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Characterisation of an avian influenza virus nucleoprotein expressed in E. coli and in insect cells.

The nucleoprotein (NP) gene from influenza virus A/Shearwater/Australia/72 has been expressed intracellularly in both E. coli and insect cells. E. coli-derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP. This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive- and negative-sense influenza neuraminidase RNA transcribed in vitro. ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector. Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.

Animals↗

An evolutionary model for the insect vitellins.

Insects can be divided into three groups based on the sizes of the polypeptide constituents of their vitellogenins and vitellins. In order to determine the relationships between these groups, antisera to the vitellins of seven insects from six taxonomic orders were used to assess immunological cross-reactivity. Antigenic relatedness was observed only between vitellins from species within the same family. Amino acid compositional data for vitellins from nine species were used to assess homology by difference matrices. The S delta Q values were similar for both intra-order and inter-order comparisons and strongly suggested relatedness. The S delta n comparisons supported the immunological data that indicated that the vitellins were evolving rapidly. For most insect vitellins there are two distinct size classes of polypeptides that seem to be derived from a single asymmetric proteolytic cleavage of a precursor. We propose a model that suggests that the different size polypeptides represent distinct domains and that in the evolution of the vitellogenin genes of the Diptera and Hymenoptera there has been domain elimination.

Amino Acid Sequence↗

Agglutinins from aquatic insects--tumor cell agglutination activity.

Agglutinins were identified in whole body extracts of aquatic insects by means of murine tumor cell agglutination, using sarcoma 180 ascites, Ehrlich, and MM-46 cells. Screening revealed agglutinins in 5 of 10 of the larvae tested, and in 2 of 6 of the water-dwelling adult insects; Gerris paludum insularis and Gyrinus japonicus. Only the agglutinin from adult G. paludum also agglutinated human erythrocytes. An ascites tumor was converted into a solid form in vivo after administration of G. paludum agglutinin. The observation that these aquatic insect agglutinins preferentially agglutinate tumor cells has considerable implications in terms of anti-tumor effects such as inhibition of cell proliferation and metastasis.

Agglutination↗

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3 beta-reductase mediated conversion to ecdysone in representative insects.

The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3 beta-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.

3-Hydroxysteroid Dehydrogenases↗

Extreme rates and heterogeneity in insect DNA evolution.

DNA-DNA hybridization studies of insects, more specifically Drosophila and cave crickets, have revealed interesting patterns of genome evolution that contrast markedly with what has been seen in other taxa, especially mammals and birds. Insect genomes are composed of sections of single-copy DNA with extreme variation in rates of evolutionary change. This variation is more extreme than between introns and exons; introns fall into the relatively conserved fraction of the genome. Attempts to calculate absolute rates of change in Drosophila DNA have all led to estimates some 5-10 times faster than those found in most vertebrates; this is true even for the more conservative part of the nuclear genome. Finally we point out that morphological similarity, chromosomal similarity, and/or ability to form interspecific hybrids is often associated with quite high levels of single-copy DNA divergence in insects as compared to mammals and birds.

Animals↗

Three-dimensional model of the insect-directed scorpion toxin from Androctonus australis Hector and its implication for the evolution of scorpion toxins in general.

The three-dimensional structure of the insect-directed toxin from the scorpion Androctonus australis Hector has been modelled using computer graphics and energy-minimization techniques. The model-building procedure was based on the known high resolution structures of two scorpion toxins of different types: toxin II from A. australis Hector, an alpha-toxin, and variant 3 from Centruroides sculpturatus Ewing that belongs to the beta-toxin structural group. Although the insect-directed toxin has one atypical disulfide bridge, the general structural features of the scorpion toxin family, including the presence of a "conserved-hydrophobic" surface, seem to be well-conserved. However, the orientation and length of some loops and regions thought to be important for toxicity are different for alpha-toxins, beta-toxins, and the insect-directed toxin. Thus, the binding of a scorpion toxin to its site on the Na+ channel seems to be based on (1) the presence of a surface containing a series of conserved and/or hydrophobic residues, more or less common to all these molecules, and (2) an adjacent area that modulates the specificity of the interaction.

Amino Acid Sequence↗

Protein quality of raw and autoclaved cowpeas: comparison between some insect resistant and susceptible varieties.

A comparative study was conducted with four improved cowpea (2 pod bug resistant and 2 susceptible) varieties to assess the effect of autoclaving at 105 degrees C under 15 psi for 30 min on their protein quality, as measured by the levels of some antinutritional factors and performance of rats fed the different cowpea diets. Also, the relationships between antinutrients in the cowpeas and protein quality indicators were examined. Results showed that autoclaving completely eliminated trypsin inhibitor, hemagglutinin and HCN, whereas it reduced significantly (p < 0.01) the levels of phytic and tannic acids in the cowpeas. In both raw and autoclaved forms, the insect susceptible cowpeas were better (p < 0.05) utilized than their resistant counterparts, a condition which was attributable to lower levels of antinutrients in the susceptible varieties. In the raw cowpeas, trypsin inhibitor, hemagglutinin and HCN were significant variables affecting cowpea protein utilization, while in autoclaved samples, tannic and phytic acids were important though nonsignificant (p > 0.05) factors. It was concluded that autoclaving at the above temperature and time improved the protein quality of the insect susceptible more than the insect resistant cowpea varieties.

Animals↗

Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin.

During postembryonic development of insects, molting cycles affect epidermal cells with alternate periods of proliferation and differentiation. Cells of the cell line established from imaginal discs of the Indian meal moth (IAL-PID2) differentiate under the action of the molting hormone, 20-hydroxyecdysone, in a manner that is meaningful in terms of the development of the tissue from which they were derived. In particular, the hormone caused an accumulation of the cells in the G2 phase of their cycle and induced the formation of epithelial-like aggregates and the synthesis of specific proteoglycans. Recent discovery of members of the insulin superfamily in insects and the role of growth factors played by this family of molecules in vertebrates led us to check for their potential effects on IAL-PID2 cell cycle regulation. On the one hand, our results showed that insulin was involved in partial resumption of the cell cycle after an arrest caused by serum deprivation, but that other growth factors present in fetal calf serum were needed for full completion of mitosis. On the other hand, the cytostatic effect of 20-hydroxyecdysone was reversible, and, prior exposure of the cells to the hormone allowed the cells to complete one cell cycle in serum-free medium. These results suggest that the production of autocrine growth factors induced by ecdysteroids could circumvent the absence of serum. This cell culture model provides potential for further study of interactions between ecdysteroids and growth factor homologs during differentiation of insect epidermal cells.

Animals↗

Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein.

The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinant Autographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established from Spodoptera frugiperda, Trichoplusia ni, and Mamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in the M. brassicae-derived MB0503 cell line was Endo H resistant. The commonly used S. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.

1-Deoxynojirimycin↗

Production of ethanol by cultured insect cells.

Proton Nuclear Magnetic Resonance (1H-NMR) Analysis of insect cell culture media used for cultivating insect cell lines derived from the fleshfly Sarcophaga peregrina, swallowtail butterfly Papilio xuthus, and cabbage armyworm Mamestra brassicae revealed that ethanol appeared in the medium as the cultures aged. By incorporating [13C-1]-glucose into the media, we pursued 13C-NMR spectrograms to show that the ethanol was derived from glucose. Thus, it became evident that the insect cells cultured in vitro produce ethanol from glucose as a metabolite.

Animals↗

Antigenic properties of recombinant glycosylated and nonglycosylated Pneumocystis carinii glycoprotein A polypeptides expressed in baculovirus-infected insect cells.

Since a continuous culture system is not yet available for the opportunistic fungal pathogen Pneumocystis carinii, obtaining suitable amounts of purified P. carinii antigens free of mammalian-host lung contaminants is difficult. Hence, production of recombinant antigen possessing epitopes found in native P. carinii antigens is critical for immunological studies. We utilized the baculovirus expression vector system (BEVS) in insect cells to determine whether B-cell epitopes present in the protein core of a native P. carinii surface glycoprotein were conserved in the recombinant polypeptide, and to investigate its glycosylation by insect cells. B-cell epitopes were retained, but the insect cells appeared to hyperglycosylate the recombinant protein.

Animals↗

Cytotoxicity and L-amino acid oxidase activity of crude insect drugs.

The cytotoxicity of crude insect drugs was measured using HeLa cells originating from human cervix and uterine cancer, using the dye uptake assay in order to find potential anticancer agents. Three kinds of extracts (buffer, methanol and ethylacetate) were prepared from 26 insects and used as raw materials for the activity assay. Among these, the buffer extracts from Tabanus, Mylabris and Huechys showed a potent anticancer activity, and those from Catharsius, Red ant, Scorpion, Tabanus and Vespae Nidus showed a strong L-amino acid oxidase (AAO) activity as well as cytotoxicity. In contrast, buffer extracts from Gryllotalpa orientalis and Apriona germari larvae showed greater/more rapid Hela cell growth than that of other insects.

Amino Acid Oxidoreductases↗

Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB.

The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by Western blot analysis. The role of the kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase). The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death. Moreover, NO-induced apoptosis involves the release of cytochrome c.

Androstadienes↗

Cycads: their evolution, toxins, herbivores and insect pollinators.

Palaeobiological evidence indicates that gymnosperms were wind-pollinated and that insect pollination began in angiosperms in the Lower Cretaceous (ca. 135 mya) leading to close associations between higher plants and their pollinators. Cycads, which were widespread and pervasive throughout the Mesozoic (250-65 mya) are among the most primitive living seed-plants found today. Because pollination by beetles and by thrips has now been detected in several modern cycads, it is attractive to speculate that some insects and cycads had already developed similar mutualistic interactions in the Triassic (250-205 mya), long before the advent of angiosperms. We also draw attention to another key factor in this insect-plant relationship, namely secondary, defensive plant substances which must always have controlled interspecific interactions. Cycads mainly produce toxic azoglucosides and neurotoxic non-protein amino acids (e.g. BMAA), which apparently are crucial elements in the development and maintenance of mutualism (pollination) and parasitism (herbivory) by cycad-linked herbivores. We now add new results on the uptake and storage of the main toxin, cycasin, of the Mexican cycad Zamia furfuracea by its pollinator, the weevil Rhopalotria mollis, and by a specialist herbivore of Zamia integrifolia, the aposematic Atala butterfly Eumaeus atala.

Animals↗

Slanted joint axes of the stick insect antenna: an adaptation to tactile acuity.

Like many flightless, obligatory walking insects, the stick insect Carausius morosus makes intensive use of active antennal movements for tactile near range exploration and orientation. The antennal joints of C. morosus have a peculiar oblique and non-orthogonal joint axis arrangement. Moreover, this arrangement is known to differ from that in crickets (Ensifera), locusts (Caelifera) and cockroaches (Blattodea), all of which have an orthogonal joint axis arrangement. Our hypothesis was that the situation found in C. morosus represents an important evolutionary trait of the order of stick and leaf insects (Phasmatodea). If this was true, it should be common to other species of the Phasmatodea. The objective of this comparative study was to resolve this question. We have measured the joint axis orientation of the head-scape and scape-pedicel joints along with other parameters that affect the tactile efficiency of the antenna. The obtained result was a complete kinematic description of the antenna. This was used to determine the size and location of kinematic out-of-reach zones, which are indicators of tactile acuity. We show that the oblique and non-orthogonal arrangement is common to eight species from six sub-families indicating that it is a synapomorphic character of the Euphasmatodea. This character can improve tactile acuity compared to the situation in crickets, locusts and cockroaches. Finally, because molecular data of a recent study indicate that the Phasmatodea may have evolved as flightless, obligatory walkers, we argue that the antennal joint axis arrangement of the Euphasmatodea reflects an evolutionary adaptation to tactile near range exploration during terrestrial locomotion.

Animals↗

Functional roles of neuropeptides in the insect central nervous system.

With the completion of the Drosophila genome sequencing project we can begin to appreciate the extent of the complexity in the components involved in signal transfer and modulation in the nervous system of an animal with reasonably complex behavior. Of all the different classes of signaling substances utilized by the nervous system, the neuropeptides are the most diverse structurally and functionally. Thus peptidergic mechanisms of action in the central nervous system need to be analyzed in the context of the neuronal circuits in which they act and generalized traits cannot be established. By taking advantage of Drosophila molecular genetics and the presence of identifiable neurons, it has been possible to interfere with peptidergic signaling in small populations of central neurons and monitor the consequences on behavior. These studies and experiments on other insects with large identifiable neurons, permitting cellular analysis of signaling mechanisms, have outlined important principles for temporal and spatial action of neuropeptides in outputs of the circadian clock and in orchestrating molting behavior. Considering the large number of neuropeptides available in each insect species and their diverse distribution patterns, it is to be expected that different neuropeptides play roles in most aspects of insect physiology and behavior.

Animals↗

Evolution and function of auditory systems in insects.

While the sensing of substrate vibrations is common among arthropods, the reception of sound pressure waves is an adaptation restricted to insects, which has arisen independently several times in different orders. Wherever studied, tympanal organs were shown to derive from chordotonal precursors, which were modified such that mechanosensitive scolopidia became attached to thin cuticular membranes backed by air-filled tracheal cavities (except in lacewings). The behavioural context in which hearing has evolved has strongly determined the design and properties of the auditory system. Hearing organs which have evolved in the context of predator avoidance are highly sensitive, preferentially in a broad range of ultrasound frequencies, which release rapid escape manoeuvres. Hearing in the context of communication does not only require recognition and discrimination of highly specific song patterns but also their localisation. Typically, the spectrum of the conspecific signals matches the best sensitivity of the receiver. Directionality is achieved by means of sophisticated peripheral structures and is further enhanced by neuronal processing. Side-specific gain control typically allows the insect to encode the loudest signal on each side. The filtered information is transmitted to the brain, where the final steps of pattern recognition and localisation occur. The outputs of such filter networks, modulated or gated by further processes (subsumed by the term motivation), trigger command neurones for specific behaviours. Altogether, the many improvements opportunistically evolved at any stage of acoustic information-processing ultimately allow insects to come up with astonishing acoustic performances similar to those achieved by vertebrates.

Animals↗

Rapid quantification of juvenile hormones and their metabolites in insect haemolymph by liquid chromatography-mass spectrometry (LC-MS).

A simple, fast and sensitive method was developed for routine determination of juvenile hormone (JH), JH diols and JH acids in insect haemolymph, by liquid chromatography-mass spectrometry (LC-MS). Sample clean-up involves the precipitation of proteins by methanol/isooctane (1:1, v/v), centrifugation and partial evaporation of the organic solvents. Since JH is bound to a carrier protein in the haemolymph, a binding protein (BP) assay was performed to ensure JH is removed during precipitation. The JH compounds were separated on a C(18) column (ReproSil-Pur ODS-3) by gradient elution with water and methanol in less than 22 min and analysed by electrospray mass spectrometry. Due to the high abundance of Na(+) in insect haemolymph, [M+Na](+) is primarily formed. The limit of detection and quantification was 6 and 20 pg for JHs, and 8 and 25 pg for JH diols, respectively. To demonstrate the applicability of the method to different insect orders, haemolymph samples from the Mediterranean field cricket ( Gryllus bimaculatus), the fall armyworm ( Spodoptera frugiperda), the pea aphid ( Acyrthosiphon pisum) and an ant species ( Myrmicaria eumenoides) were analysed.

Animals↗