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PubMed2006

A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly.

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides in flies. A C-terminal aldehyde insect kinin analog, Fmoc-RFFPWG-H (R-LK-CHO), demonstrates stimulation of Malpighian tubule fluid secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h from flies injected with 1 microL of distilled water by almost 50%. The analog not only inhibits stimulation of housefly fluid secretion by the native kinin Musdo-K, but also by thapsigargin, a SERCA inhibitor, and by ionomycin, a calcium ionophore. The activity of R-LK-CHO is selective, however, as related C-terminal aldehyde analogs do not demonstrate an inhibitory response on housefly fluid secretion. The selective inhibitory activity of R-LK-CHO on housefly tubules represents an important lead in the development of environmentally friendly insect management agents based on the insect kinins.

Aldehydes↗
PubMed2006

Discontinuous gas exchange in insects.

Insect respiratory physiology has been studied for many years, and interest in this area of insect biology has become revitalized recently for a number of reasons. Technical advances have greatly improved the precision, accuracy and ease with which gas exchange can be measured in insects. This has made it possible to go beyond classic models such as lepidopteran pupae and examine a far greater diversity of species. One striking result of recent work is the realization that insect gas exchange patterns are much more diverse than formerly recognized. Current work has also benefited from the inclusion of comparative methods that rigorously incorporate phylogenetic, ecological and life history information. We discuss these advances in the context of the classic respiratory pattern of insects, discontinuous gas exchange. This mode of gas exchange was exhaustively described in moth pupae in the 1950s and 1960s. Early workers concluded that discontinuous gas exchange was an adaptation to reduce respiratory water loss. This idea is no longer universally accepted, and several competing hypotheses have been proposed. We discuss the genesis of these alternative hypotheses, and we identify some of the predictions that might be used to test them. We are pleased to report that what was once a mature discipline, in which the broad parameters and adaptive significance of discontinuous gas exchange were thought to be well understood, is now a thriving and vigorous field of research.

Animals↗
PubMed2006

Continuity versus split and reconstitution: exploring the molecular developmental corollaries of insect eye primordium evolution.

Holometabolous insects like Drosophila proceed through two phases of visual system development. The embryonic phase generates simple eyes of the larva. The postembryonic phase produces the adult specific compound eyes during late larval development and pupation. In primitive insects, by contrast, eye development persists seemingly continuously from embryogenesis through the end of postembryogenesis. Comparative literature suggests that the evolutionary transition from continuous to biphasic eye development occurred via transient developmental arrest. This review investigates how the developmental arrest model relates to the gene networks regulating larval and adult eye development in Drosophila, and embryonic compound eye development in primitive insects. Consistent with the developmental arrest model, the available data suggest that the determination of the anlage of the rudimentary Drosophila larval eye is homologous to the embryonic specification of the juvenile compound eye in directly developing insects while the Drosophila compound eye primordium is evolutionarily related to the yet little studied stem cell based postembryonic eye primordium of primitive insects.

Animals↗
PubMed1980

Measurement of the pyroelectric coefficient and permittivity on Rhododendron and Encephalartos leaves and on the insect Periplaneta americana.

Pyroelectric (PE) voltage responses to single square radiation signals absorbed in epidermal layers of the leaves of Encephalartos villosus and Rhododendron catawbiense and in the integument of the insect Periplaneta americana L. were investigated on dry preparations as well as on whole fresh leaves and live insects. Measurements of the initial slopes of the responses, the rise times, and the peak voltages were carried out with 10 interchangeable load resistors from 5M omega to 10 G omega, connected parallel to the samples. In encephalartos leaves this yielded for the permittivity epsilon and the PE coefficient lambda in the upper/lower epidermis, epsilon = 30/60 and lambda = 2/6 pC/cm2K, respectively, and in rhododendron, epsilon = 60/200 and lambda = 3/15 pC/cm2K. In periplaneta we found epsilon = 300 and lambda = 20 pC/cm2K for the abdomen rings, and epsilon = 1,000 and lambda = 350 pC/cm2K for the thorax of live insects. Generally, dry preparations of leaves, and especially of insects, showed smaller PE responses. The outer epidermis of live plant leaves and the integument of live insects appear to be polar and to exhibit pronounced PE properties.

Animals↗
PubMed1998

Carbonyl reduction of an anti-insect agent imidazole analogue of metyrapone in soil bacteria, invertebrate and vertebrate species.

Carbonyl reduction to the respective alcohol metabolites of the anti-insect agent imidazole analogue of metyrapone, NKI 42255 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone) and its parent compound metyrapone was characterized in subcellular fractions previously described bacterial and mammalian hydroxysteroid dehydrogenases/carbonyl from soil bacteria, as well as insect, invertebrate and teleost species. The enzymes involved in this metabolic step were characterized with respect to their cosubstrate specificities, inhibitor susceptibilities, and immunological crossreactivities with antibodies directed against reductases (HSD/CR). All fractions investigated rapidly reduced metyrapone, with highest specific activities found in insect, invertebrate and vertebrate fractions. Except for the insect fractions, all species examined reduced the NKI compound. Cosubstrate dependence and inhibitor specificities suggest that the enzymes described belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR). Immunological crossreactions to the previously established subgroup of HSD/CRs were found in trout liver microsomes and insect homogenates, but not in all bacterial extracts or earthworm microsomes. These findings suggest that the high CR activities found in these fractions belong to different subgroups of SDR or AKR.

Alcohol Oxidoreductases↗
PubMed2000

Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus.

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517-518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.

Animals↗
PubMed2000

Insect cells as HLA-restricted antigen-presenting cells for the IFN-gamma elispot assay.

Measurement of specific cellular immune responses in patients undergoing immunotherapy is difficult. Established approaches, including cytotoxicity (e.g., 51Cr release) and cytokine release assays, require in vitro culturing for several weeks or more of patients' peripheral blood mononuclear cells (PBMC) and the addition of exogenous cytokines. Therefore, the immunological response does not reflect in vivo conditions. To address these disadvantages, we have used an interferon-gamma (IFN-gamma) Elispot assay for detecting peptide-specific CD8(+) lymphocytes in PBMC. A limitation of this assay is the lack of a reproducible source of antigen-presenting cells (APCs). Currently available APCs often lead to significant background levels. It has been shown that transfected insect cells can express empty MHC class I molecules on their surface. We have transfected Drosophila melanogaster S2 cells and the Lepidopteran line Sf9 with the gene encoding human HLA-A2.1. We demonstrate that insect cells expressing a human HLA molecule effectively function as APCs in the IFN-gamma Elispot assay. Initially the feasibility of the assay was assessed using CD8(+) T cells from HLA-A2.1(+) donors with known reactivity against an HLA-A2.1-binding epitope of the influenza matrix protein. Use of insect cells as APCs abrogated background spots, increasing sensitivity. We further observed that a short-term prestimulation of PBMC with peptide-pulsed insect cells markedly enhanced the frequency of peptide-specific T cells that could be measured in the Elispot assay without increasing the background. This approach was then used to measure CD8(+) T cell reactivity to a peptide from tyrosinase, an antigen that is processed and presented by melanoma cells. Insect cells expressing human HLA molecules provide a standard APC for monitoring CD8(+) T cell responses to tumor and viral peptides during immunotherapy.

Animals↗
PubMed2000

Insect cellular reactions to the lipopolysaccharide component of the bacterium Serratia marcescens are mediated by eicosanoids.

Nodulation, which begins with the formation of cellular microaggregates, is the predominant cellular defense reaction to bacterial infections in insects. We suggested that these reactions to bacterial infections are mediated by eicosanoids. The lipopolysaccharide (LPS) component of some bacterial cells stimulates defense reactions in mammals and insects. Here, we report on experiments designed to test the hypothesis that eicosanoids mediate microaggregation reactions to LPS. Injections of LPS (purified from the bacterium, Serratia marcescens) into larvae of the tenebrionid beetle, Zophobas atratus, stimulated microaggregation reactions in a dose-dependent manner. Treatments with eicosanoid-biosynthesis inhibitors immediately prior to LPS challenge sharply reduced the microaggregation responses. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase and lipoxygenase reduced microaggregation, supporting our view that microaggregate formation involves lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 30min after injection, and microaggregation was significantly reduced, relative to control insects, over the following 90min. The dexamethasone effects were reversed by treating LPS-injected insects with the eicosanoid precursor, arachidonic acid. These findings indicate that cellular defense reactions to a specific component of bacterial cells are mediated by eicosanoids, and open up new possibilities for dissecting detailed hemocytic actions in insect immune reactions to bacterial infections.

Journal Article↗
PubMed2000

Organizational and activational effects of hormones on insect behavior.

The concepts of hormone organization and activation provide a framework for thinking about the influence of hormones on development, brain, and behavior in vertebrates. There is good evidence for activational effects of hormones on the nervous system and behavior in insects, but organizational effects are almost never discussed in the insect literature. This paper explores the utility of the concepts of hormonal organization and activation of behavior in insects. We describe the two concepts as developed from studies of vertebrates, review some insect examples that appear to fit this classification scheme, and consider how explicit use of the concept of organization might benefit studies of the insect brain and behavior.

Journal Article↗
PubMed2002

The toxin Tx4(6-1) from the spider Phoneutria nigriventer slows down Na(+) current inactivation in insect CNS via binding to receptor site 3.

Tx4(6-1) a neurotoxic peptide from the venom of the aggressive South American 'armed' spider Phoneutria nigriventer, has been previously isolated and sequenced. It shows no detectable activity in mice but affects the peripheral nervous system of insects by stimulating glutamate release at the neuromuscular junction. Here we investigate possible interactions of the toxin with voltage-activated sodium channels (Na(v)). We confirm that it is ineffective on mammalian Na(v) channels, and establish that it competes with the alpha-like toxin 125I-Bom IV, for binding on the site 3 of insect Na(v) channel (IC(50) value around 25nM). The physiological consequences of this binding to the insect Na(v) channel are shown by electrophysiology: Tx4(6-1) prolongs evoked axonal action potentials (APs) (<500&mgr;s duration in control). Prolonged 8-10ms or 'plateau' 500-800ms APs accompanied by repetitive firing at 80-150Hz are recorded after 4-8min of toxin action. This modification of evoked activity is due to a slowing down of sodium current inactivation. Effects of Tx4(6-1) on sodium current are compared with those of a typical scorpion alpha-toxin and of some other spider toxins active on insect Na(v) channels. At the end of long voltage pulses, the maintained inward sodium current may represent 50% of the peak current after scorpion alpha-toxin but only about 8-10% after spider toxins. To understand the slight differences in the effects of alpha-scorpion and spider toxins on the insect Na(v) channel, structural studies of toxin-channels interactions would be necessary.

Journal Article↗
PubMed1997

Purification, structure and activity of three insect toxins from Buthus occitanus tunetanus venom.

One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.

Action Potentials↗
PubMed1997

Spatio-temporal properties of motion detectors matched to low image velocities in hovering insects.

Our recent study [O'Carroll et al. (1996). Nature 382, 63-66) described a correlation between the spatio-temporal properties of motion detecting neurons in the optic lobes of flying insects and behaviour. We consider here theoretical properties of insect motion detectors at very low image velocities and measure spatial and temporal sensitivity of neurons in the lobula complex of two specialised hovering insects, the bee-fly Bombylius and the hummingbird hawkmoth, Macroglossum. The spatio-temporal optima of direction-selective neurons in these insects lie at lower velocities than those of other insects which we have studied, including large syrphid flies, which are also excellent hoverers. We argue that spatio-temporal optima reflect a compromise between the demands of diverse behaviour, which can involve prolonged periods of stationary, hovering flight followed by spectacular high speed pursuits of conspecifics. Males of the syrphid Eristalis which engage in such behaviour, have higher temporal frequency optima than females. High contrast sensitivity in these flies nevertheless results in reliable responses at very low image velocities. Neurons of Bombylius have two distinct velocity optima, suggesting that they sum inputs from two classes of motion correlator with different time constants. This also provides sensitivity to a large range of velocities.

Animals↗
PubMed2002

A method for recording behavior and multineuronal CNS activity from tethered insects flying in virtual space.

We describe a low cost, novel virtual reality-based insect flight simulator that combines visual, olfactory and mechanosensory stimuli with multichannel neurophysiological recording techniques. Three-dimensional visual environments were created using customized modifications of a first person flight simulator computer game. Experiments could be performed in open-loop, where the flying insect's movement through the environment is 'driven' by the human operator, or in closed-loop where the movement of the environment is controlled by optically sensed movements of the insect's abdomen. During flight, we recorded multineuronal activity from the ventral nerve cord between the brain and thoracic ganglia. Results show that in open-loop conditions, induced turns of the environment evoked characteristic compensatory optomotor responses. Coordination of wing and body kinematics was similar to that observed in free flight. In closed-loop conditions, the insect was able to navigate through the simulated environment and produce flight tracks in response to presentation of pheromone that resemble those observed in free flight. We discuss the effectiveness of this preparation and its utility for addressing specific questions of insect flight as well as general questions in neuroethology.

Animals↗
PubMed1997

Building a brain: developmental insights in insects.

Understanding the cellular, molecular and genetic mechanisms involved in building the brain remains one of the most challenging problems of neurobiology. In this article, we review recent work on the developmental mechanisms that generate the embryonic brain in insects. We compare some of the early developmental events that occur in the insect brain with those that operate during brain development in vertebrates and find that numerous parallels are present at both the cellular and the molecular levels. Thus, the roles of glial cells in prefiguring axon pathways, the function of pioneer neurons in establishing axon pathways, and the formation of a primary axon scaffolding are features of embryonic brain development in both insects and vertebrates. Moreover, at the molecular genetic level homologous regulatory genes control morphogenesis, regionalization and patterning during embryonic brain development in both insects and vertebrates. This indicates that there might be universal mechanisms for brain development, and that knowledge gained from Drosophila and other insects is relevant to our understanding of brain development in other more complex organisms, including man.

Animals↗
PubMed1997

Molecular biology of insect neuronal GABA receptors.

Ionotropic gamma-aminobutyric acid (GABA) receptors are distributed throughout the nervous systems of many insect species. As with their vertebrate counterparts, GABAA receptors and GABAC receptors, the binding of GABA to ionotropic insect receptors elicits a rapid, transient opening of anion-selective ion channels which is generally inhibitory. Although insect and vertebrate GABA receptors share a number of structural and functional similarities, their pharmacology differs in several aspects. Recent studies of cloned Drosophila melanogaster GABA receptors have clarified the contribution of particular subunits to these differences. Insect ionotropic GABA receptors are also the target of numerous insecticides and an insecticide-resistant form of a Drosophila GABA-receptor subunit has enhanced our understanding of the structure-function relationship of one aspect of pharmacology common to both insect and vertebrate GABA receptors, namely antagonism by the plant-derived toxin picrotoxinin.

Animals↗
PubMed1999

Insect immune activation by recombinant Galleria mellonella apolipophorin III(1).

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein. Its function, as currently understood, lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. Recent studies with native Galleria mellonella-apoLp-III gave first indications of an unexpected role of that protein in insect immune activation. Here we report the immune activation by the recombinant protein, documenting a newly discovered correlation between lipid physiology and immune defense in insects. The complete cDNA sequence of G. mellonella-apoLp-III was identified by mixed oligonucleotide-primed amplification of cDNA (MOPAC), 3'-RACE-PCR, and cRACE-PCR. The sequence coding for the native protein was ligated into a pET-vector; this construct was transfected into Escherichia coli and overexpressed in the bacteria. Photometric turbidity assays with human low density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs revealed the full functionality of the isolated recombinant apoLp-III with regard to its lipid-association ability. For proving its immune-stimulating capacity, apoLp-III was injected into the hemocoel of last instar G. mellonella larvae and the antibacterial activity in cell-free hemolymph was determined 24 h later. As a result, the hemolymph samples of injected insects contained strongly increased antibacterial activities against E. coli as well as clearly enhanced lysozyme-like activities. From Northern blot analysis of total RNA from insects injected with apoLp-III or the bacterial immune provocator lipopolysaccharide, it could be concluded that the transcription rate of apoLp-III mRNA does not vary in comparison to untreated last instar larvae.

Amino Acid Sequence↗
PubMed2003

Improvement of glycosylation in insect cells with mammalian glycosyltransferases.

The N-glycans of recombinant glycoproteins expressed in insect cells mainly contain high mannose or tri-mannose structures, which are truncated forms of the sialylated N-glycans found in mammalian cells. Because asialylated glycoproteins have a shorter half-life in blood circulation, we investigated if sialylated therapeutic glycoprotein can be produced from insect cells by enhancing the N-glycosylation machinery of the cells. We co-expressed in two insect cell lines, Sf9 and Ea4, the human alpha1-antitrypsin (halpha1AT) protein with a series of key glycosyltransferases, including GlcNAc transferase II (GnT2), beta1,4-galactosyltransferase (beta14GT), and alpha2,6-sialyltransferase (alpha26ST) by a single recombinant baculovirus. We demonstrated that the enhancement of N-glycosylation is cell type-dependent and is more efficient in Ea4 than Sf9 cells. Glycan analysis indicated that sialylated halpha1AT proteins were produced in Ea4 insect cells expressing the above-mentioned exogenous glycosyltransferases. Therefore, our expression strategy may simplify the production of humanized therapeutic glycoproteins by improving the N-glycosylation pathway in specific insect cells, with an ensemble of exogenous glycosyltransferases in a single recombinant baculovirus.

Animals↗
PubMed2002

Occurrence of insect kinins in the flesh fly, stable fly and horn fly-mass spectrometric identification from single nerves and diuretic activity.

MALDI-TOF mass spectrometric analysis of single lateral abdominal nerves (LANs) demonstrate the presence of the insect kinin Musdo-K in the housefly Musca domestica, and identify heretofore unknown insect kinins in two other Dipteran species as Musdo-K in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans. The insect kinin native to the flesh fly Neobellieria bullata is identified as Drome-K. Musdo-K and Drome-K are identical save for the conservative substitution of Ser for Thr in position 2. The sequences of the insect kinins are, therefore, remarkably conserved throughout Dipterans. The in vitro Malpighian tubule fluid secretion activity of Musdo-K in the stable fly is similar to that in the housefly, whereas that of Drome-K is 30-fold more potent in the flesh fly than in the fruit fly. Given the structural identities of the kinins and CRF-like diuretic hormones of these Dipteran species, the housefly can serve as a model insect for the study of diuretic peptides and their functions in the stable fly and horn fly, both livestock pests.

Amino Acid Sequence↗