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PubMed2003

The xenobiotic transporter, MRP2, in epithelia from insects, sharks, and the human breast: implications for health and disease.

A large number of mechanisms, including special excretory transporters, have evolved to help organisms excrete deleterious xenobiotics and endogenous molecules. We have examined the xenobiotic transport function of a putative multidrug resistance associated protein, MRP2, in three different epithelia: the insect renal (Malpighian) tubules, the secretory tubule of the shark rectal gland, and in ductules of the human breast. In the case of the insect and shark, transporter activity occurs in epithelia capable of great fluid transport. In the case of the insect Malpighian tubule, understanding the underlying mechanisms of this transporter may help with efforts to control populations of disease-carrying agriculturally important insects. In striking contrast, ductule architecture in nonlactating human breast ductules is that of an epithelium with a closed lumen. Immunocytochemical studies show that MRP2 is localized in the apical region of the ductule epithelial cells. In this unique case, MRP2 substrates transported into the lumen could possibly be concentrated. Transport substrates of MRP2 include carcinogens as well as antioxidants and other salutary molecules. Thus, in the breast ductule, MRP2 may play a significant role in breast epithelial cell health and cancer carcinogenesis.

Animals↗
PubMed2001

Structure and diversity of insect nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) is an agonist-regulated ion-channel complex responsible for rapid neurotransmission. The vertebrate nAChR, assembled from five homologous subunits, penetrates the synaptic membrane. Different subunit combinations lead to receptor subtypes with distinctive pharmacological profiles. In comparison with mammalian nAChRs, the insect receptor is poorly understood relative to functional architecture and diversity. Several genes for Drosophila, Locusta and Myzus encoding insect nAChR subunits have been identified, although the functional assembly and presence of different subtypes of these receptors are not defined. The insect nAChR is the primary target site for the neonicotinoid insecticides, thereby providing an incentive to explore its functional architecture with neonicotinoid radioligands, photoaffinity probes and affinity chromatography matrices. This review considers the current understanding of the structure and diversity of insect nAChRs based mainly on recent studies in molecular biology and protein biochemistry.

Animals↗
PubMed2001

Relationships between sulfonylurea herbicide treatment of host plants and the performance of herbivorous insects.

Previous work had shown that the sulfonylurea herbicide chlorsulfuron affected the survival of a herbivorous insect species dwelling on a sub-lethally exposed host plant. Further experiments have been conducted to establish whether this negative effect was a single occurrence characteristics for the specific insect-plant interaction and the specific herbicide tested. Three insect-plant interactions were tested for the effects of selected sulfonylurea herbicides, i.e. metsulfuron-methyl, chlorsulfuron and tribenuron-methyl. The species pairs tested were Pieris brassicae/Brassica napus, Gastrophysa polygoni/Fallopia convolvulus and Sitobium avenae/Triticum aestivium. No significant effects on survival and relative growth rate of P brassicae or G polygoni were found when treating the host plants with sulfonylurea herbicides. However, the host plants had a significantly reduced root and shoot growth rate when treated with herbicide. Treating T aestivium with the recommended field rate of metasulfuron-methyl did not cause any change in development time, growth rate or fecundity of S avenae feeding on the host plants. The data presented suggest that the increased mortality observed for G polygoni larvae feeding on chlorsulfuron-treated host plants observed earlier was characteristic for this herbicide and for the specific plant-insect interaction only.

Analysis of Variance↗
PubMed2002

Activation of octopaminergic receptors by essential oil constituents isolated from aromatic plants: possible mode of action against insect pests.

As a result of screening a large number of essential oils from Israeli aromatic plants and their biologically active constituents, we isolated two oils with high activity against several stored-product insects. In this study the effect of these compounds on the acetylcholinesterase and the octopamine systems in insects was studied in order to elucidate their mode of action. Inhibition of acetylcholinesterase activity in vitro was evident only at high concentrations (10(-3) M) and could not account effectively for the low-dose mortality for some stored-product insects observed in vivo. However, the essential oil constituents were found to cause a significant increase in the levels of the intracellular messenger, cyclic AMP of abdominal epidermal tissue in the model insect, Helicoverpa armigera Hübn. The effect was significant even at low, physiological concentrations (10(-8) M) when tested directly on abdominal epidermal tissue preparations in vitro. This intracellular response was found to resemble closely the significant increases in the levels of the cyclic AMP of abdominal epidermal tissue due to treatment with the neurotransmitter/neuromodulator, octopamine. Subsequent treatment with the octopaminergic antagonist, phentolamine, effectively inhibited the cyclic AMP levels induced by essential oil treatment, indicating possible competitive activation of octopaminergic receptors by essential oil constituents.

Acetylcholinesterase↗
PubMedDate not supplied

United States Department of Agriculture-Agricultural Research Service research on managing insect resistance to insecticides.

Insecticide resistance has developed within many classes of pesticide, and over 500 species of insects and mites are resistant to one or more insecticides. Insecticide resistance and the consequent losses of food and fiber caused by failure to control insect and mite pests causes economic losses of several billion dollars worldwide each year. It is the goal of insect resistance management (IRM) to preserve useful pesticides by slowing, preventing or reversing development of resistance in pests. Important aspects of this goal are understanding the development of resistance and monitoring to determine ways to prevent its development. We describe programs specific to missions of the US Department of Agriculture, Agricultural Research Service, which are designed to characterize insecticide resistance in insects and mites with the goal of managing pests in an ecologically acceptable manner. Resistance management of cotton, potatoes, vegetables, melons, ornamentals, greenhouse crops, corn, stored grains, livestock, honeybees and mites, as well as management of transgenic crops are evaluated. We conclude that IRM is a vital part of stewardship of any pest management product and must be a combined effort of manufacturers, growers, consultants, extension services and grower organizations, working closely with regulators, to achieve logistically and economically feasible systems that prolong the effectiveness of all pest-control products.

Agriculture↗
PubMedDate not supplied

United States Department of Agriculture-Agricultural Research Service: advances in the molecular genetic analysis of insects and their application to pest management.

USDA-ARS scientists have made important contributions to the molecular genetic analysis of agriculturally important insects, and have been in the forefront of using this information for the development of new pest management strategies. Advances have been made in the identification and analysis of genetic systems involved in insect development, reproduction and behavior which enable the identification of new targets for control, as well as the development of highly specific insecticidal products. Other studies have been on the leading edge of developing gene transfer technology to better elucidate these biological processes though functional genomics and to develop new transgenic strains for biological control. Important contributions have also been made to the development and use of molecular markers and methodologies to identify and track insect populations. The use of molecular genetic technology and strategies will become increasingly important to pest management as genomic sequencing information becomes available from important pest insects, their targets and other associated organisms.

Agriculture↗
PubMed2007

A simple purification protocol for the detection of peptide hormones in the hemolymph of individual insects by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The endocrine system of insects is largely based on peptide hormones. Nevertheless, an unequivocal chemical demonstration of the occurence in the hemolymph (the 'insect blood') is still lacking for most if not all insect peptide hormones, although this is the only way to prove their hormonal status. Focusing on peptides released during ecdysis behavior of the tobacco hornworm Manduca sexta, we developed a purification protocol based on ultrafiltration and a single reversed-phase high-performance liquid chromatography (RP-HPLC) step that for the first time allowed the mass spectrometric and chemical identification of a peptide hormone in the hemolymph of single specimens. Since this method is simple, relatively cheap and fast, it should be useful for routine endocrinological analyses and for monitoring peptide release during different physiological conditions and behaviors in insects.

Animals↗
PubMed1997

Viral infection. II. Hemin induces overexpression of p67 as it partially prevents appearance of an active p67-deglycosylase in baculovirus-infected insect cells.

The roles of p67-deglycosylase (p67-DG) in the regulation of protein synthesis in baculovirus-infected insect cells were studied. Like vaccinia viral infection, baculovirus infection of insect cells also induced the appearance of a p67-DG. However, p67-DG activity could not be detected because these cells do not contain a detectable level of p67. The baculovirus expression vector system (BEVS), however, promotes significant expression of cloned p67-cDNA. The expression of p67 was significantly enhanced by the addition of hemin to the growth medium. Maximum enhancement was observed at 5 microM hemin. Data suggest that hemin prevents the activation of latent p67-DG inside the cell and does not have any effect on p67 gene transcription. To gain a better understanding of the mechanism of p67-DG activation and hemin stimulation of p67 synthesis, we have now purified p67-DG from baculovirus-infected insect cells. We prepared antibodies against this protein. These antibodies reacted with a 105-kDa protein in cell extracts from the uninfected insect cells (Sf9), KRC-7, and L929 (animal cells). In addition, these antibodies reacted with an additional 60-kDa protein in the cell extracts of baculovirus-infected Sf9 cells and vaccinia virus-infected KRC-7 and L929 cells. Data are also presented to show that the antibodies against p67-DG reacted more efficiently (40%) with the 60-kDa protein in both hemin-deficient reticulocyte lysate and hemin-deficient baculovirus-infected cells. We suggest that hemin prevents the conversion of an inactive p67-DG into an active form possibly by covalent modification such as protein phosphorylation or protein glycosylation. The active form is more efficiently recognized by the p67-DG antibodies since these antibodies were prepared against the active form of p67-DG.

Acetylglucosaminidase↗
PubMed1997

Characterization of insect cell lines: heteroduplex analysis employing a mitochondrial 16S ribosomal RNA gene fragment.

Routine cell line characterization procedures are not adequate for characterizing the cell lines of insect origin. Ribosomal RNA (rRNA) gene sequences and their comparisons have been used successfully for delineating species and phylogenetic analysis. Using similar principles, we have standardized a protocol for the confirmation of species identity of insect cell lines. The procedure includes PCR amplification of the mitochondrial 16S rRNA gene fragment from the cell line and larvae of known insect species and heteroduplex analysis to detect the sequence variation in the PCR-amplified rRNA gene fragments. If the PCR fragment of the cell lines yields a homoduplex with the larvae of known species, then the cell line is conspecific with the larvae. If the larvae and cell line are of two different species, then the analysis exhibits multiple bands of heteroduplexes. The technique also allows detection of cross-contamination of culture having two insect cell lines belonging to two different species.

Aedes↗
PubMed2000

The evolution of polyandry: multiple mating and female fitness in insects.

Theory suggests that male fitness generally increases steadily with mating rate, while one or a few matings are sufficient for females to maximize their reproductive success. Contrary to these predictions, however, females of the majority of insects mate multiply. We performed a meta-analysis of 122 experimental studies addressing the direct effects of multiple mating on female fitness in insects. Our results clearly show that females gain directly from multiple matings in terms of increased lifetime offspring production. Despite a negative effect of remating on female longevity in species without nuptial feeding, the positive effects (increased egg production rate and fertility) more than outweigh this negative effect for moderate mating rates. The average direct net fitness gain of multiple mating was as high as 30-70%. Therefore, the evolutionary maintenance of polyandry in insects can be understood solely in terms of direct effects. However, our results also strongly support the existence of an intermediate optimal female mating rate, beyond which a further elevated mating rate is deleterious. The existence of such optima implies that sexual conflict over the mating rate should be very common in insects, and that sexually antagonistic coevolution plays a key role in the evolution of mating systems and of many reproductive traits. We discuss the origin and maintenance of nuptial feeing in the light of our findings, and suggest that elaborate and nutritional ejaculates may be the result of sexually antagonistic coevolution. Future research should aim at gaining a quantitative understanding of the evolution of female mating rates. Copyright 2000 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed2000

Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells.

YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Cellular toxicities and membrane binding characteristics of insecticidal crystal proteins from Bacillus thuringiensis toward cultured insect cells.

The pattern of in vitro toxicity of activated toxins from several classes of entomocidal inclusion genes from Bacillus thuringiensis was measured using eight established cell lines from lepidopteran insects. Protoxins representing CryIA(b), CryIA(c), and a mixture of all three CryIA toxins (subtypes a, b, and c; from B. thuringiensis subsp. kurstaki HD-1) were compared with the protoxin representing CryIC in a bioassay which measured the viability of cultured insect cells upon exposure to entomocidal toxin proteins. The responses of the various cell lines were very specific toward the individual toxin proteins. CryIC activated protoxin was toxic for cells of Manduca sexta, Plodia interpunctella, and to a lesser extent Spodoptera frugiperda. CryIA(b) and CryIA(c) proteins were toxic toward M. sexta but relatively non-toxic for P. interpunctella or S. frugiperda. The toxicity of CryIA(b), CryIA(c), and the composite CryIA activated toxins toward cells of Choristoneura fumiferana varied substantially, with the CryIA mixture being slightly more toxic than CryIA(c) alone. CryIC toxin had no effect toward C. fumiferana cells. Probit regression analysis of dose-response relationships between insect species and crystal protein composition demonstrated specific patterns of toxicity which may be related to membrane-receptor site binding by specific toxins. Membrane binding analysis of 125I-labeled CryIA(b), CryIA(c), and CryIC toxins to insect cells from three of the cell lines yielded high specific binding only with M. sexta cells toward CryIA(c) toxin. Lower levels of binding were observed with CryIA(b) and CryIC toward cells of C. fumiferana and P. interpunctella. Although relatively low binding levels for CryIC were observed with P. interpunctella cells, toxicity was high for these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

A nonpermissive entomophthoralean fungal infection increases activation of insect prophenoloxidase

Entomophaga maimaiga and Entomophaga aulicae are entomopathogenic fungi that show species-specific infection in Lepidoptera. These fungi grow as protoplasts in the hemolymph of permissive insect hosts. E. maimaiga infects gypsy moth larvae, Lymantria dispar, and E. aulicae infects hemlock looper, Lambdina fiscellaria. Cross-infections do not occur and are referred to as a nonpermissive response. We circumvented cuticular barriers and injected E. aulicae protoplasts into L. dispar and investigated factors which could potentially curtail entomophthoralean fungal infection in a nonpermissive host insect. There was no evidence of (i) restricted fungal growth in cell-free hemolymph of the nonpermissive host, (ii) fungal toxins specific to host insect, (iii) hemocyte encapsulation of fungal protoplasts in a nonpermissive or permissive infection, or (iv) fungal-specific induction of plasma proteins in L. dispar. However, higher levels of phenoloxidase activity for up to 96 h postchallenge, as well as a prophenoloxidase-activating trypsin activity, were observed for L. dispar challenged with E. aulicae when compared to an E. maimaiga challenge. Three isoforms of phenoloxidase (pI 5.0-5.5) and at least six isoforms of trypsin activity (four basic trypsins pI 8-10 and two acidic trypsins pI 4-6) with preferences for small amino acid residues were activated in L. dispar after challenge. In vitro prophenoloxidase activation experiments showed that treatment of L. dispar hemolymph with E. aulicae protoplast plasma membranes consistently resulted in higher prophenoloxidase activation than E. maimaiga. We suggest that differences in protoplastic components of Entomophaga species, such as the surface glycoproteins, are implicated in activation of zymogenic trypsins in the insect which in turn activate the prophenoloxidase cascade as a nonpermissive response. Copyright 1998 Academic Press.

Journal Article↗
PubMed1999

Aggregation of entomopathogenic nematodes, heterorhabditis spp. and steinernema spp., among host insects at 9 and 20 degrees C and effects on efficacy

The negative binomial distribution (NBD) with density-dependent k was used to test and describe the distribution of nematodes in Otiorhynchus sulcatus, Galleria mellonella, and Spodoptera exigua at 9 and 20 degrees C. Aggregation was greater in O. sulcatus than in the other two hosts. In case of G. mellonella exposed to the heterorhabditid HF85, the distribution of nematodes in the insects was random. There was only a small effect of low temperature on aggregation; this effect was stronger for O. sulcatus than for the other two hosts. Aggregation of nematodes among insects in a population increases the infection chance of already infected insects and decreases the infection chance of uninfected insects. Consequently, more nematodes will have to penetrate O. sulcatus than G. mellonella or S. exigua to cause the same proportion of infection in a population. The relevance of aggregation of nematodes for control O. sulcatus is discussed. Copyright 1999 Academic Press.

Journal Article↗
PubMed1996

Three-dimensional structure of nucleotide-bearing crossbridges in situ: oblique section reconstruction of insect flight muscle in AMPPNP at 23 degrees C.

We have explored the three-dimensional structure of myosin crossbridges in situ in order to define the structural changes that occur when nucleotide binds to the myosin motor. When AMPPNP binds to rigor insect flight muscle, each half sarcomere lengthens by approximately 2.0 nm and tension is reduced by approximately 70% without a reduction in stiffness, suggesting partial reversal of the power stroke. We have obtained averaged oblique section three-dimensional reconstructions of mechanically monitored insect flight muscle in AMPPNP that permit simultaneous examination of all myosin crossbridges within the unit cell and direct comparison of calculated transforms with X-ray diagrams of the native fibers. Transforms calculated from the oblique section reconstruction of AMPPNP insect flight muscle at 23 degrees C show good agreement with native X-ray diagrams, suggesting that the average crossbridge forms in the reconstruction reflect the native structure. In contrast to the rigor lead and rear crossbridges in the double chevrons, the averaged reconstruction of AMPPNP fibers show only one crossbridge class, in the position of the rigor lead bridge. The portion of the crossbridge close to the thick filament appears broader than in rigor, and shows a small 0.5 to 1.0 nm M-ward shift of the regulatory domain region of myosin. In transverse view, AMPPNP "lead" crossbridges are less azimuthally bent than rigor. Fitting the atomic model of actomyosin subfragment 1 to the averaged crossbridges shows that the detectable differences between rigor bridges and between rigor and AMPPNP bridges occur in the alignment and angles of the regulatory domains and suggests that rear bridges are more strained than lead crossbridges. The apparent absence of rear bridges in AMPPNP in averaged reconstructions indicates detachment of a number of force-bearing bridges, which conflicts with the maintained stiffness of the fibers used for the reconstruction. This conflict may be explained if rigor rear bridges become distributed irregularly over more actin sites in AMPPNP, so that their average density is too low to appear in the averaged reconstructions. The reconstructions indicate that in insect flight muscle the response of in situ rigor crossbridges to AMPPNP binding is not uniform. Lead bridges persist but have altered structure in the light chain domain, whereas rear bridges detach and possibly redistribute. Shape changes in attached myosin heads within the myofibrillar lattice are in the appropriate direction and of the appropriate magnitude needed to explain the sarcomere lengthening. This could be a direct response to nucleotide binding, a passive response to rear bridge detachment, or a combination of both.

Adenosine Triphosphate↗
PubMed1999

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells.

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.

Adaptation, Physiological↗
PubMed2001

On mutualists and exploiters: plant-insect coevolution in pollinating seed-parasite systems.

We investigate the coevolution of time of flowering and time of pollinator emergence in an obligate association between a plant and an insect that both pollinates and parasitizes flowers. Numerical analysis shows that the system in general evolves towards a time of flowering different from the time favoured by the abiotic environment. The equilibrium towards which the system evolves is a local fitness maximum (an ESS) with respect to mutational variation in flowering time but, for the insect, it can be a local fitness minimum at which selection on mutational variation in the time of insect emergence is disruptive. A consequence of evolutionary convergence to a fitness minimum is that pollinators having an earlier phenology can coexist with pollinators having a later phenology. Since late emerging insects are more likely to encounter and oviposit within previously pollinated flowers, their effect on the plant is more exploitative, leading them to function as cheaters within the system. Thus, in the long term, pollinators and exploiters are likely to be found in stable coexistence in pollinating seed-parasite systems.

Animals↗
PubMed1996

Expression purification and characterization of recombinant human inducible prostaglandin G/H synthase from baculovirus-infected insect cells.

The inducible isoform of human prostaglandin G/H synthase (human cyclooxygenase; hCOX2) has been produced in Sf21 insect cells using the baculovirus expression system. The full-length gene for hCOX2 was placed under the control of the hybrid pCap/PolH promoter and recombinant virus generated by homologous recombination. Insect cells infected with recombinant virus synthesized active hCOX2 at levels exceeding 5% of total cellular protein 72 h postinfection. Optimal production on a preparative scale and high activity yields were attained in 8-liter spinner flasks using a supplemented Grace's medium containing 10% FCS. The apo-enzyme was purified to homogeneity by detergent extraction and ion exchange chromatography and functionally reconstituted with heme to form the holo-enzyme. The purified enzyme from insect cells was identified as hCOX2 by enzymatic activity, Western immunoassay, and N-terminal sequence analysis; the latter also indicated correct processing of the hCOX2 signal sequence. Insect recombinant hCOX2 displays high specific activity for both cyclooxygenase and peroxidase activities at levels indistinguishable from mammalian derived enzyme. Spectroscopic analysis suggests that the recombinant enzyme adopts native-like secondary and tertiary structure. The data presented here demonstrate that this system is capable of providing high yields of active enzyme for biochemical, biophysical, and pharmacological investigations.

Animals↗