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Unusual tension reception in an insect.

Muscle tension receptors in animals monitor the tension generated by muscles. This information is important for the initiation and control of movements and for muscle tone in relation to spatial orientation and gravity. Vertebrates have tendon organs located at the musculo-tendinous junction. The number of muscle fibers attached to one receptor is in the range of 3 to 25. In insects by contrast, only a few examples are known where muscle tension is measured by only single receptors embedded in the muscle. All other muscle activity is monitored by a range of other receptors that detect strains on the cuticle or movements of the joints. Here we describe a set of approximately 200 receptor cells located on a single insect muscle. These receptor cells are associated with ovipositor muscle fibers and were preferentially responsive to muscle tension and not muscle length. Although single receptors may respond differently, their summed response to altered muscle tension characterized them as phasic-tonic type receptors. Experimental activation of muscle receptors in animals producing a basic oviposition motor pattern inhibited homonymous muscle activity without resetting the phase of the rhythm. These results suggest a potential role of tension receptors in regulating ovipositor muscle activity and in particular preventing excessive muscle tension during oviposition. The muscle receptors presented here provide the first example of tension measurement in insects by a few hundred receptor cells associated with a single muscle. Their role in motor control and relation to other tension receptors in vertebrates and invertebrates are discussed.

Animals↗

A method for controlling body wall turgor during electrophysiological recording from central neurons in ecdysing insects.

A method for obtaining intracellular recordings from central neurons of insects during the performance of ecdysial behaviors is described. Body wall turgor must be restored to high levels in order to provide the CNS with appropriate sensory feedback required for the generation of motor output. This is accomplished by installing the insect in a decompression chamber so that an opening in the animal's ventral surface communicates with the atmosphere. Reduction of chamber pressure causes expansion of the insect's body wall. It is suggested that adaptations of this technique to invertebrates with hydrostatic skeletons is possible and would permit electrophysiological studies of behaviors which normally require high blood pressures for their performance.

Animals↗

Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects.

The olfactory receptors of terrestrial animals exist in an aqueous environment, yet detect odorants that are primarily hydrophobic. The aqueous solubility of hydrophobic odorants is thought to be greatly enhanced via odorant binding proteins (OBP) which exist in the extracellular fluid surrounding the odorant receptors. We have isolated and partially sequenced 14 candidate OBPs from six insect (moth) species. All 14 represent a single homologous family based on conserved sequence domains. The 14 proteins can be divided into three subfamilies based on differences in tissue specific expression and similarities in amino acid sequences. All 14 proteins are specifically expressed in antennal olfactory tissue. Subfamily I represents previously described pheromone binding proteins (PBP), which are male-specific, associate with pheromone-sensitive neurons, and are highly variable in their sequences when compared among species. Subfamilies II and III are expressed in both male and female antennae, appear to associate with general-odorant-sensitive neurons, and are highly conserved when compared among species. The properties of the subfamily II and III proteins suggest these are general-odorant binding proteins (GOBP). The properties of the respective insect OBP subfamilies suggest that they have different odorant binding specificities. The association of different insect OBP subfamilies with distinct classes of olfactory neurons having different odorant specificities suggests that OBPs can act as selective signal filters, peripheral to the actual receptor proteins.

Amino Acid Sequence↗

Inhibition of insect acetylcholinesterase by the potato glycoalkaloid alpha-chaconine.

Homogenates from several insect species were assayed for inhibition of acetylcholinesterase by the potato glycoalkaloid alpha-chaconine. Colorado potato beetle acetylcholinesterase was up to 150-fold less sensitive than other species tested. Acetylcholinesterase from an insecticide-resistant strain of Colorado potato beetles was more sensitive to inhibition than the susceptible strain. Most insect species tested had inhibitory concentrations causing a 50% reduction in activity in the 5 to 40 microM range. Sensitive insect acetylcholinesterases were similar to mammalian cholinesterases in their response to alpha-chaconine. The results indicate that pesticides and host plant resistance factors may interact at the same target. Changes in the target due to selection pressure from either pesticides or host plant resistance factors could affect the efficacy of both control strategies.

Adaptation, Physiological↗

Improved efficacy of ethyl formate against stored grain insects by combination with carbon dioxide in a 'dynamic' application.

Ethyl formate is being evaluated as a fumigant for stored grain as it is a potential alternative to the ozone-depleting fumigant methyl bromide and to phosphine, which is under pressure owing to the development of strong resistance in stored grain insects. However, use of ethyl formate faces significant challenges, such as poor penetration through grain, significant losses to grain sorption, high concentrations of fumigant required to control insects, and flammability risks, which have limited its further development. In this study it was found that the combination of carbon dioxide (5-20%) with ethyl formate significantly enhanced efficacy of the fumigant against external living stages of the rice weevil, Sitophilus oryzae (L.), the lesser grain borer, Rhyzopertha dominica F., and the flour beetle, Tribolium castaneum (Herbst). Dynamic application of ethyl formate and carbon dioxide mixture (100 mg litre-1 ethyl formate, 20% CO2) pumped through a model silo containing wheat (50 kg) for one gas exchange was also investigated. A flow rate of 6 litres min-1 gave a relatively even distribution of fumigant throughout the grain column and similar mortality levels among cultures of S. oryzae and T. castaneum placed at three positions, the top, middle and bottom of the column. Mortality of 99.8% of mixed stage cultures of T. castaneum and 95.1% of S. oryzae was achieved in 3 h exposures to 111 and 185 mg ethyl formate h litre-1 respectively applied by the dynamic method. It is concluded that the combination of carbon dioxide with ethyl formate and dynamic application enhances distribution and efficacy of the fumigant against stored grain insects.

Animals↗

Insect resistance to Nilaparvata lugens and Cnaphalocrocis medinalis in transgenic indica rice and the inheritance of gna+sbti transgenes.

Molecular genetic analysis and insect bioassay of transgenic indica rice 'Zhuxian B' plants carrying snowdrop lectin gene (gna) and soybean trypsin inhibitor gene (sbti) were investigated in detail. PCR, 'dot' blot and PCR-Southern blot analysis showed that both transgenes had been incorporated into the rice genome and transmitted up to R3 progeny in most lines tested. Some transgenic lines exhibited Mendelian segregation, but the other showed either 1:1 (positive: negative for the transgenes) or other aberrant segregation patterns. The segregation patterns of gna gene crossed between R2 and R3 progeny. In half of transgenic R3 lines, gna and sbti transgenes co-segregated. Two independent homozygous lines expressing double transgenes were identified in R3 progeny. Southern blot analysis demonstrated that the copy numbers of integrated gna and sbti transgenes varied from one to ten in different lines. Insect bioassay data showed that most transgenic plants had better resistance to both Nilaparvata lugens (Stahl) and Cnaphalocrocis medinalis (Guenee) than wild-type plants. The insect resistance of transgenic lines increased with the increase in transgene positive ratio in most of the transgenic lines. In all, we obtained nine lines of R3 transgenic plants, including one pure line, which had better resistance to both N lugens and C medinalis than wild-type plants.

Animals↗

High-level expression of active human cyclooxygenase-2 in insect cells.

Active human cyclooxygenase-2 (Cox-2) was expressed at high levels in insect cells using a recombinant baculovirus. The specific activity of Cox-2 in the microsomes of infected cells was 0.51 mumol O2/min/mg and was comparable to that obtained for partially purified Cox-2 from ovine placenta (0.55 mumol O2/min/mg). The Cox-2 enzyme expressed in insect cells was glycosylated to varying extents and most of the cyclooxygenase activity was in the high-speed microsomal pellet. The insect-cell-expressed enzyme also showed characteristic 15-hydroxyeicosa-tetraenoic acid production after aspirin treatment and had typical inhibition profiles with a number of known nonsteroidal antiinflammatory drugs.

Animals↗

Production of mature human apolipoprotein A-I in a baculovirus-insect cell system: propeptide is not essential for intracellular processing but may assist rapid secretion.

To achieve expression of human mature apolipoprotein A-I (apoA-I) in the baculovirus-insect cell expression system, the propeptide encoding region of full-length preproapoA-I was deleted using polymerase chain reaction and the resulting cDNA was cloned into BacPak8 plasmid. After transfection into Sf21 insect cells and plaque purification, mature human apoA-I was secreted by the infected cells into the medium as determined by immunoblotting, amino-terminal sequencing, and molecular weight determination. In both monolayer cell cultures, and in suspension cell culture, maximum expression was achieved by the fifth day. For the first 4 days, 50 to 70% of the synthesized apoA-I was retained in the cells. This intracellular apoA-I was represented by mature apoA-I as shown by immunoblotting and amino-terminal sequencing. Further incubation resulted in a sharp decrease in the cell apoA-I content without a corresponding increase in protein in the medium and most likely represents intracellular degradation of the protein. We conclude that the deletion of the propeptide, while not preventing the correct cleavage of prepeptide during intracellular processing, results in reduced secretion of mature apoA-I. The baculovirus-insect cell expression system described in this study provides a useful method for producing recombinant mature apoA-I and is a potential tool for understanding the function of propeptide in intracellular transport and secretion of apoA-I from cells.

Amino Acid Sequence↗

Fluorometric microplate assay to measure glutathione S-transferase activity in insects and mites using monochlorobimane.

Elevated levels of glutathione S-transferases (GSTs) play a major role as a mechanism of resistance to insecticides and acaricides in resistant pest insects and mites, respectively. Such compounds are either detoxicated directly via phase I metabolism or detoxicated by phase II metabolism of metabolites as formed by microsomal monooxygenases. Here we used monochlorobimane (MCB) as an artificial substrate and glutathione to determine total GST activity in equivalents of single pest insects and spider mites in a sensitive 96-well plate-based assay system by measuring the enzymatic conversion of MCB to its fluorescent bimane-glutathione adduct. The differentiation by their GST activity between several strains of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), with different degrees of resistance to numerous acaricides was more sensitive with MCB compared to the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Compared to an acaricide-susceptible reference strain, one field population of T. urticae showed a more than 10-fold higher GST activity measured with MCB, in contrast to a less than 2-fold higher activity when CDNB was used. Furthermore, we showed that GST activity can be sensitively assessed with MCB in homogenates of pest insects such as Heliothis virescens, Spodoptera frugiperda (Lepidoptera: Noctuidae), Plutella xylostella (Lepidoptera: Yponomeutidae), and Myzus persicae (Hemiptera: Aphididae).

Animals↗

Purification and characterization of a scorpion defensin, a 4kDa antibacterial peptide presenting structural similarities with insect defensins and scorpion toxins.

Insect defensins are a group of inducible small-sized antibacterial peptides with three intramolecular disulfide bridges. NMR studies have recently shown that they share striking structural similarities with scorpion toxins. We have investigated in a scorpion species, Leiurus quinquestriatus, the potential presence of antibacterial molecules and report the isolation and structural characterization of a novel insect defensin homologue, which we refer to as scorpion defensin. This peptide shows a remarkably high degree of sequence homology with a defensin recently characterized in a species belonging to the ancient insect order of the Odonata with which it defines a novel ancient subclass of defensins. The scorpion defensin has in common with the scorpion toxins a consensus sequence Cys-[...]-Cys-Xaa-Xaa-Xaa-Cys-[...]-Gly-Xaa-Cys-[...]-Cys-Xaa-Cys present in all scorpion toxins characterized so far.

Amino Acid Sequence↗

Isolation and characterization of a new member of the insect defensin family from a beetle, Allomyrina dichotoma.

A new family member of insect defensin, an antibacterial peptide, has been isolated from larvae of a beetle, Allomyrina dichotoma. The peptide consisted of 43 amino acids and 6 cystein residues were conserved in the same position as that of other insect defensins. The new defensin was found to be inducible by bacterial injection. Analysis of the antibacterial spectrum of A. dichotoma defensin indicated that this peptide showed antibacterial activity against Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis but not against Gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa, indicating a typical spectrum of the insect defensin family. In addition, A. dichotoma defensin also exhibited antibacterial activity against methicillin-resistant S. aureus (MRSA) isolated from a patient.

Amino Acid Sequence↗

Expression of functional melanocortin 1 receptors in insect cells.

We expressed epitope-tagged human melanocortin 1 receptor (MC1R) in insect cells using two different recombinant baculovirus constructs; one of which encoded MC1R with an N-terminal Flag epitope and a C-terminal polyHis tag, while the other encoded the MC1R with a C-terminal Myc tag. The constructs were used to infect Sf9 insect cells. For both constructs, immunoblotting with tag-specific antibodies demonstrated the presence of the receptor in the infected cells. The infected Sf9 cells were characterized by radioligand binding using [125I][Nle4,D-Phe7]alpha-MSH. Both saturation and competition analysis, using alpha-, beta-, and gamma 1-MSH on the tagged MC1R expressed in the insect cells, gave binding constants and potency orders that were undistinguishable from those obtained on MC1R expressed in COS cells. The expression level obtained (in the order of pmoles of binding sites per mg of protein) will now facilitate attempts to purify the receptor. This is the first report that demonstrates a functional expression of recombinant melanocortin receptor in nonmammalian cells.

Amino Acid Sequence↗

Absence of short-term regulation over gluconeogenesis by glucose in the insect Manduca sexta L.

In vivo gluconeogenesis from (3-13 C)alanine was evident in terminal instar Manduca sexta larvae from the selective fractional 13C enrichment in trehalose, a disaccharide of glucose and the major blood sugar of insects. De novo glucose synthesis was observed in insects fed a low carbohydrate diet for 1 or more days. Gluconeogenesis was not inhibited by a single injection of glucose nor by short-term feeding on glucose-supplemented diet. Reduced fractional 13C enrichment in trehalose was demonstrated upon glucose administration, but was explained by isotopic dilution following direct synthesis of trehalose from the unlabeled glucose. Isotopic dilution was also quantified by analysis of the 13C labeling pattern in trehalose synthesized following injection of (1,2-13C2)glucose. The results suggest the absence of short-term regulation over gluconeogenesis by glucose and may partially explain why blood sugar level in M. sexta and other insects fluctuates over a wide concentration range. Although glucose had no observable effects on gluconeogenesis, injection of or feeding glucose resulted in a significantly increased activity of the pentose phosphate pathway.

Alanine↗

Overexpression of hepatitis delta antigen protects insect cells from baculovirus-induced cytolysis.

Hepatitis delta virus (HDV) is a human pathogen causing fulminant hepatitis and liver cirrhosis. HDV has a circular single-stranded RNA genome, which encodes only a single protein, hepatitis delta antigen (HDAg), from the antigenomic strand. Although the functional roles of HDAg have been extensively studied, the molecular mechanism of persistent infection and pathogenesis of HDV are not yet understood. Here we report that overexpressed HDAg protects cells from death in baculovirus-infected insect cells. Using both wild-type and recombinant baculovirus-infected insect cells, we have demonstrated that HDAg inhibited wild-type baculovirus-induced cytolysis and thus extended the survival of virus-infected insect cells. By deletion analysis, we show that N-terminal 25 amino acids are essential for this function. From these data, we suggest that HDAg may play a major role in persistent infection of HDV.

Animals↗

Paraquat inhibits the processing of human manganese-dependent superoxide dismutase by SF-9 insect cell mitochondria.

Precursor human manganese-dependent superoxide dismutase (hMn-SOD) was expressed using the baculovirus system in Spodoptera fungiperda (Sf-9) insect cells. Following infection of Sf-9 cells with hMn-SOD-expressing baculovirus, mature hMn-SOD was expressed at 15-25% of total cellular protein, with the recombinant protein localized in the mitochondrial matrix. Partial amino acid sequencing and SOD activity assays indicated that the hMn-SOD was correctly processed and assembled by insect mitochondria. This expression system was used to study the effects of paraquat and menadione, two intracellular superoxide generators, on processing of precursor hMn-SOD by insect mitochondria. Paraquat was found to potently inhibit mitochondrial processing of hMn-SOD, leading to the accumulation of precursor hMn-SOD and a decrease in measurable Mn-SOD activity. In contrast, menadione treatment was not found to affect the ratio of precursor to mature Mn-SOD. Paraquat did not lead to lower total production of hMn-SOD or cellular toxicity at the concentrations which were found to block processing of precursor hMn-SOD. These results indicate that mitochondrial processing and import of the precursor protein hMn-SOD are early events susceptible to dysfunction induced by the redox-cycling agent paraquat. These results also emphasize that mitochondrial processing of precursor proteins represents a parameter of cellular function which may be compromised, preceding the appearance of more generalized deterioration of cellular function, under certain toxic or pathological conditions.

Animals↗

Integration of endocrine signals that regulate insect ecdysis.

The extremely large number of insects and members of allied groups alive today suggests that molting--shedding of an old cuticle--may be one of the most commonly performed behaviors on our planet. Removal of an old cuticle in insects is associated with stereotyped, species-specific patterns of behavior referred to as ecdysis. It has been recognized for decades that the initiation of ecdysis is under hormonal control, but until recently many of the key peptides that regulate ecdysis were unknown. The report in 1996 of a new ecdysis-triggering hormone (ETH) sparked an era of significant advances in our understanding of the regulation of molting. This article summarizes the current model of peptide regulation of ecdysis, a model that is based on a positive feedback loop between ETH and a brain peptide, eclosion hormone. Then the relationship of these regulatory peptides to the neural circuitry that is the ultimate driver of the behavior are described. Because insects can undergo both status quo (larval-larval) and metamorphic (larval-pupal and pupal-adult) molts, differences in ecdysis behavior at different life stages are described and potential sources of these differences are identified. Most of the work described is based on studies of ecdysis in the hawkmoth, Manduca sexta, but results from studies of ecdysis in the fruit fly Drosophila melanogaster are also discussed.

Animals↗

Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells.

Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells. TPO was sequentially extracted from insect cells using various buffers and the protein was purified to homogeneity on a C4 reversed-phase semipreparative column using high-performance liquid chromatography. The purified protein was identified as hTPO by enzyme-linked immunosorbent assay, Western blot, and amino acid sequence analyses. Carbohydrate analysis of the recombinant hTPO showed that the protein is glycosylated and mannose is the major oligosaccharide. We have extended the carbohydrate analysis by establishing the occurrence of N-acetyl galactosamine which suggested that the recombinant hTPO might contain O-glycosyl moieties. Purified hTPO reacted specifically with sera from patients with Hashimoto's thyroiditis. Crude as well as purified hTPO did not show any enzymatic activity when produced in Sf9 insect cells grown in serum free medium. In contrast, hTPO produced in the presence of 10% fetal bovine serum containing 1 microgram/ml of haematin was enzymatically active. However, the enzymatic activity of the recombinant hTPO was lower than that often found with hTPO purified from thyroid tissue. Availability of purified hTPO in relatively large quantities should allow further structural and immunological studies.

Amino Acid Sequence↗