a and CfCDA2b. Transcriptional analysis using isoform-specific RT-PCR primers indicated that both isoforms were upregulated during the molt. Interestingly, CfCDA2b transcripts were most abundant in the head during the molting stage while those of CfCDA2a were predominant in the epidermis during the feeding period. Injection of CfCDA2-specific dsRNA into C. fumiferana larvae or pre-pupae induced both abnormal phenotypes and high mortality, which resulted from an inability to shed the old cuticle. These results suggest that CfCDA2 plays an important role in the molting process, and that the two alternatively spliced transcripts have different functions during insect development. This is the first detailed characterization of lepidopteran chitin deacetylase gene.
  Graphical abstract
  
  PubDate: 2013-04-28T06:28:52Z
FISH identification of Helicoverpa armigera and Mamestra brassicae chromosomes by BAC and fosmid probes
- Abstract: Publication date: Available online 27 April 2013
  Source:Insect Biochemistry and Molecular Biology
  Author(s): Ken Sahara , Atsuo Yoshido , Fukashi Shibata , Noriko Fujikawa , Takuya Okabe , Makiko Tanaka-Okuyama , Yuji Yasukochi
  Since the Bombyx mori genome sequence was published, conserved synteny between B. mori and some other lepidoperan species has been revealed by either FISH (fluorescence in situ hybridization) with BAC (bacterial artificial chromosome) probes or linkage analysis. However, no species belonging to the Noctuidae, the largest lepidopteran family which includes serious polyphagous pests, has been analyzed so far with respect to genome-wide conserved synteny and gene order. For that purpose, we selected the noctuid species Helicoverpa armigera and Mamestra brassicae, both with n = 31 chromosomes. Gene-defined fosmid clones from M. brassicae and BAC clones from a closely related species of H. armigera, Heliothis virescens, were used for a FISH analysis on pachytene chromosomes. We recognized all H. armigera chromosomes from specific cross-hybridization signals of 146 BAC probes. With 100 fosmid clones we identified and characterized all 31 bivalents of M. brassicae. Synteny and gene order were well conserved between the two noctuid species. The comparison with the model species B. mori (n = 28) showed the same phenomenon for 25 of the 28 chromosomes. Three chromosomes (#11, #23 and #24) had two counterparts each in H. armigera and M. brassicae. Since n = 31 is the modal chromosome number in Lepidoptera, the noctuid chromosomes probably represent an ancestral genome organization of Lepidoptera. This is the first identification of a full karyotype in Lepidoptera by means of BAC cross-hybridization between species. The technique shows the potential to expand the range of analyzed species efficiently.
  Graphical abstract
  
  PubDate: 2013-04-28T06:28:52Z
Editorial Board
- Abstract: Publication date: May 2013
  Source:Insect Biochemistry and Molecular Biology, Volume 43, Issue 5
  
  PubDate: 2013-04-20T21:42:07Z
Resistance to Bt maize in Mythimna unipuncta (Lepidoptera: Noctuidae) is mediated by alteration in Cry1Ab protein activation
- Abstract: Publication date: Available online 18 April 2013
  Source:Insect Biochemistry and Molecular Biology
  Author(s): Joel González-Cabrera , Matías García , Pedro Hernández-Crespo , Gema P. Farinós , Félix Ortego , Pedro Castañera
  Bt maize cultivars based on the event MON810 (expressing Cry 1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.
  Graphical abstract
  
  PubDate: 2013-04-20T21:42:07Z
Manduca sexta Serpin-7, a Putative Regulator of Hemolymph Prophenoloxidase Activation
- Abstract: Publication date: Available online 6 April 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Serpins regulate various physiological reactions in humans and insects, including certain immune responses, primarily through inhibition of serine proteases. Six serpins have previously been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a full-length cDNA sequence of another Manduca serpin, named serpin-7. The open reading frame of serpin-7 encodes a polypeptide of 400 amino acid residues with a predicted signal peptide of the first 15 residues. Multiple protein sequence alignment of the reactive center loop region of the M. sexta serpins indicated that serpin-7 contains Arg-Ile at the position of the predicted scissile bond cleaved by protease in the serpin inhibition mechanism. The same residues occur in the scissile bond of the reactive center loop in M sexta serpin-4 and serpin-5, which are protease inhibitors that can block prophenoloxidase activation in plasma. Serpin-7 transcript was detected in hemocytes and fat body, and its expression increased in fat body after injection of larvae with Micrococcus luteus. Recombinant serpin-7 added to larval plasma inhibited spontaneous melanization and decreased prophenoloxidase activation stimulated by bacteria. Serpin-7 inhibited prophenoloxidase-activating protease-3 (PAP3), forming a stable serpin-protease complex. Considering that serpin-3 and serpin-6 are also efficient inhibitors of PAP3, it appears that multiple serpins present in plasma may have redundant or overlapping functions. We conclude that serpin-7 has serine protease inhibitory activity and is likely involved in regulation of proPO activation or other protease-mediated aspects of innate immunity in M. sexta.
  Graphical abstract
  
  PubDate: 2013-04-09T21:32:20Z
Sterol/steroid metabolism and absorption in a generalist and specialist caterpillar: Effects of dietary sterol/steroid structure, mixture and ratio
- Abstract: Publication date: Available online 6 April 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Insects cannot synthesize sterols de novo, so they typically require a dietary source. Cholesterol is the dominant sterol in most insects, but because plants contain only small amounts of cholesterol, plant-feeding insects generate most of their cholesterol by metabolizing plant sterols. Plants almost always contain mixtures of different sterols, but some are not readily metabolized to cholesterol. Here we explore, in two separate experiments, how dietary phytosterols and phytosteroids, in different mixtures, ratios, and amounts, affect insect herbivore sterol/steroid metabolism and absorption; we use two caterpillars species – one a generalist (Heliothis virescens), the other a specialist (Manduca sexta). In our first experiment caterpillars were reared on two tobacco lines – one expressing a typical phystosterol profile, the other expressing high amounts/ratios of stanols and 3-ketosteroids. Caterpillar reared on the control tobacco contained mostly cholesterol, but those reared on the modified tobacco had reduced amounts of cholesterol, and lower total sterol/steroid body profiles. In our second experiment, caterpillars were reared on artificial diets containing known amounts of cholesterol, stigmasterol, cholestanol and/or cholestanone, either singly or in various combinations and ratios. Cholesterol and stigmasterol-reared moths were mostly cholesterol, while cholestanol-reared moths were mostly cholestanol. Moth tissue cholesterol concentration tended to decrease as the ratio of dietary cholestanol and/or cholestanone increased. In both moths cholestanone was metabolized into cholestanol and epicholestanol. Interestingly, M. sexta generated much more cholestanol than epicholestanol, while H. virescens did the opposite. Finally, total tissue steroid levels were significantly reduced in moths reared on diets containing very high levels of cholestanol. We discuss how dietary sterol/steroid structural differences are important with respect to sterol/steroid metabolism and uptake, including species-specific differences.
  Graphical abstract
  
  PubDate: 2013-04-09T21:32:20Z
Gloverins of the silkworm Bombyx mori: Structural and Binding Properties and Activities
- Abstract: Publication date: Available online 6 April 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Gloverins are basic, glycine-rich and heat-stable antibacterial proteins (∼14-kDa) in lepidopteran insects with activity against Escherichia coli, Gram-positive bacteria, fungi and a virus. Hyalophora gloveri gloverin adopts a random coil structure in aqueous solution but has α-helical structure in membrane-like environment, and it may interact with the lipid A moiety of lipopolysaccharide (LPS). Manduca sexta gloverin binds to the O-specific antigen and outer core carbohydrate of LPS. In the silkworm Bombyx mori, there are four gloverins with slightly acidic to neutral isoelectric points. In this study, we investigate structural and binding properties and activities of B. mori gloverins (BmGlvs), as well as correlations between structure, binding property and activity. Recombinant BmGlv1-4 were expressed in bacteria and purified. Circular dichroism (CD) spectra showed that all four BmGlvs mainly adopted random coli structure (>50%) in aqueous solution in regardless of pH, but contained α-helical structure in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), smooth and rough mutants (Ra, Rc and Re) of LPS and lipid A. Plate ELISA assay showed that BmGlvs at pH 5.0 bound to rough mutants of LPS and lipid A but not to smooth LPS. Antibacterial activity assay showed that positively charged BmGlvs (at pH 5.0) were active against E. coli mutant strains containing rough LPS but inactive against E. coli with smooth LPS. Our results suggest that binding to rough LPS is the prerequisite for the activity of BmGlvs against E. coli.
  Graphical abstract
  
  PubDate: 2013-04-09T21:32:20Z
Albino (al) is a tetrahydrobiopterin (BH4)-deficient mutant of the silkworm Bombyx mori
- Abstract: Publication date: Available online 6 April 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Albino (al) is a lethal mutant of Bombyx mori that exhibits a colourless cuticle after the first ecdysis and dies without feeding on mulberry. Previous studies have indicated that sclerotisation was insufficient because of defective phenylalanine and tyrosine metabolism in albino larvae. However, the genetic mechanism underlying the albino phenotype has not been determined. Dopamine plays a central role in insect cuticle colouration and sclerotisation. The pathway for dopamine biosynthesis from phenylalanine involves phenylalanine hydroxylase (PAH; EC 1.14.16.1) and tyrosine hydroxylase (TH; EC 1.14.16.2). Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases, including PAH and TH. Thus, BH4 is indispensable for cuticle colouration and sclerotisation. Here we report on identifying mutations in the gene that encodes for the Bombyx homolog of 6-pyruvoyl-tetrahydropterin synthase (PTS) which is involved in the biosynthesis of BH4, in 2 strains with different al alleles. In strain a60 (al), a transposable element was inserted in exon 2 of BmPTS. In strain a61 (al 2 ), an 11-bp deletion was identified in the exon 2 region of BmPTS. After oral administration of BH4 to the al 2 larvae, the survival rate was effectively increased and the larval integument was pigmented. These results indicated that BmPTS was responsible for the albino mutants of B. mori. We conclude that (i) a mutation in BmPTS leads to an insufficient supply of BH4 and results in defective dopamine biosynthesis and (ii) lack of dopamine results in cuticle colouration and sclerotisation failure. Lemon (lem) is a BH4-deficient mutant. It has been reported that de novo synthesis of zygotic BH4 was indispensable for viability of the embryo in eggs laid by lem (lem/lem l ) females. We found that lem/lem, al 2 /al 2 larvae produced by lem (lem/lem) females were viable during the first instar stage, suggesting that al 2 /al 2 embryo could synthesis BH4 by using maternally transmitted BmPTS.
  Graphical abstract
  
  PubDate: 2013-04-09T21:32:20Z
Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm, Bombyx mori
- Abstract: Publication date: Available online 6 April 2013
  Source:Insect Biochemistry and Molecular Biology
  
  ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.
  Graphical abstract
  
  PubDate: 2013-04-09T21:32:20Z
Silverfish silk is formed by entanglement of randomly coiled protein chains
- Abstract: Publication date: Available online 9 April 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Silks are semi-crystalline solids in which protein chains are associated by intermolecular hydrogen bonding within ordered crystallites, and by entanglement within unordered regions. By varying the type of protein secondary structure within crystallites and the overall degree of molecular order within fibers, arthropods produce fibers with a variety of physical properties suited to many purposes. We characterized silk produced as a tactile stimulus during mating by the grey silverfish (Ctenolepisma longicaudata) using Fourier transform infrared spectroscopy, polarized Raman spectroscopy, gel electrophoresis and amino acid analysis. Fibers were proteinaceous—the main component being a 220 kDa protein—and were rich in Gln/Glu, Leu, and Lys. The protein structure present was predominantly random coil, with a lesser amount of beta-structure. Silk fibers could readily be solubilized in aqueous solutions of a mild chaotrope, sodium dodecyl sulfate, indicating protein chains were not cross-linked by disulfide or other covalent bonds. We conclude that entanglement is the major mechanism by which these silk proteins cohere into a solid material. We propose silks used as short-term tactile cues are subject to less stringent requirements for molecular order relative to other silks, allowing the random coil structure to be favored as an adaptation promoting maximal entanglement and adhesion.
  Graphical abstract
  
  PubDate: 2013-04-09T21:32:20Z
Extensive inter- and intraspecific venom variation in closely related parasites targeting the same host: The case of Leptopilina parasitoids of Drosophila
- Abstract: Available online 2 April 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  The arms race between immune suppressive parasites that produce virulence factors and hosts that evolve resistance to these factors is suggested to be a key driver for the diversification of both partners. However, little is known regarding the diversity of virulence factors in closely related parasites or the mechanisms underlying the variation of virulence. One of the best-described model to address this issue is the interaction between Leptopilina parasitic wasps and their Drosophila hosts, in which variation of virulence is well documented. Thanks to a combined transcriptomic and proteomic approach, we have identified the main secreted proteins in the venom of Leptopilina heterotoma (Gotheron strain, 66 proteins) and of two well-characterized strains of L. boulardi, ISm and ISy (65 and 49 proteins, respectively). Results revealed significant quantitative differences in venom components between the L. boulardi strains, in agreement with their different virulence properties. Strikingly, the two related Leptopilina species did not share any abundant venom protein. The main identified proteins in L. boulardi were RhoGAPs and serpins while an aspartylglucosaminidase was found abundant in L. heterotoma. The extensive quantitative variation observed between these species may be related with their use of different virulence strategies and/or to differences in their host range (specialist versus generalist). Altogether, our data suggests that parasitoid venom can quickly evolve, mainly through rapid changes in regulation of gene expression. It also evidences venom evolutionary processes largely described in other venomous animals i.e. the convergent recruitment of venom proteins between phylogenetically unrelated organisms, and the role of duplications in the emergence of multigenic families of virulence factors.
  Graphical abstract
  
  PubDate: 2013-04-06T11:00:18Z
The insulin/TOR signal transduction pathway is involved in the nutritional regulation of juvenile hormone synthesis in Aedes aegypti
- Abstract: Available online 26 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Juvenile hormone (JH) levels must be modulated to permit the normal progress of development and reproductive maturation in mosquitoes. JH is part of a transduction system that assesses nutritional information and controls reproduction in mosquitoes. Adult female Aedes aegypti show nutritionally-dependent dynamic changes in corpora allata (CA) JH biosynthetic activities. A coordinated expression of most JH biosynthetic enzymes has been described in female pupae and adult mosquitoes; increases or decreases in transcript levels for all the enzymes were concurrent with increases or decreases in JH synthesis; suggesting that transcriptional changes are at least partially responsible for the dynamic changes of JH biosynthesis. The goal of the present study is to identify signaling network components responsible for the nutritional-dependent changes of JH synthesis in the CA of mosquitoes. The insulin/TOR signaling network plays a central role in the transduction of nutritional signals that regulate cell growth and metabolism in insects. These pathways have also been suggested as a link between nutritional signals and JH synthesis regulation in the CA of cockroaches and flies. We used a combination of in vitro studies and in vivo genetic knockdown experiments to explore nutritional signaling pathways in the CA. Our results suggest that the insulin/TOR pathway plays a role in the transduction of the nutritional information that regulates JH synthesis in mosquitoes. Transcriptional regulation of the genes encoding JH biosynthetic enzymes is at least partially responsible for these nutritionally modulated changes of JH biosynthesis.
  Graphical abstract
  
  PubDate: 2013-03-29T02:25:22Z
Subcellular localization of the fatty acyl reductase involved in pheromone biosynthesis in the tobacco budworm, Heliothis virescens (Noctuidae: Lepidoptera)
- Abstract: Available online 26 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Sex pheromone components are produced in specialized glands of female moths via well-characterized biosynthetic pathways, where a Fatty Acyl Reductase (FAR) is often essential for producing the specific ratio of the different pheromone components. The subcellular localization and membrane topology of FARs is important for understanding how pheromones are synthesized and exported to the exterior for release. We investigated the subcellular localization of HvFAR from the noctuid moth Heliothis virescens by producing recombinant fusion proteins with green fluorescent protein (GFP) in yeast. A C-terminally tagged construct was localized to the endoplasmic reticulum (ER) and retained full reductive activity on a broad range of saturated and unsaturated fatty acyl precursors. In contrast, an N-terminally-tagged construct was poorly expressed in the cytoplasm and was not enzymatically active, indicating that HvFAR requires a free N-terminal for both proper targeting and catalytic activity. A series of truncations of the N-and C-termini of HvFAR was conducted based on in silico-predicted hydrophobic domains and transmembrane regions. The N-terminally truncated protein was found in the cytoplasm and did not retain activity, emphasizing the importance of the N-terminal for FAR function. In addition, the orientation in the membrane of the C-terminus-tagged HvFAR-GFP construct was analyzed using a fluorescence protease protection (FPP) assay, implying that the C-terminal of HvFAR is orientated towards the cytoplasm. These results, together with previous data on the localization of desaturases, confirm the importance of the ER as a subcellular site of pheromone production.
  Graphical abstract
  
  PubDate: 2013-03-29T02:25:22Z
Insights into the different functions of multiple peptidoglycan recognition proteins in the immune response against bacteria in the mosquito, Armigeres subalbatus
- Abstract: Available online 26 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Peptidoglycan recognition proteins (PGRPs) are a group of proteins that recognize and/or bind to peptidoglycan on the surface of a number of pathogens. To understand the roles of multiple PGRPs in the mosquito Armigeres subalbatus (AsPGRPs), we studied the effects of infection of two bacteria, the gram negative Escherichia coli and the gram positive Micrococcus luteus, on the transcriptional expression of AsPGRPs and RNA interference (RNAi) of AsPGRPs on the immune responses of mosquitoes against the two bacteria. Injection of E. coli or M. luteus into adult mosquitoes both significantly increased the transcription of AsPGRP-S1, but not the other AsPGRPs. A mosquito survival assay using injection of E. coli or M. luteus into AsPGRP double-stranded RNA (dsRNA) injected mosquitoes showed that RNAi of AsPGRPs had different impacts on the survival abilities of mosquitoes, and that AsPGRP-LCs seem to be the most critical ones. Real-time Polymerase Chain Reaction (real-time PCR) analysis indicated that the expression of four antimicrobial peptides (AMPs) was dramatically changed after AsPGRP-LB and AsPGRP-LC RNAi, although AsPGRP-S1 and AsPGRP-LE had slight, but significant, effects, suggesting that the changes in survival abilities were potentially due to the changes in AMP expression after AsPGRP RNAi. In addition, bacterial challenges following AsPGRP-LC RNAi did not induce the expression of AMPs to their normal level as in control experiments. An in vivo assay indicated that AsPGRP-LC RNAi had no significant effects on the phagocytic ability of the hemocytes, suggesting that AsPGRP-LC is not a key factor mediating phagocytosis of bacteria in this mosquito.
  Graphical abstract
  
  PubDate: 2013-03-29T02:25:22Z
Calcitonin receptor 1 (AedaeGPCRCAL1) hindgut expression and direct role in myotropic action in females of the mosquito Aedes aegypti (L.)
- Abstract: Available online 22 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  In anautogenous mosquitoes such as Aedes aegypti females the calcitonin-like diuretic hormone 31 (DH31) stimulates natriuretic fluid excretion from the Malpighian tubules (MTs) after a blood meal. We previously cloned and functionally characterized AedaeGPCRcal1 from A. aegypti, the ortholog of the Drosophila melanogaster DH31 receptor and immunolocalized it in the MTs. However, localization of the calcitonin receptor-like receptor 1 (GPCRCAL1) in the hindgut of any insect is unknown, and specifically, knowledge on its role in hindgut contraction in response to Aedae-DH31 peptide is lacking. We analyzed the expression of AedaeGPCRCAL1 in hindgut by western blot and immunohistochemisty, and evaluated its role in hindgut contractility by application of Aedae-DH31 before and after receptor RNA interference (RNAi). The receptor was detected as a 73 kDa band in western blots of hindgut and immunofluorescence revealed the receptor was expressed in hindgut circular and longitudinal muscles but not in the hindgut epithelial cells. In vitro, incubation in 1 μM solution of Aedae-DH31 peptide significantly increased the hindgut contraction frequency in normal mosquitoes. Hindguts from females treated with AedaeGPCRcal1 dsRNA and incubated with DH31 showed a reduction of 50% percent in their contraction frequency with respect to controls. These results suggest that DH31 hormone released from the brain post-blood meal has a direct and coordinative action on the excretory system, MTs and hindgut, by which AedaeGPCRCAL1 signaling stimulates MT primary urine secretion and hindgut contraction resulting in rapid postprandial fluid excretion.
  Graphical abstract
  
  PubDate: 2013-03-25T22:38:25Z
Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen, but not its corresponding enol
- Abstract: Available online 21 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase). In this study, we investigated two genetically distant spider mite strains with high resistance to spirodiclofen. Despite the strong resistance levels to spirodiclofen (up to 680-fold), only limited cross-resistance with other members of this group such as spiromesifen and spirotetramat could be detected. Amplification and sequencing of the ACCase gene from resistant and susceptible strains did not reveal common non-synonymous mutations, and expression levels of ACCase were similar in both resistant and susceptible strains, indicating the absence of target-site resistance. Furthermore, we collected genome-wide expression data of susceptible and resistant T. urticae strains using microarray technology. Analysis of differentially expressed genes revealed a broad response, but within the overlap of two resistant strains, several cytochrome P450s were prominent. Quantitative PCR confirmed the constitutive over-expression of CYP392E7 and CYP392E10 in resistant strains, and CYP392E10 expression was highly induced by spirodiclofen. Furthermore, stage specific expression profiling revealed that expression levels were not significantly different between developing stages, but very low in eggs, matching the age-dependent resistance pattern previously observed. Functional expression of CYP392E7 and CYP392E10 confirmed that CYP392E10 (but not CYP392E7) metabolizes spirodiclofen by hydroxylation as identified by LC-MS/MS, and revealed cooperative substrate binding and a Km of 43μM spirodiclofen. CYP392E10 also metabolizes spiromesifen, but not spirotetramat. Surprisingly, no metabolism of the hydrolyzed spirodiclofen-enol metabolite could be detected. These findings are discussed in the light of a likely resistance mechanism.
  Graphical abstract
  
  PubDate: 2013-03-22T03:05:05Z
Effects of period RNAi on V-ATPase expression and rhythmic pH changes in the vas deferens of Spodoptera littoralis (Lepidoptera: Noctuidae)
- Abstract: Available online 14 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Circadian clocks (oscillators) regulate multiple aspects of insect behaviour and physiology. The circadian system located in the male reproductive tract of Lepidoptera orchestrates rhythmic sperm release from testis and sperm maturation in the upper vas deferens (UVD). Our previous research on the cotton leafworm, Spodoptera littoralis, suggested rhythmic changes in the V-ATPase levels in the UVD epithelium, which correlated with rhythmic pH fluctuations in the UVD lumen. However, it was not known whether UVD cells contain clock mechanism that generates these daily fluctuations. In the current paper, we show circadian rhythm in the expression of clock gene period at the mRNA and protein level in the UVD epithelium. To determine the role of PER in V-ATPase and pH regulation, testes–UVD complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment, which transiently lowered per mRNA and protein in the UVD, altered expression of V-ATPase c subunit. In addition, per RNAi caused a significant delay in the UVD lumen acidification. These data demonstrate that the UVD molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic V-ATPase activity and periodic acidification of the UVD lumen.
  Graphical abstract Highlights ► Epithelium of the upper vas deferens (UVD) contains an oscillator with PERIOD protein (PER) as the key regulatory component. ► Rhythmical expression of period gene can be transiently reduced in cultured male reproductive organs using the RNAi method. ► Expression of V-ATPase subunits c in the UVD epithelium is rhythmic and directly dependent on the PER protein. ► PER-based oscillator in the UVD epithelium drives rhythmic pH changes by affecting V-ATPase expression.
  
  PubDate: 2013-03-18T03:05:14Z
Analysis of a digestive prolyl carboxypeptidase in Tenebrio molitor larvae
- Abstract: Available online 14 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP.
  Graphical abstract Highlights ► The first prolyl carboxypeptidase (PRCP) from the midgut of Tenebrio molitor larvae was purified and characterized. ► The gene encoding the PRCP was cloned and sequenced. ► Digestion of proline-rich dietary proteins was determined to be a new function of PRCP in a stored product pest. ► Evolution of digestive complex in insects with slightly acidic midgut pH may include incorporation of lysosomal peptidases.
  
  PubDate: 2013-03-18T03:05:14Z
Molecular characterization and functional significance of the Vti family of SNARE proteins in tick salivary glands
- Abstract: Available online 14 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and Amblyomma americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases.
  Graphical abstract Highlights Physiology of a Tick feeding (left) and the general morphology of salivary gland acini (top-right). Secretory granules (present in salivary gland acini) undergo vesicle fusion events driven in part by the interactions with VTI proteins syntaxins (bottom-right). ► Functional significance of the VTI family of SNARE proteins, AmVti1a, AmVti1b, and AaVti1b was determined. ► The qRT-PCR analysis revealed the highest expression of the VTI family in unfed salivary glands. ► The VTI family of SNARE proteins is localized differently in unfed and partially fed salivary glands. ► Knockdown of the VTI family of SNARE proteins suggests an important role in tick blood feeding, survival and oviposit.
  
  PubDate: 2013-03-18T03:05:14Z
Impact of environment on mosquito response to pyrethroid insecticides: Facts, evidences and prospects
- Abstract: April 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology, Volume 43, Issue 4
  
  By transmitting major human diseases such as malaria, dengue fever and filariasis, mosquito species represent a serious threat worldwide in terms of public health, and pose a significant economic burden for the African continent and developing tropical regions. Most vector control programmes aiming at controlling life-threatening mosquitoes rely on the use of chemical insecticides, mainly belonging to the pyrethroid class. However, resistance of mosquito populations to pyrethroids is increasing at a dramatic rate, threatening the efficacy of control programmes throughout insecticide-treated areas, where mosquito-borne diseases are still prevalent. In the absence of new insecticides and efficient alternative vector control methods, resistance management strategies are therefore critical, but these require a deep understanding of adaptive mechanisms underlying resistance. Although insecticide resistance mechanisms are intensively studied in mosquitoes, such adaptation is often considered as the unique result of the selection pressure caused by insecticides used for vector control. Indeed, additional environmental parameters, such as insecticides/pesticides usage in agriculture, the presence of anthropogenic or natural xenobiotics, and biotic interactions between vectors and other organisms, may affect both the overall mosquito responses to pyrethroids and the selection of resistance mechanisms. In this context, the present work aims at updating current knowledge on pyrethroid resistance mechanisms in mosquitoes and compiling available data, often from different research fields, on the impact of the environment on mosquito response to pyrethroids. Key environmental factors, such as the presence of urban or agricultural pollutants and biotic interactions between mosquitoes and their microbiome are discussed, and research perspectives to fill in knowledge gaps are suggested.
  Graphical abstract Highlights ► Impact of environment on mosquito response to pyrethroids is reviewed. ► Main known pyrethroid resistance mechanisms are described. ► Environmental factors potentially affecting these mechanisms are identified. ► Research perspectives are proposed.
  
  PubDate: 2013-03-18T03:05:14Z
An insect trypsin-like serine protease as a target of microRNA: Utilization of microRNA mimics and inhibitors by oral feeding
- Abstract: April 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology, Volume 43, Issue 4
  
  MicroRNAs (miRNAs) are non-coding small RNAs (18–25 nt) that play crucial roles in various biological processes, including development, as gene regulators. In this study, we identified a miRNA, har-miR-2002b, that is specifically expressed during larval stages of Helicoverpa armigera (cotton bollworm). For the first time, we explored the possibility of utilizing synthetic inhibitor and mimic of a miRNA by oral feeding and confirmed that the inhibitor and mimic can successfully inhibit/oversupply har-miR-2002b in H. armigera larvae. Bioassays by oral feeding indicated 70% reduction in fecundity and 40% larval mortality in the presence of har-miR-2002b mimic. In addition, a large proportion of those larvae that made it to the pupal stage were deformed and only few moths emerged from them. This implied an important role of har-miR-2002b in larval development and adult fecundity. A trypsin-like serine protease (Ha-TLP) was identified as a target of har-miR-2002b. In vivo and in vitro studies revealed that in the presence of the miRNA mimic Ha-TLP transcript/protein levels and enzyme activity were significantly reduced. In contrast, inhibition of har-miR-2002b led to significant up-regulation of Ha-TLP transcript levels. This is the first study which shows the potential of utilization of miRNA mimics and inhibitors in pest control by targeting specific insect genes.
  Graphical abstract Highlights ► An insect microRNA (miRNA) targets a trypsin-like serine protease. ► Oral feeding of the synthetic miRNA mimic reduced survival, growth and fecundity. ► miRNA mimics/inhibitors remained functional through oral feeding and reduced/increased the serine protease transcript levels. ► miRNA mimics/inhibitors can be utilized by oral feeding to study miRNA-target interactions.
  
  PubDate: 2013-03-18T03:05:14Z
Editorial Board
- Abstract: April 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology, Volume 43, Issue 4
  
  PubDate: 2013-03-18T03:05:14Z
MicroRNA functions in insects
- Abstract: April 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology, Volume 43, Issue 4
  
  MicroRNAs (miRNAs) are small non-coding RNAs that are generated in all eukaryotes and viruses. Their role as master regulators of gene expression in various biological processes has only been fully appreciated over the last decade. Accumulating evidence suggests that alterations in the expression of miRNAs may lead to disorders, including developmental defects, diseases and cancer. Here, I review what is currently known about miRNA functions in insects to provide an insight into their diverse roles in insect biology.
  Graphical abstract Highlights ► MicroRNAs are ∼22 nucleotide non-coding RNAs produced in all eukaryotes. ► MicroRNAs are key regulators of gene expression at the post-transcriptional level. ► In insects, microRNAs are involved in various functions, including development, immunity and host–pathogen interactions.
  
  PubDate: 2013-03-18T03:05:14Z
Bacterial infection activates the immune system response and dysregulates microRNA expression in honey bees
- Abstract: Available online 13 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  In insects, a rapid and massive synthesis of antimicrobial peptides (AMPs) is activated through signaling pathways (Toll and Imd) to combat invading microbial pathogens. However, it is still unclear whether different types of bacteria provoke specific responses. Immune response mechanisms and the activation of specific genes were investigated by challenging Apis mellifera workers with the Gram-negative bacterium Serratia marcescens or the Gram-positive bacterium Micrococcus luteus. The immune system responded by activating most genes of the Toll and Imd pathways, particularly AMP genes. However, genes specifically regulated by M. luteus or S. marcescens were not detected, suggesting an interaction between the signaling pathways that lead to immune effectors synthesis. Despite this finding, kappaB motifs in the 5´-UTRs of selected genes suggest a pathway-specific control of AMP and transferrin-1 gene expression. Regulation by miRNAs was also investigated and revealed a number of candidates for the post-transcriptional regulation of immune genes in bees.
  Graphical abstract Highlights ► Specific immune response provoked by Micrococcus luteus and Serratia marcescens was investigated in honey bees. ► The immune system responded by activating genes of the Toll and Imd pathways. ► Immune-related genes exclusively regulated by M. luteus or S. marcescens were not detected. ► A pathway-specific regulation of immune-related genes was suggested by the presence of KappaB motifs in their 5’- UTR. ► MiRNA dysregulation was detected in honey bees following S. marcescens infection.
  
  PubDate: 2013-03-14T04:31:15Z
Dpp/BMP transport mechanism is required for wing venation in the sawfly Athalia rosae
- Abstract: Available online 7 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  The pattern of wing venation varies considerably among different groups of insects and has been used as a means of species-specific identification. However, little is known about how wing venation is established and diversified among insects. The decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling pathway plays a critical role in wing vein formation during the pupal stages in Drosophila melanogaster. A key mechanism is BMP transport from the longitudinal veins (LVs) to the posterior crossvein (PCV) by the BMP-binding proteins, short gastrulation (Sog) and twisted gastrulation2/crossveinless (Tsg2/Cv). To investigate whether the BMP transport mechanism is utilized to specify insect wing vein patterns in other than Drosophila, we used the sawfly Athalia rosae as a model, which has distinct venation patterns in the fore- and hindwings. Here, we show that Ar-dpp is ubiquitously expressed in both the fore- and hindwings, but is required for localized BMP signaling that reflects distinct wing vein patterns between the fore- and hindwings. By isolating Ar-tsg/cv in the sawfly, we found that Ar-Tsg/Cv is also required for BMP signaling in wing vein formation and retains the ability to transport Dpp. These data suggest that the BMP transport system is widely used to redistribute Dpp to specify wing venation and may be a basal mechanism underlying diversified wing vein patterns among insects.
  Graphical abstract Highlights ► The sawfly serves as an excellent model for wing vein development. ► BMP signaling reflects wing vein pattern in the fore- and hindwings, although Ar-dpp is ubiquitously expressed. ► Ar-Tsg/Cv is required for BMP signaling in the wing vein primordia and wing vein formation. ► Redistribution of Dpp/BMP via transport mechanism may be widely involved in specifying insect wing venation. ► The mechanisms for wing vein formation appear to be evolutionarily conserved between Drosophila and the sawfly.
  
  PubDate: 2013-03-10T03:05:23Z
SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda
- Abstract: Available online 5 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with amolecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X.
  Graphical abstract Highlights ► ? An initiator caspase, SfDronc, was identified in Spodoptera frugiperda. ? SfDronc is orthologous to the apoptotic initiator caspase Dronc from Drosophila melanogaster. ? Silencing SfDronc inhibits apoptosis in Sf9 cells, and SfDronc cleaves and activates the effector caspase Sf-caspase-1. ? SfDronc is inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related inhibitor AcP35. ? SfDronc is likely to be Sf-caspase-X, the long-sought apoptotic initiator caspase in S. frugiperda.
  
  PubDate: 2013-03-06T03:08:25Z
Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in E. coli: purification, activity, and binding properties toward multiple pesticides
- Abstract: Available online 5 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmoles L−1 of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC50 values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects.
  Graphical abstract Highlights ► The insect P450 CYP6G1 is linked to resistance to agriculturally-important pesticides. ► When heterologously expressed in E. coli, CYP6G1 is present in both soluble and membrane-bound fractions. ► Recombinant CYP6G1 binds to several, structurally-dissimilar pesticide compounds including DDT, imidacloprid and, endosulfan. ► CYP6G1 activity towards the probe substrate p-nitroanisole is inhibited by multiple pesticide ligands.
  
  PubDate: 2013-03-06T03:08:25Z
Molecular characterization of the flightin gene in the wing-dimorphic planthopper, Nilaparvata lugens, and its evolution in Pancrustacea
- Abstract: Available online 1 March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Flightin was initially identified in Drosophila melanogaster. Previous work has shown that Drosophila flightin plays a key role in indirect flight muscle (IFM) function and has limited expression in the IFM. In this study, we demonstrated that flightin is conserved across the Pancrustacea species, including winged insects, non-winged insects, non-insect hexapods and several crustaceans. The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a long-distance migration insect with wing dimorphism, is the most destructive rice pest in Asia. We showed that flightin was one of the most differentially expressed genes in macropterous and brachypterous BPH adults. In female BPHs, flightin was expressed in the IFM of macropterous adults, no expression was detected in brachypterous ones; while in male BPHs, flightin was not only expressed in the IFM of macropterous adults, but also in the dorsal longitudinal muscle (DLM) in the basal two abdominal segments of both macropterous and brachypterous ones. RNAi and transmission electron microscopy results showed that flightin played key roles in maintaining IFM and male DLM structure, which drive wing movements in macropterous adults and the vibration of the male-specific tymbal, respectively. Using Daphnia magna as an example of a crustacean species, we observed that flightin was expressed in juvenile instars and adults, and was localized in the antenna muscles. These results illustrate the functional variations of flightin in insects and other arthropod species and provide clues as to how insects with flight apparatuses evolved from ancient pancrustaceans.
  Graphical abstract Highlights ► Flightin is conserved in Pancrustacea, which includes hexapods and crustaceans. ► Flightin was found in the tymbal muscles of the planthopper male adult. ► Flightin was found in the indirect flight muscles of the long-winged planthopper adult. ► Flightin was not detected in the thorax of the short-winged planthopper adult. ► Flightin localized to Daphnia magna antenna muscles.
  
  PubDate: 2013-03-02T22:41:44Z
Diversity and expression of P450 genes from Dendroctonus valens LeConte (Curculionidae: Scolytinae) in response to different kairomones
- Abstract: Available online 27 February 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Bark beetles (Curculionidae: Scolytinae) are major cause of woody plants death in the world. They colonize the stem and other parts of trees recognizing host-produced specific compounds (kairomones) and insect pheromones. Bark beetle’s antennae and alimentary canal participate in the host selection identifying chemical compounds produced by trees and insects, and also in the metabolism and detoxification of these compounds. The red turpentine beetle (RTB), Dendroctonus valens LeConte, is a unaggressive species that colonize > 40 pine species (Pinaceae) in North and Central America. Several studies suggest that bark beetle cytochrome P450 enzymes are involved in monoterpene oxidation. In this study we identified by means of PCR, cloning, sequencing, and bioinformatic analysis, eleven full-length genes: five CYP4, four CYP6, and two CYP9 in the antennae and gut region of RTB, after stimulation with vapors of monoterpenes: (±)-α- pinene, (R)-(+)-α-pinene, (S)-(-)-β-pinene, (S)-(-)-α-pinene and (+)-3-carene; pine trees volatiles used by RTB as kairomones. The recovered cDNA of these genes vary from 1.5 kb to 1.8 kb and the open frame encodes from 496 to 562 amino acid proteins. The bioinformatic analysis suggests that the majority of P450 proteins encoded by these genes are membrane anchored in the endoplasmic reticulum. RT-qPCR assays showed differential expression of all CYP genes between male and female. The gene expression was dependent of monoterpenes and exposure time, with some of them sex, antennae and gut region specific. Significant differences among monoterpenes, gut region, antennae and exposure time were found. Our results suggest that some of these genes may be involved in the detoxification process of these compounds during tree colonization.
  Graphical abstract Highlights ► Eleven full-length CYP genes were identified in Dendroctonus valens’ antenna and gut. ► The expression of some genes in the antenna was dependent upon sex and treatment. ► The CYP genes in the gut showed a different expression respet to monoterpene assayed. ► Over-expression of CYP6 and CYP9 gene suggests a role in the detoxification process.
  
  PubDate: 2013-03-02T22:41:44Z
Editorial Board
- Abstract: March 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology, Volume 43, Issue 3
  
  PubDate: 2013-02-16T07:07:03Z
Meta-diamide insecticides acting on distinct sites of RDL GABA receptor from those for conventional noncompetitive antagonists
- Abstract: Available online 14 February 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  The RDL GABA receptor is an attractive target of insecticides. Here we demonstrate that meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor antagonists showing high insecticidal activity against Spodoptera litura. We also suggest that the mode of action of the meta-diamides is distinct from that of conventional noncompetitive antagonists (NCAs), such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. Using a membrane potential assay, we examined the effects of the meta-diamide 3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7) and NCAs on mutant Drosophila RDL GABA receptors expressed in Drosophila Mel-2 cells. NCAs had little or no inhibitory activity against at least one of the three mutant receptors (A2′S, A2′G, and A2′N), which were reported to confer resistance to NCAs. In contrast, meta-diamide 7 inhibited all three A2′ mutant receptors, at levels comparable to its activity with the wild-type receptor. Furthermore, the A2′S・T6′V mutation almost abolished the inhibitory effects of all NCAs. However, meta-diamide 7 inhibited the A2′S・T6′S mutant receptor at the same level as its activity with the wild-type receptor. In contrast, a G336M mutation in the third transmembrane domain of the RDL GABA receptor abolished the inhibitory activities of meta-diamide 7, although the G336M mutation had little effect on the inhibitory activities of conventional NCAs. Molecular modeling studies also suggested that the binding site of meta-diamides was different from those of NCAs. Meta-diamide insecticides are expected to be prominent insecticides effective against A2′ mutant RDL GABA receptors with a different mode of action.
  Graphical abstract Highlights ► Meta-diamides showed high insecticidal activity against Spodoptera litura. ► Meta-diamide 7 inhibited A2′ mutant RDL GABA receptors. ► G336M mutation abolished the inhibitory activitiy of meta-diamide 7. ► Distinct mode of action of meta-diamides was shown.
  
  PubDate: 2013-02-16T07:07:03Z
Proteomic profiling of thermal acclimation in Drosophila melanogaster
- Abstract: Available online 13 February 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Thermal acclimation drastically alters thermotolerance of ectotherms, but the mechanisms determining this plastic response are not fully understood. The present study investigates the proteomic response (2D-DIGE) of adult Drosophila melanogaster acclimated at 11, 25 or 31 °C. As expected 11 °C-acclimation improved cold tolerance and 31 °C-acclimation improved heat tolerance. We hypothesized that the marked organismal responses to acclimation could be detected at the proteomic level assuming that changes in the abundance of specific proteins are linked to the physiological changes underlying the phenotypic response. The 31 °C-acclimated flies displayed a particular divergent proteomic profile where molecular chaperones made up a large number of the proteins that were modulated during heat acclimation. Many other proteins showed significant modulation during acclimation including proteins involved in iron ion and cell redox homeostasis, carbohydrate and energy metabolism, chromatin remodeling and translation, and contractile machinery. Interestingly the changes in protein abundance were often unrelated to transcriptional activity of the genes coding for the proteins, except for the most strongly expressed proteins (e.g. Hsp70). The 11 °C-acclimation evoked weak proteomic response despite the marked effect on the organismal phenotype. Thus the acquired cold tolerance observed here may involve regulatory process such as posttranslational regulation rather than de novo protein synthesis.
  Graphical abstract Highlights ► Molecular mechanisms of thermal acclimation. ► Heat acclimation considerably affects the proteome. ► Cold acclimation shows weak proteomic response.
  
  PubDate: 2013-02-16T07:07:03Z
Steroid hormone 20-hydroxyecdysone regulation of the very-high-density lipoprotein (VHDL) receptor phosphorylation for VHDL uptake
- Abstract: Available online 13 February 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  During the metamorphic stage of holometabolous insects, the biosynthetic precursors needed for the synthesis of a large number of adult proteins are acquired from the selective absorption of storage proteins. The very-high-density lipoprotein (VHDL), a non-hexameric storage protein, is consumed by the fat body from the hemolymph through VHDL receptor (VHDL-R)-mediated endocytosis. However, the mechanism of the uptake of VHDL by a VHDL-R remains unclear. In this study, a VHDL-R from Helicoverpa armigera was found to be involved in 20E-regulated VHDL uptake through the regulation of steroid hormone 20-hydroxyecdysone (20E). The transcripts of VHDL-R were detected mainly in the fat body and integument during the wandering stage. The transcription of VHDL-R was upregulated by 20E through the ecdysteroid receptor (EcRB1) and Ultraspiracle (USP1). In addition, 20E stimulates the phosphorylation of VHDL-R through protein kinase C for ligand binding. VHDL-R knockdown in larvae results the inhibition of development to adulthood. These data imply that 20E regulates VHDL-R on both transcriptional and posttranslational levels for VHDL absorption.
  Graphical abstract Highlights ► The transcription of VHDL-R was upregulated by 20E through ecdysteroid receptor (EcRB1) and Ultraspiracle protein (USP1). ► 20E phosphorylates VHDL-R through protein kinase C for ligand binding. ► VHDL-R knockdown in larvae results the inhibition of development to adulthood.
  
  PubDate: 2013-02-16T07:07:03Z
Identification of a cuticle protein with unique repeated motifs in the silkworm, Bombyx mori
- Abstract: Available online 29 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  The insect cuticle is non-cellular matrix secreted from a monolayer of epidermal cells. After abrasion of the larval cuticle of the silkworm, Bombyx mori, a protein with molecular mass of 135 kDa is newly detected in the cuticle. Mass spectrometric analysis of the tryptic fragments from this protein revealed that the 135-kDa protein is encoded by the Cb10 gene. In the predicted amino acid sequence of Cb10, three repeated motifs with [YxGGFGGppG(L/V)L] sequence are found in the C-terminal region. In addition to the repeated motifs, Cb10 has seventeen CxxxxC motifs randomly distributed throughout the polypeptide chain and serine rich region at N-terminal region. The Cb10 gene is strongly expressed in epidermal cells after pupal ecdysis, and its expression in the larval epidermal cells is induced not only by cuticular abrasion, but also by bacterial infection. These expression patterns suggest some specific roles of this protein in pupal cuticle formation and defense reactions.
  Graphical abstract Highlights ► In the abraded cuticle of the silkworm, Bombyx mori, a protein of 135 kDa appears. ► The accumulation of the 135-kDa protein is observed only in the abraded part. ► The accumulation in the cuticle also occurs after bacterial infection. ► The 135-kDa protein is a product of the Cb10 gene, and [YxGGFGGppG(L/V)L] motif was newly identified in the sequence.
  
  PubDate: 2013-02-01T03:07:25Z
Molecular cloning and characterization of the α-glucosidase II from Bombyx mori and Spodoptera frugiperda
- Abstract: Available online 30 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  The α-glucosidase II (GII) is a heterodimer of α- and β-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of α-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the α-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII α- and β-subunit genes and the GII α-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII α-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl α-D-glucopyranoside (pNP-αGlc) but were inactive toward N-glycan. Although the B. mori GII β-subunit was not required for the hydrolysis of pNP-αGlc, a B. mori GII complex of the α- and β-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII α-subunit protein was less sensitive to α-glucosidase inhibitors than was the S. frugiperda GII α-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of α-glucosidase inhibitors from mulberry leaves.
  Graphical abstract Highlights ► We cloned glucosidase II subunit genes from Bombyx mori (BmGlcIIα and BmGlcIIβ) and Spodoptera frugiperda (SfGlcIIα). ► Recombinant glucosidase II subunits (rBmGlcIIα, rBmGlcIIβ, and rSfGlcIIα) were produced by a baculovirus expression system. ► rBmGlcIIα and rSfGlcIIα hydrolyzed p-nitrophenyl α-D-glucopyranoside but not N-glycan. ► The formation of a rBmGlcIIα/rBmGlcIIβ complex was required for N-glycan cleavage. ► rBmGlcIIα was less sensitive than rSfGlcIIα to α-glucosidase inhibitors in mulberry leaves.
  
  PubDate: 2013-02-01T03:07:25Z
Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations
- Abstract: Available online 31 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Ips pini bark beetles use myrcene hydroxylases to produce the aggregation pheromone component, ipsdienol, from myrcene. The enantiomeric ratio of pheromonal ipsdienol is an important prezygotic mating isolation mechanism of I. pini and differs among geographically distinct populations. We explored the substrate and product ranges of myrcene hydroxylases (CYP9T2 and CYP9T3) from reproductively-isolated western and eastern I. pini. The two cytochromes P450 share 94% amino acid identity. CYP9T2 mRNA levels were not induced in adults exposed to myrcene-saturated atmosphere. Functional assays of recombinant enzymes showed both hydroxylated myrcene, (+)- and (–––)-α-pinene, 3-carene, and R-(+)-limonene, but not α-phellandrene, (–)-β-pinene, γ-terpinene, or terpinolene, with evidence that CYP9T2 strongly preferred myrcene over other substrates. They differed in the enantiomeric ratios of ipsdienol produced from myrcene, and in the products resulting from different α-pinene enantiomers. These data provide new information regarding bark beetle pheromone evolution and factors affecting cytochrome P450 structure-function relationships.
  Graphical abstract Highlights ► Myrcene hydroxylases (CYP9T2, CYP9T3) from eastern and western Ips pini populations have 94% amino acid identity. ► Both enzymes accept myrcene, α-pinene, 3-carene, and (R)-(+)-limonene as substrates. ► CYP9T2 expression is not induced by its preferred substrate, myrcene.
  
  PubDate: 2013-02-01T03:07:25Z
Receptors for the Neuropeptides, Myoinhibitory Peptide and SIFamide, in Control of the Salivary Glands of the Blacklegged Tick Ixodes scapularis
- Abstract: Available online 25 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Tick salivary glands are important organs that enable the hematophagous feeding of the tick. We previously described the innervation of the salivary gland acini types II and III by a pair of protocerebral salivary gland neurons that produce both myoinhibitory peptide (MIP) and SIFamide (Šimo et al., 2009b). In this study we identified authentic receptors expressed in the salivary glands for these neuropeptides. Homology-based searches for these receptors in the Ixodes scapularis genome sequence were followed by gene cloning and functional expression of the receptors. Both receptors were activated by low nanomolar concentrations of their respective ligands. The temporal expression patterns of the two ligands and their respective receptors suggest that the SIFamide signaling system pre-exists in unfed salivary glands, while the MIP system is activated upon initiation of feeding. Immunoreactivity for the SIFamide receptor in the salivary gland was detected in acini types II and III, surrounding the acinar valve and extending to the basal region of the acinar lumen. The location of the SIFamide receptor in the salivary glands suggests three potential target cell types and their probable functions: myoepithelial cells that may function in the contraction of the acini and/or the control of the valve; large, basally located dopaminergic granular cells for regulation of paracrine dopamine; and neck cells that may be involved in the control of the acinar duct and its valve.
  Graphical abstract Highlights ► Receptors for two neuropeptides, MIP and SIFamide, were identified and characterized in the tick salivary glands. ► Immunohistochemistry of SIFamide receptor revealed three candidate target cells of the SIFamide. ► The target cells of the SIFamide are proposed to be myoepithelial cell, basal granular cell, and neck cells
  
  PubDate: 2013-01-28T22:26:37Z
Identification of microRNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum
- Abstract: Available online 23 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of miRNAs in a global key pest Plutella xylostella as well as comparative analysis of miRNA expression profile of the insect in association with parasitization by Diadegma semiclausum. Combining the deep sequencing data and bioinformatics, 235 miRNAs were identified from P. xylostella. Differential expression of host cellular miRNAs in response to parasitism was examined by making small RNA libraries from parasitized and naive second instar larvae of P. xylostella. Bantam, miR-276*, miR-10, miR-31 and miR-184 were detected as five most abundant miRNAs in both libraries and 96 miRNAs were identified that were differentially expressed after parasitization. Bantam*, miR-184 and miR-281* were significantly down-regulated and two miRNAs miR-279b and miR-2944b* were highly induced in parasitized larvae. Interestingly, high copy numbers and differential expression of several miRNA passenger strands (miRNA*) suggest their potential roles in host-parasitoid interaction. In conclusion, expression profiling of miRNAs provided insights into their possible involvement in insect immune response to parasitism and offer an important resource for further studies.
  Graphical abstract Highlights ► 235 conserved and novel miRNAs were identified from P. xylostella larvae using deep sequencing. ► A number of miRNAs were identified to be differentially expressed following parasitization with D. semiclausum. ► The differentially expressed miRNAs are presumed to be involved in immune pathways.
  
  PubDate: 2013-01-25T17:16:24Z
Maize toxin degrades peritrophic matrix proteins and stimulates compensatory transcriptome responses in fall armyworm midgut
- Abstract: Available online 7 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function.
  Graphical abstract Highlights ► Maize insect resistant 1-cysteine protease (Mir1-CP) is expressed in resistant Mp708 maize. ► Insect Intestinal Mucin in the peritrophic matrix (PM) is a key target of Mir1-CP. ► Genes encoding PM structural proteins are upregulated in midguts (MGs) from caterpillars fed on Mp708. ► Likewise for genes encoding digestive enzymes compared with MGs from caterpillars fed on diets lacking Mir1-CP. ► Transcriptional regulation of MG genes is a means by which insects counter the Mir1-CP resistance trait of Mp708 maize
  
  PubDate: 2013-01-07T22:08:06Z
Sex pheromone recognition and immunolocalization of three pheromone binding proteins in the black cutworm moth Agrotis ipsilon
- Abstract: Available online 5 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Insect pheromone binding proteins (PBPs) are believed to solubilize and transport hydrophobic sex pheromones across sensillum lymph to membrane-associated pheromone receptors. To address the molecular mechanisms of PBPs in insect pheromone perception, we undertook a systemic study on the PBPs of the black cutworm Agrotis ipsilon at transcript as well as protein level from tissue distribution and cellular localization to pheromone binding affinity. We cloned three full-length PBP genes AipsPBP1-3 from A. ipsilon antennae, and demonstrated that AipsPBP1–3 transcripts were highly expressed in male antennae. The electron microscopic examinations revealed at least six types of olfactory sensilla on male and female antenna: trichodea, chaetica, basiconica, coeloconica, squamiformia and Böhm bristles. The immunocytochemistry results demonstrated that AipsPBP1-3 proteins were strongly expressed in the sensillum lymph of the trichoid sensilla of male moth. The binding assays showed that AipsPBP1 had high binding affinities with the major sex pheromone components Z7-12:Ac and Z9-14:Ac among five related chemicals and was clustered together with the long trichoid sensilla-expressing LdisPBPs of Lymantria dispar. AipsPBP2 showed high binding affinities also with Z11-16:Ac. AipsPBP3 displayed a high affinity only with Z11-16:Ac. Our studies provide further detail evidences for the involvement of moth PBPs in pheromone discrimination and selective recognition of specific components of the female sex pheromone blends.
  Graphical abstract Highlights ► We report the cloning, male antenna expression of PBP genes of Agrotis ipsilon. ► The PBPs are strongly expressed in the sensillum lymph of the sensilla trichodea. ► AipsPBP1 show high binding affinities to Z7-12:Ac and Z9-14:Ac. ► AipsPBP2 show high binding affinities to Z7-12:Ac, Z9-14:Ac and Z11-16:Ac. ► AipsPBP3 specifically binds to the Z11-16:Ac with high binding ability.
  
  PubDate: 2013-01-07T22:08:06Z
Neuropeptide F regulates male reproductive processes in the desert locust, Schistocerca gregaria
- Abstract: Available online 4 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  Although Neuropeptide F (NPF) has been identified in different insect species, its function has mainly been studied in the fruit fly, Drosophila melanogaster, where it regulates diverse physiological processes, such as learning, stress responses and male courtship behavior. In locusts, only a truncated form of the “full-length” NPF (the biologically active “trNPF”) has been isolated. This 9 AA peptide stimulates oocyte maturation, food intake and weight increase in adult desert locusts (Schistocerca gregaria [Forskål]). In this study, we investigated whether this peptide is also involved in the regulation of male reproductive physiology in this orthopteran species. Daily injections of trNPF in adult males resulted in proportionally heavier testes and seminal vesicles, while RNAi-mediated knockdown of the Schgr-NPF precursor transcript gave rise to proportionally lighter testes and seminal vesicles. Furthermore, adult males precociously displayed courtship behavior when injected daily with trNPF, while this behavior was inhibited or delayed by RNAi knockdown of the Schgr-NPF precursor transcript. In order to further analyze these effects of trNPF on male reproductive physiology, fertility of males was tested by analyzing progeny numbers following copulation with untreated females. In this way, we showed that daily trNPF injection in adult males resulted in a larger egg pod size and a higher percentage of hatched eggs per egg pod after copulation, while RNAi knockdown caused the opposite effects. Taken together, we provide clear evidence for a role of trNPF in the regulation of reproductive physiology in adult males of the desert locust, S. gregaria. Possible modes of action of trNPF in influencing these reproductive processes in male locusts are discussed.
  Graphical abstract Highlights ► Injection of trNPF in adult males results in proportionally heavier testes and seminal vesicles. ► When injected with trNPF, adult males precociously display courtship behavior. ► TrNPF injection positively influences post-copulatory fertility of adult males.
  
  PubDate: 2013-01-04T09:04:17Z
The interaction between Trypanosoma rangeli and the nitrophorins in the salivary glands of the triatomine Rhodnius prolixus (Hemiptera; Reduviidae)
- Abstract: Available online 4 January 2013
  Publication year: 2013
  Source:Insect Biochemistry and Molecular Biology
  
  The parasite Trypanosoma rangeli develops in the intestinal tract of triatomines and, particularly in species of the genus Rhodnius, invades the hemolymph and salivary glands, where subsequent metacyclogenesis takes place. Many aspects of the interaction between T. rangeli and triatomines are still unclear, especially concerning the development of the parasite in the salivary glands and how the parasite interacts with the saliva. In this work, we describe new findings on the process of T. rangeli infection of the salivary glands and the impact of infection on the saliva composition. To ensure a complete infection (intestinal tract, hemolymph and salivary glands), 3th instar Rhodnius prolixus nymphs were fed on blood containing T. rangeli epimastigotes using an artificial feeder. After molt to the 4th instar, the nymphs were inoculated with epimastigotes in the hemolymph. The results showed that the flagellates started to invade the salivary glands by the 7th day after the injection. The percentage of trypomastigotes inside the salivary glands continuously increased until the 25th day, at which time the trypomastigotes were more than 95% of the T. rangeli forms present. The salivary contents from T. rangeli-infected insects showed a pH that was significantly more acidic (
  PubDate: 2013-01-04T09:04:17Z
Cloning and characterization of a microsomal epoxide hydrolase from Heliothis virescens
- Abstract: Available online 28 December 2012
  Publication year: 2012
  Source:Insect Biochemistry and Molecular Biology
  
  Epoxide hydrolases (EHs) are α/β-hydrolase fold superfamily enzymes that convert epoxides to 1,2-trans diols. In insects EHs play critical roles in the metabolism of toxic compounds and allelochemicals found in the diet and for the regulation of endogenous juvenile hormones (JHs). In this study we obtained a full-length cDNA, hvmeh1, from the generalist feeder Heliothis virescens that encoded a highly active EH, Hv-mEH1. Of the 10 different EH substrates that were tested, Hv-mEH1 showed the highest specific activity (1,180 nmol min−1 mg−1) for a 1,2-disubstituted epoxide-containing fluorescent substrate. This specific activity was more than 25- and 3,900-fold higher than that for the general EH substrates cis-stilbene oxide and trans-stilbene oxide, respectively. Although phylogenetic analysis placed Hv-mEH1 in a clade with some lepidopteran JH metabolizing EHs (JHEHs), JH III was a relatively poor substrate for Hv-mEH1. Hv-mEH1 showed a unique substrate selectivity profile for the substrates tested in comparison to those of MsJHEH, a well-characterized JHEH from M. sexta, and hmEH, a human microsomal EH. Hv-mEH1 also showed unique enzyme inhibition profiles to JH-like urea, JH-like secondary amide, JH-like primary amide, and non-JH-like primary amide compounds in comparison to MsJHEH and hmEH. Although Hv-mEH1 is capable of metabolizing JH III, our findings suggest that this enzymatic activity does not play a significant role in the metabolism of JH in the caterpillar. The ability of Hv-mEH1 to rapidly hydrolyze 1,2-disubstituted epoxides suggests that it may play roles in the metabolism of fatty acid epoxides such as those that are commonly found in the diet of Heliothis.
  Highlights ► A microsomal epoxide hydrolase (i.e., Hv-mEH1) encoding cDNA was cloned from the tobacco budworm Heliothis virescens. ► A recombinant Hv-mEH1 was expressed and its ability to hydrolyze general and fluorescent EH substrates, and juvenile hormone III (JH III) was determined. ► The ability of urea and amide compounds, including JH-like ureas and amides, to inhibit Hv-mEH1 was tested. ► Biochemical characterization suggested that JH III is a poor substrate of Hv-mEH1
  
  PubDate: 2012-12-31T10:15:44Z
Detection of the Wolbachia-encoded DNA binding protein, HU beta, in mosquito gonads
- Abstract: Available online 31 December 2012
  Publication year: 2012
  Source:Insect Biochemistry and Molecular Biology
  
  Wolbachia are obligate intracellular bacteria that cause cytoplasmic incompatibility in mosquitoes. In an incompatible cross, eggs of uninfected females fail to hatch when fertilized by sperm from infected males. We used polyacrylamide gel electrophoresis and tandem mass spectrometry to identify Wolbachia proteins in infected mosquito gonads. These included surface proteins with masses of 25 and 18 kDa and the DNA binding protein, HU beta. Using reverse transcriptase polymerase chain reaction, we showed that the HU gene is transcribed in Wolbachia-infected Culex pipiens and Aedes albopictus mosquitoes. We sequenced HU genes from four Wolbachia strains and compared deduced protein sequences with additional homologs from the databases. Among the Rickettsiales, Wolbachia HU has distinct N- and C-terminal basic/acidic amino acid motifs as well as a pair of conserved, cysteine residues.
  Graphical abstract Highlights ► Wolbachia express a DNA binding protein, HU, in mosquito testes and ovaries. ► HU abundance is comparable to that of Wolbachia surface proteins. ► Wolbachia HU has unique amino acid motifs absent in E. coli homologs. ► HU is the first Wolbachia DNA binding protein detected by mass spectrometry.
  
  PubDate: 2012-12-31T10:15:44Z
Biochemical properties, expression profiles, and tissue localization of orthologous acetylcholinesterase-2 in the mosquito, Anopheles gambiae
- Abstract: Available online 23 December 2012
  Publication year: 2012
  Source:Insect Biochemistry and Molecular Biology
  
  Acetylcholinesterases (AChEs) catalyze the hydrolysis of acetylcholine, a neurotransmitter for cholinergic neurotransmission in animals. Most insects studied so far possess two AChE genes: ace-1 paralogous and ace-2 orthologous to Drosophila melanogaster ace. We characterized the catalytic domain of Anopheles gambiae AChE1 in a previous study (Jiang et al., 2009) and report here biochemical properties of A. gambiae AChE2 expressed in Sf9 cells. An unknown protease in the expression system cleaved the recombinant AChE2 next to Arg110, yielding two non-covalently associated polypeptides. A mixture of the intact and cleaved AChE2 had a specific activity of 72.3 U/mg, much lower than that of A. gambiae AChE1 (523 U/mg). The order of V max/K M values for the model substrates was acetylthiocholine > propionylthiocholine ≈ acetyl-(β-methyl)thiocholine > butyrylthiocholine. The IC50’s for eserine, carbaryl, BW284C51, paraoxon and malaoxon were 1.32, 13.6, 26.8, 192 and 294 nM, respectively. A. gambiae AChE2 bound eserine and carbaryl stronger than paraoxon and malaoxon, whereas eserine and malaoxon modified the active site Ser232 faster than carbaryl or paraoxon did. Consequently, the k i’s were 1.173, 0.245, 0.029 and 0.018 μM−1min−1 for eserine, carbaryl, paraoxon and malaoxon, respectively. Quantitative polymerase chain reactions showed a similar pattern of ace-1 and ace-2 expression. Their mRNAs were abundant in early embryos, greatly decreased in late embryos, larvae, pupae, and pharate adult, and became abundant again in adults. Both transcripts were higher in head and abdomen than thorax of adults and higher in male than female mosquitos. Transcript levels of ace-1 were 1.9- to 361.8-fold higher than those of ace-2, depending on developmental stages and body parts. Cross-reacting polyclonal antibodies detected AChEs in adult brains, thoracic ganglia, and genital/rectal area. Activity assays, immunoblotting, and tandem mass spectrometric analysis indicated that A. gambiae AChE1 is responsible for most of acetylthiocholine hydrolysis in the head extracts. Taken together, these data indicate that A. gambiae AChE2 may play a less significant role than AChE1 does in the mosquito nervous system.
  Graphical abstract Highlights ► Characterization of A. gambiae AChE2 using model substrates. ► Proteolysis next to Arg110 and its possible role in enzyme regulation. ► Inhibition kinetics of the enzyme and comparison with A. gambiae AChE1’s. ► Expression profiles of ace-1 and ace-2 mRNAs in the mosquito. ► Localization of A. gambiae AChEs in adult tissues.
  
  PubDate: 2012-12-23T22:13:06Z