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Chromosomal response of insect and mammalian cells to streptonigrin: a comparative study.

We assessed the chromosomal response of insect (mosquito, Aedes albopictus) and mammalian (Chinese hamster ovary, CHO) cells to streptonigrin (SN). Both types of cells were pulse-treated for 20 min with increasing doses of SN and the frequency of chromosome aberrations and sister chromatid exchanges (SCEs) for each SN dose was determined. Our results show that the SN doses inducing remarkable chromosome damage (expressed as frequency of aberrations per cell and per chromosome) in CHO cells fail to produce a significant increase of aberrations in mosquito chromosomes. Moreover, CHO cells exhibited a dose-dependent increase in SCEs which was not observed in mosquito cells. Our results show that while mammalian cells are very sensitive, insect cells are very resistant to SN at the chromosome level. It is possible that variations in the chromatin fibril structure and in the intracellular antioxidant pool may be responsible for the differential response of insect and mammalian chromosomes to SN.

Aedes↗

A new type of highly polymerized yolk protein from the cochineal insect Dactylopius confusus.

A female specific protein was isolated from eggs and female hemolymph of cochineal insects, using density gradient ultracentrifugation, ammonium sulfate precipitation, and size exclusion column chromatography. The protein was found to consist of four different subunits with apparent molecular weights (Mr) 45,000, 49,000, 53,000, and 56,000, respectively. All four subunits were found to be glycosylated; no association of lipids was detected. Size exclusion column chromatography and non-denaturing polyacrylamide gel electrophoresis demonstrated that the native yolk protein exists as large polymers. Electron microscopy showed that these molecules are long, helical ribbons of variable size which are found in both hemolymph and eggs. Using cryo-electron microscopy, it was shown that the ribbons were 14.6 +/- 1.5 nm wide; the helix they form has a repeat distance of 104.9 +/- 11.3 nm and a diameter of 42.1 +/- 5 nm. A clear substructure of the ribbons was recognized. The newly identified protein is the major yolk protein of Dactylopius confusus and no other proteins resembling the more familiar vitellins of other insect species were detected. Moreover, the D. confusus yolk protein appears to be unique both in its subunit structure and in its polymerizing qualities. Thus, the cochineal yolk protein (CYP) is suggested to represent a new type of insect yolk protein.

Amino Acid Sequence↗

Quantification of periviscerokinin-1 in the nervous system of the American cockroach, Periplaneta americana. An insect neuropeptide with unusual distribution.

This study was undertaken to reveal the quantitative distribution of the insect neuropeptide periviscerokinin-1 (Pea-PVK-1) in the central nervous system of Periplaneta americana and to demonstrate that neurons stained in a previous immunohistochemical study contain authentic Pea-PVK-1. For this, we combined ELISA, HPLC, and MALDI-TOF mass spectrometry. The high specificity of the used antiserum enabled the quantification of Pea-PVK-1 in unseparated tissue extracts. No cross-reactivities with other insect neuropeptides were detected in ELISA. Only two immunoreactive fractions, coeluting with synthetic Pea-PVK-1 in its oxidized and nonoxidized form, were found in HPLC-separated extracts of the brain, suboesophageal ganglion, metathoracic ganglion, second abdominal ganglion with or without perisympathetic organ, and terminal ganglion. By using MALDI-TOF mass spectrometry, we were able to confirm the existence of authentic Pea-PVK-1 in these fractions. The abdominal perisympathetic organs contained 6.3 pmol Pea-PVK-1 per animal; another 1.3 pmol were found in the abdominal ganglia. More than 90% of the total 8.2 pmol in the central nervous system was found in the abdominal ganglia and their perisympathetic organs. The corpora cardiaca and corpora allata did not contain immunoreactive material, suggesting that Pea-PVK-1 is not released by the cephalic neurohaemal system. The quantitative distribution of Pea-PVK-1 differs considerably from that of other known insect neuropeptides.

Animals↗

Rhythmic autocrine activity in cultured insect epidermal cells.

It is now well established that ecdysteroids can be produced in insects in the absence of prothoracic glands. In this respect, it has been shown that cells in culture can produce ecdysteroids. Our aims were: (1) to determine whether ecdysteroid target cells of epidermal origin could also be the source of ecdysteroids; (2) to monitor more accurately the kinetics of ecdysteroid production; and (3) to check for possible relationships between this synthetic activity and dynamics of cell division. An insect cell line (IAL-PID2) established from imaginal discs of the Indian meal moth, Plodia interpunctella, with wild-type sensitivity to ecdysteroids was used in our study. Our results showed that the Plodia cell line exhibited autocrine activity. When division of IAL-PID2 cells was synchronized, a rhythmic production of ecdysteroids was observed. However, further experiments indicated that this rhythmicity could be cell autonomous. This led us to anticipate the existence of two cell subpopulations that would be able to produce ecdysteroids rhythmically, a minor one that would be cell cycle serum-independent population, and a major population that would need serum growth factors to proliferate and produce ecdysteroids. Qualitative study of the ecdysteroid content of the media clearly showed that ecdysone was the major immunoreactive product. Taken together, our findings clearly show that an insect cell line of epidermal origin is capable of rhythmic autocrine production of ecdysteroids. These results support the hypothesis that alternate sites for ecdysteroid production in vivo may exist and could play a role in local regulation of development. We now plan to determine the cellular basis of this rhythmic autocrine activity and to confirm the existence of growth factor-autonomous cells in the culture as well as the potent role played by ecdysteroids in the cross-talk between various cell subpopulations.

Animals↗

BmK AS: new scorpion neurotoxin binds to distinct receptor sites of mammal and insect voltage-gated sodium channels.

This study was undertaken to assess the binding properties of BmK AS on both mammal and insect excitable cell membranes. It was found that BmK AS bound specifically to a single class of non-interacting binding sites on both rat brain and cockroach nerve cord synaptosomes with high affinity (K(d) = 1.49 +/- 0.14 and 0.79 +/- 0.29 nM) and low capacity (B(max) = 1.39 +/- 0.09 and 6.60 +/- 1.25 pmol/mg protein), respectively. Binding kinetics showed that BmK AS could bind and reach equilibrium quickly, and dissociate partially from its binding sites on both kinds of synaptosomes. The binding of BmK AS was independent of membrane potential. Veratridine could not modify the binding of BmK AS. The competitive binding assay showed that specific binding of (125)I-BmK AS could be significantly inhibited by native BmK AS, BmK AS-1, BmK IT2 and BmK IT on both synaptosomes. Unexpectedly, only about 20-30% binding of BmK AS on mammal synaptosomes was inhibited by BmK I at 10(-5)-10(-9) M, but not on insect synaptosomes. It thus suggests that BmK AS type neurotoxins might bind to a distinct receptor site of sodium channels on mammal and insect excitable cell membranes with a manner similar to beta-scorpion toxins.

Animals↗

Candidate target mechanisms of the growth inhibitor cyromazine: studies of phenylalanine hydroxylase, puparial amino acids, and dihydrofolate reductase in dipteran insects.

Cyromazine, an insect growth regulator, affects larval and pupal cuticles in dipterans and some other insects. The mode of action of this aminotriazine is not known yet, though it has been shown not to inhibit the synthesis of chitin and cuticular proteins. Cyromazine may, however, act on some step(s) of sclerotization of the cuticle. In the present study, we have analyzed the key enzyme for the production of sclerotization agents, phenylalanine hydroxylase (PAH), using the enzyme from Drosophila, a cyromazine-sensitive insect. PAH was studied in vitro with cyromazine and three biologically less active derivatives at concentrations ranging from 1 microM to 1 mM. None of the compounds did significantly affect PAH activity. Nor did cyromazine, fed to last instar larvae of Musca domestica, change the relative content of phenylalanine and tyrosine, or the total amount and profile of amino acids of puparial cuticles, which showed a larviform shape typical for cyromazine intoxication. Taken together, this study does not support the hypothesis that phenylalanine hydroxylase represents a target site of cyromazine. In additional studies, the conflicting results, as reported by others, on in vitro inhibition of dihydrofolate reductase (DHFR) by cyromazine were re-examined using the enzymes from larvae of the blowfly, Protophormia terraenovae, and from hen liver. There was no significant inhibition of either DHFR at 100 microM by cyromazine as well as by dicylanil, a pyrimidine analog that is biologically more active than cyromazine. In conclusion, the mode of action of cyromazine remains completely open.

Amino Acids↗

Gene expression and the evolution of insect polyphenisms.

Polyphenic differences between individuals arise not through differences at the genome level but as a result of specific cues received during development. Polyphenisms often involve entire suites of characters, as shown dramatically by the polyphenic castes found in many social insect colonies. An understanding of the genetic architecture behind polyphenisms provides a novel means of studying the interplay between genomes, gene expression and phenotypes. Here we discuss polyphenisms and molecular genetic tools now available to unravel their developmental bases in insects. We focus on several recent studies that have tracked gene-expression patterns during social insect caste determination. BioEssays 23:62-68, 2001. Published 2001 John Wiley & Sons, Inc.

Animals↗

Synthesis of antifeedants for insects: novel behaviour-modifying chemicals from plants.

The need to protect our food supply from predatory insect attack using more ecologically acceptable methods has led to a rapidly growing interest in behavior-modifying chemicals from natural sources. Compounds which deter insects from feeding (antifeedants) are attracting special attention owing to their potential use in integrated pest control management systems. We have been studying the synthesis of plant-derived compounds that display antifeedant properties. The aim of the work is to understand more precisely the feeding mechanisms of insects at a molecular level so as ultimately to be able to design simpler compounds capable of mimicking the activity of the natural products. Our synthetic studies on the sesquiterpenoid antifeedants, polygodial and warburganal, and on the diterpenoid clerodane, ajurgarin I, have shown the promise of this approach. Current effort is directed towards structure-activity studies and synthesis of the extraordinarily potent antifeedant and growth-disrupting agent azadirachtin, isolated from the Neem tree, Azadirachta indica (A. Juss). This work has led to the correct structure assignment for azadirachtin and afforded many compounds for biological evaluation. It is of special significance that incorporation of the hydroxydihydrofuran portion of azadirachtin in a simple model system formed a compound with antifeedant activity comparable to that of the natural product.

Animals↗

Plant production of volatile semiochemicals in response to insect-derived elicitors.

An increase in the release of volatile compounds by plants in response to insect feeding is triggered by interaction of elicitors in the oral secretions of insect herbivores with damaged plant tissues. This herbivore damage triggers de novo biosynthesis of volatile plant metabolites derived from several different biochemical pathways. Natural enemies of herbivores use these volatile semiochemicals to locate their hosts. Although some volatile compounds are released from storage in plants immediately whenever damage to cells or glands occurs, the induced compounds are only synthesized and released during the light period. This often results in a delay between feeding damage and release of volatiles. Plants release the induced compounds from undamaged as well as damaged leaves. Thus, damage to only a few leaves results in a systemic response and release of volatiles by the entire plant. We propose that plants respond differently to individual herbivore species at least in part due to the composition of insect elicitors that come in contact with the plant. Specialist parasitoids can differentiate the volatile blends released due to damage by hosts from those resulting from non-host damage as well as from mechanical damage, thereby facilitating host location for the parasitoid. Elicitors in the oral secretions of beet armyworm caterpillars have been identified and synthesized.

Animals↗

Adaptation of insects to insecticides.

Insects have successfully adapted to most insecticides by becoming resistant to them. This adaptation, of recent origin, has evolved rapidly and independently in a large number of species and is of serious economic and medical importance. The origins of resistance are still obscure, but resistance is assumed to be pre-adaptive, arising through recurrent mutation of existing alleles. However, in at least one well-researched case it probably originated by gene duplication. Resistance can be monofactorial or multifactorial. When several independent mechanisms confer resistance to the same group of insecticides the order in which these mechanisms are selected may reflect their effectiveness in protecting insects from the toxic effects of the different compounds. Our concept of resistance is changing as insects continue to adapt to the insecticide-containing environment. Criteria for defining resistance in both fundamental and practical terms are re-examined here in the light of recent work.

Adaptation, Biological↗

Tool use in insect foraging by the chimpanzees of Ngogo, Kibale National Park, Uganda.

Chimpanzee tool use for resource acquisition has been reported at numerous research sites. The chimpanzees of the Kibale Forest, western Uganda, have not previously been observed to use tools in foraging for insects. Here I report the first observation of tool use by the chimpanzees of the Ngogo community of Kibale National Park, Uganda, in insect foraging. Three adult females, one adolescent male, and one juvenile male were observed making and using tools to probe into a fallen dead tree to collect insect and wood pieces. I discuss the importance of this observation, and the behavioral similarities with chimpanzees from other sites.

Animals↗

Antisense inhibition of neuronal nicotinic receptors in the tobacco-feeding insect, Manduca sexta.

Acetylcholine is the predominant excitatory transmitter in the insect central nervous system with many of its effects mediated by nicotinic acetylcholine receptors. These receptors are present at very high density and are structurally heterogeneous, although little is known about functional distinctions between them. An interesting system for examining these receptors is the larval stage of Manduca sexta, a nicotine-resistant tobacco-feeding insect. The nicotinic responses of cultured neurons were found to be blocked by mecamylamine and curare but highly resistant to alpha-bungarotoxin. The responses were also unaffected by the reducing agent dithiothreitol and the alkylating agent bromoacetylcholine suggesting that the alpha-subunit dicysteine agonist binding site is protected. To begin determining the functional roles of different subunits in these receptors, cultured neurons were treated with oligonucleotides based on the gene sequence of the alpha subunit, MARA1. Antisense DNA caused a significant downward shift in the amplitude distribution of nicotinic responses compared to sense or reverse antisense treatments. These treatments did not affect currents mediated by the application of GABA. The reduction in the nicotinic depolarization and inward currents did not affect the rate of current onset or recovery, suggesting that antisense MARA1 causes a partial block of all nicotinic responses in these neurons. These results demonstrate that receptor gene expression in insect neurons can be manipulated in a sequence-specific manner by antisense treatment and they provide evidence that MARA1 is important for normal nicotinic responses in Manduca.

Animals↗

Pharmacological approaches to nitric oxide signalling during neural development of locusts and other model insects.

A novel aspect of cellular signalling during the formation of the nervous system is the involvement of the messenger molecule nitric oxide (NO), which has been discovered in the mammalian vascular system as mediator of smooth muscle relaxation. NO is a membrane-permeant molecule, which activates soluble guanylyl cyclase (sGC) and leads to the formation of cyclic GMP (cGMP) in target cells. The analysis of specific cell types in model insects such as Locusta, Schistocerca, Acheta, Manduca, and Drosophila shows that the NO/cGMP pathway is required for the stabilization of photoreceptor growth cones at the start of synaptic assembly in the optic lobe, for regulation of cell proliferation, and for correct outgrowth of pioneer neurons. Inhibition of the NOS and sGC enzymes combined with rescue experiments show that NO, and potentially also another atypical messenger, carbon monoxide (CO), orchestrate cell migration of enteric neurons. Cultured insect embryos are accessible model systems in which the molecular pathways linking cytoskeletal rearrangement to directed cell movements can be analyzed in natural settings. Based on the results obtained from the insect models, I discuss current evidence for NO and cGMP as essential signalling molecules for the development of vertebrate brains.

Animals↗

Insect detoxifying enzymes: their importance in pesticide synergism and resistance.

Pyrethroid esterases of Trichoplusia ni, Spodoptera littoralis and Bemisia tabaci hydrolyze the trans-isomers of various pyrethroids more extensively than the cis-isomers. Profenofos fed to T. ni larvae at a level inhibiting the gut pyrethroid esterases by 65% with trans-permethrin and of 95% with cis-cypermethrin increased the toxicity of topically applied trans-permethrin by fourfold and cis-cypermethrin by 20-fold. Similar assays with S. littoralis resulted in an increase of about threefold in the toxicity of both compounds. Monocrotophos, profenofos, acephate, and methidathion inhibited pyrethroid esterase activity in B. tabaci and synergized considerably the toxicity of cypermethrin. The remarkable tolerance of the predator Chrysopa carnea to pyrethroids is attributed to the presence of a high level of pyrethroid esterase activity with a unique specificity for hydrolyzing the cis-isomer. Phenyl saligenin cyclic phosphonate, a potent inhibitor for larval pyrethroid esterases synergized the toxicity of trans-permethrin by 68-fold from an LD50 of 17,000 micrograms/g to 250 micrograms/g. In contrast, oxidase inhibitors such as piperonyl butoxide, SV-1, and MPP synergized considerably the toxicity of pyrethroids in Tribolium castaneum and Musca domestica. Hence the predominant pathway for pyrethroid detoxification in insects, whether hydrolytic or oxidative, depends largely on the insect species. The high toxicity of the recent developed acylureas results from their high retention in the insects. Assays using radiolabeled diflubenzuron and chlorfluazuron applied to fourth instar T. castaneum larvae revealed a rapid elimination of diflubenzuron (T1/2 approximately equal to 7 h) as compared with chlorfluazuron (T1/2 > 100 h). Addition of 100 ppm DEF to the diet increased both the retention time and the toxicity of diflubenzuron in both T. castaneum and S. littoralis, which was due probably to the inhibition of diflubenzuron hydrolase activity. Esterases, hydrolyzing pyrethroids, and acylureas may serve as tools for evaluating potential synergists and for monitoring resistance in various agricultural pests due to increased metabolism.

Animals↗

Expression of hornet genes encoding venom allergen antigen 5 in insects.

Antigen 5, also known as Dol m V, is a major allergen found in the venom of the baldfaced hornet, Dolichovespula maculata. We have inserted the f10 and f17 cDNAs, which encode hornet antigen 5 (HA5) forms 2 (Dol m; VB) and 3 (Dol m VA), respectively, into the genome of the baculovirus, AcMNPV, to produce the recombinant baculovirus gene expression vectors, vEV-HA5f10 and vEV-HA5f17. Insect cells infected with either vEV-HA5f10 or vEV-HA5f17 produce and secrete a novel protein with an electrophoretic mobility which is similar if not identical to authentic mature Dol m V. The gene products also react specifically with a polyclonal antiserum raised to Dol m VB as expected. Dol m V gene products were not acutely toxic when injected into insect larvae. However, infection of fifth instar larvae with vEV-HA5f17 resulted in premature melanization of the larvae and lower weight gain than infection with control virus. Thus, the Dol m V gene product has a subtle, possibly cytotoxic or biochemical effect on insects. The expression systems may prove useful in further structural and functional characterization of these proteins.

Allergens↗

Plant coloration undermines herbivorous insect camouflage.

The main point of our hypothesis "coloration undermines camouflage" is that many color patterns in plants undermine the camouflage of invertebrate herbivores, especially insects, thus exposing them to predation and causing them to avoid plant organs with unsuitable coloration, to the benefit of the plants. This is a common case of "the enemy of my enemy is my friend" and a visual parallel of the chemical signals that plants emit to call wasps when attacked by caterpillars. Moreover, this is also a common natural version of the well-known case of industrial melanism, which illustrates the great importance of plant-based camouflage for herbivorous insects and can serve as an independent test for our hypothesis. We claim that the enormous variations in coloration of leaves, petioles and stems as well as of flowers and fruits undermine the camouflage of invertebrate herbivores, especially insects. We assume that the same principle might operate in certain animal-parasite interactions. Our hypothesis, however, does not contrast or exclude other previous or future explanations of specific types of plant coloration. Traits such as coloration that have more than one type of benefit may be selected for by several agents and evolve more rapidly than ones with a single type of advantage.

Animals↗

Molecular and cellular organization of insect chemosensory neurons.

Animals use their chemosensory systems to detect and discriminate among chemical cues in the environment. Remarkable progress has recently been made in our knowledge of the molecular and cellular basis of chemosensory perception in insects, based largely on studies in Drosophila. This progress has been possible due to the identification of gene families for olfactory and gustatory receptors, the use of electro-physiological recording techniques on sensory neurons, the multitude of genetic manipulations that are available in this species, and insights from several insect model systems. Recent studies show that the superfamily of chemoreceptor proteins represent the essential elements in chemosensory coding, endowing chemosensory neurons with their abilities to respond to specific sets of odorants, tastants or pheromones. Investigating how insects detect chemicals in their environment can show us how receptor protein structures relate to ligand binding, how nervous systems process complex information, and how chemosensory systems and genes evolve.

Animals↗

Plants on red alert: do insects pay attention?

Two recent hypotheses have proposed that non-green plant colouration evolved as a defence against herbivores, either as protective colouration promoting handicap signals indicating plant fitness or by undermining their crypsis. The handicap hypothesis posits a co-evolutionary process between plants and herbivores, whereas the anti-crypsis hypothesis suggests that an arms race between insects and plants is the evolutionary mechanism. Both explanations assume that insects are the evolutionary origin causing plants' colouration. Here, we propose a different hypothesis, termed the "Defence Indication hypothesis". This idea focuses on the multiple protective functions of anthocyanins and carotenoids as pigments, and suggests that plant colouration evolved primarily in response to various stressors. Because pigments and defensive compounds share a common biosynthesis, the production of pigments also provides elevated defensive strengths against herbivores, a process termed priming. In effect, the Defence Indication hypothesis predicts that pleiotropic effects of the pigments and, more generally, plants' shared defence responses, explain why insects might react to plant colouration.

Animals↗